Provisional Patent Application Serial Number 60/526,764, filed December 3,2003.

FIELD OF THE INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

BACKGROUND OF THE INVENTION Detergent and other cleaning compositions typically include a complex combination of active ingredients. For example, most cleaning products include a surfactant system, enzymes for cleaning, bleaching agents, builders, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes.

Despite the complexity of current detergents, there are many stains that are difficult to completely remove. Furthermore, there is often residue build-up, which results in

discoloration (e. g. , yellowing) and diminished aesthetics due to incomplete cleaning.

These problems are compounded by the increased use of low (e. g., cold water) wash temperatures and shorter washing cycles. Moreover, many stains are composed of complex mixtures of fibrous material, mainly incorporating carbohydrates and carbohydrate derivatives, fiber, and cell wall components (e. g. , plant material, wood, mud/clay based soil, and fruit). These stains present difficult challenges to the formulation and use of cleaning compositions.

In addition, colored garments tend to wear and show appearance losses. A portion of this color loss is due to abrasion in the laundering process, particularly in automated washing and drying machines. Moreover, tensile strength loss of fabric appears to be an unavoidable result of mechanical and chemical action due to use, wearing, and/or washing and drying. Thus, a means to efficiently and effectively wash colored garments so that these appearance losses are minimized is needed.

Cleaning compositions that comprise esterases, lipases and cutinases are well- known in the art. However, these enzymes have a very low ratio of perhydrolysis to hydrolysis. This results in the conversion of most of the ester substrate into acid, instead of the more desirable peracid. This is a serious drawback, since formula space and cost considerations render it feasible to include only a limited amount of substrate.

In sum, despite improvements in the capabilities of cleaning compositions, there remains a need in the art for detergents that remove stains, maintain fabric color and appearance, and prevent dye transfer. In addition, there remains a need for detergent and/or fabric care compositions that provide and/or restore tensile strength, as well as provide anti-wrinkle, anti-bobbling, and/or anti-shrinkage properties to fabrics, as well as provide static control, fabric softness, maintain the desired color appearance, and fabric anti-wear properties and benefits. In particular, there remains a need for the inclusion of compositions that are capable of removing the colored components of stains, which often remain attached to the fabric being laundered. In addition, there remains a need for

improved methods and compositions suitable for textile bleaching.

In addition to the fabric and garment cleaning area, bleaching is commonly used in the pulp and paper industry. Prior to production of paper, pulp is typically treated to remove undesirable colored contaminants. This provides pulp that is suitable for production of paper of higher quality than pulp that is not treated to remove colored contaminants and other undesirable components present in pulp. For example, in the paper recycling industry, removal of ink is necessary. Although standard methods are suitable for deinking paper with oil or water-based inks, the increased use of electrostatic inks has made deinking problematic, as these inks are much more difficult to remove.

There are various methods available for deinking paper, including the use of enzymes (See e. g., U. S. Patent No. 5,370, 770). However, there remains a need in the art for efficient, cost-effective methods for treatment of pulp for paper (recycled and new) product production.

Bleaching is also commonly used in the personal care market (e. g., dental whiteners, hair bleachers, etc. ). Although personal care bleaching products have improved over the years, there remains a need for mild, easy to use, cost-effective bleaching methods for this setting.

SUMMARY OF THE INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

In some embodiments, the present invention provides compositions comprising at least one perhydrolase, wherein the perhydrolase exhibits a perhydrolysis to hydrolysis

ratio that is greater than 1.

The present invention also provides isolated perhydrolases, wherein the perhydrolases exhibit a perhydrolysis to hydrolysis ratio that is greater than 1. In some preferred embodiments, the perhydrolase is M. smegmatis perhydrolase. In alternative preferred embodiments, the perhydrolase is at least approximately about 35% homologous to M. smegmatis perhydrolase. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO : 2. In some preferred embodiments, the perhydrolases have immunological cross- reactivity with M. smegmatis perhydrolase. In still further embodiments, the perhydrolase is at least a portion of M. smegmatis perhydrolase, wherein the perhydrolase has a perhydrolysis to hydrolysis ration that is greater than 1. In alternative embodiments, the perhydrolase is a structural homologue of M. smegmatis perhydrolase, in which the active site is homologous to at least one amino acid selected from the group consisting of Sl l, D192, andH195 ofthe M. smegmatis perhydrolase.

The present invention also provides isolated perhydrolase variants having amino acid sequences comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2. In some embodiments, at least one modification is made at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein the modified amino acid is selected from the group consisting of Cys7, AsplO, Serl l, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thrl59, Thrl86, Ilel92, Ilel94, and Phel96. In further embodiments, the modification comprises at least one substitution at an amino acid position equivalent to a

position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of Ml, K3, R4, I5, L6, C7, D10, Sll, L12, T13, W14, W16, G15, V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31, V32, R33, W34, T35, G36, L38, Q40, Q41, D45, L42, G43, A44, F46, E47, V48, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, I60, D61, D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77, L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97, Y99F100, R101, R102, P104, L105, D106, I107, A108, L109, 0110, Mill, S112, V113, L114, V115, T116, Q117, V118, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P146, P148, W149, F150, I153, F154, I194, and F196.

In some preferred embodiments, the variant perhydrolase exhibits a change in peracid hydrolysis compared to the wild-type perhydrolase. In some embodiments, the change in peracid hydrolysis is a decrease, while in other embodiments, the change in peracid hydrolysis is an increase.

In some alternative preferred embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.1 or less, in comparison with wild-type perhydrolase. In alternative preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of R4, L12, G15, P18, R27, W34L38, A44, E51, G52, L53, S54, T58, R67, L68, S72, A79, T80, D85, L86, V87, N94, K97, R101, V118, Ll l9, G124, G126, and I194.

In further alternative embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.2 or less, in comparison with wild-type perhydrolase. In yet additional embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in

M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of R4, I5, D10, L12, W14, G15, P18, V19, T25, R27, W34, L38, A44, I49, E50, E51, G52, L53, S54, A55, R56, T58, N59, D62, T64, D65, R67, L68, N69, S72, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, R101, L82, P83, L86, V87, N94, T96, K97, F100, R101, L109, Ml 11, L114, VI 18, L119, A122, G124, G126, T127, Y129, W149, and I194.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.3 or less, in comparison with wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of R4, I5, D10, L12, W14, G15, L12, P18, V19, G22, A23, T25, E26, R27, W34, G36, L38, Q41, L42, G43, A44, I49, E50, E51, G52, L53, S54, A55, R56, T57, N59, T58, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, Y99, F100, R101, R102, P104, L109, G110, Mlll, L114, V118, Lll9, A122, G124, V125, G126, T127, Y129, W149, F154, and I194.

In yet further embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.4 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of R4, I5, L6, D10, Sll, L12, W14, G15, W16, P18, V19, G22, A23, T25, E26, R27, F28, W34, T35, G36, L38, Q41, L42, G43, A44, D45, E47, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, T58, I60, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76,

C77, A79, T80, L82, P83, D85, L86, V87, N94, P66, T96, K97, Y99, F100, R101, R102, P104, I107, L109, G110, M111, S112, L114, V118, L119, S121, A122, G124, V125, G126, T127, Y129, W149, F150, F154, I194, and F196.

In some embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.5 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S 112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, LI 19, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, S112, S121, S54, S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In additional embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis of about 0.6 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in

M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, LI 19, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, Ml 11, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, S112, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, V125, V19, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In yet additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.7 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, Gl 10, G124, G43, L109, L119,

L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, Ml 11, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, S112, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, Sl 1, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84, L86, Ml 11, N59, P146, P24, P66, Q41, R102, R27, R56, Sl 12, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In still further embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0. 8 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119,

L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, Mill, L114, L119, W149, Yld29, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, S11, S112, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84, L86, Mill, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52, I107, I194, I49, I5, I60, I89, L114, L42, L53, L68, L78, L84, Mill, N59, N94, P146, P24, P30, P63, P66, P83, Q117, R101, R4, S112, S121, S72, T116, T120, T127, T13, T57, T96, V113, V125, V17, V19, V32, V87, W149, Y129, Y73, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 1.5 or greater, in comparison with wild-type perhydrolase. In some

preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, LI 19, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, Sl 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, LI 19, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, Ml 11, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, S112, S121, S54, S72, T25, T35, T57, T58, Vl 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84, L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52, I107, I194, I49, I5, I60, I89, L114, L42, L53, L68, L78, L84, Mill, N59, N94, P146, P24, P30, P63, P66, P83, Q117,

R101, R4, Sl 12, S121, S72, Tl 16, T120, T127, T13, T57, T96, V113, V125, V17, V19, V32, V87, W149, Y129, and Y73, Y99, A108, A44, C7, D10, D106, D31, D61, D85, E26, E51, F100, F28, F46, G110, G22, G36, G43, G52, G70, I107, I153, I49, I5, I89, K3, L105, L53, L6, L78, L86, M1, N69, P104, P146, P18, P24, P30, P83, Q117, Q40, Q41, R102, R27, R33, R4, S121, S72, S76, T120, T128, T13, T35, T80, T96, V115, V118, V32V48, V87, W34, G190, V191, G193, T197, E198, A199, R202, D203, G205, V206, A209, E210, Q211, S214, and L215.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis between about 1.2 and about 1.5, in comparison with wild-type perhydrolase.

In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, C7, D106, D31, D61, D85, E26, E50, E51, F100, F150, F28, F46, G110, G126, G22, G70, I107, K3, L105, L42, L6, L78, Mill, N59, N69, P104, P146, P148, P18, P30, P63, Q117, Q40, Q41, R102, R27, R33, R4, S54, S76, T116, T120, T128, T64, T80, T96, V113, V115, V118, W34, and Y73.

In yet further embodiments, the present invention provides variant perhydrolases in which the variant perhydrolases exhibit a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is at least about 1.2. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, I49, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95,

K97, R101, T103, P104, L105, D106, I107, L109, Mlll, V113, Q117, V118, S121, G124, V125, G126, T127, P148, F150, I153, F154, and F196.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0. 8 or less. In some embodiments, the variant perhydrolase comprising at least one modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D45, D61, D62, D65, D85, E26, E47, E50, E51, F100, F150, F154, F196, F28, F46, G110, G124, G126, G15, G22, G36, G52, G70, I107, I153, I194, I49, I5, 160, I89, K3, K97, L105, L109, L114, L119, L12, L38, L42, L53, L6, L68, L78, L82, L84, K86, Ml, Mill, N59N94, P146, P18, P24, P30, P66, P83, Q40, Q41, R101, R102, R27, R33, R4, R56, R67, S11, S112, S54, S72, S76, T103, T116, T120, T127, T128, T13, T25, T35, T57, T64, T80, T96, V113, V115, V118, V125, V17, V19, V32, V48, V87, W13, W149, W16, W34, Y129, Y73, and Y99.

In alternative embodiments, the present invention provides variant perhydrolases comprising at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D31, D45, D61, D62, D65, D85, E26, E47, E50, E51, F100, F150, F154F196, F28, F46, G110, G124, G126, G15, G22, G36, G43, G52, G70, I107, I153, I194, I49, I5, I60, I89, K3, K97, L105, L109, L114, L119, L12, L38, L42, L53, L6, L68, L78, L82, L84, L86, M1, M111, N59, N69, N94, P104, P146, P148, P18, P24, P30, P63, P66, P83, Q117, Q40, Q41, R101, R102, R27, R33, R4, R56, R67, Sll, S112, S121, S54, S72, S76, T103, T116, T120, T127, T128, T13, T25, T35, T57, T58, T64, T80, T96,

V113, V115, V118, V125, V17, V19, V32, V48, V87, W14, W149, W16, W34, Y129, Y73, and Y99.

In yet additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 1.2 and about 2. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, I49, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, R101, T103, P104, L105, D106, I107, L109, Mill, VI 13, Q117, VI 18, S121, G124, V125, G126, T127, P148, F150, I153, F154, F196, G190, E198, A199, R202, D203, V206, A209, E210, Q211, and V212.

In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2 and about 2.5. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A44, C7, D10, D85, D95, E26, E47, I107, L12, L42, P104, P148, S54, Q40, Q117, D203, V206, E210.

In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2.5 and about 3. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at

an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A44, C7, I107, K97, L12, L78, P104, Q40, and V125.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 3.0 and about 5. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of D10, D85, L53, L78, and S54.

In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.1 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sl l, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, and W34.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.2 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from

the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, Il 10, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, and Y73.

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.3 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sl 1, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, Mill, N94, P146, Q41, R102, R27, R4, R56, Sl 1, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, and Y129.

In yet additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.4 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is

selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, Mill, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, and V87.

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.5 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, Mill, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76,

T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, Mill, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, and Y129.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.6 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising t least one substitution at an amino acid position equivalent to a position in M. srreegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, Mil 1, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, I107, I5, I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115, V118, V32, V48, V87, W149, and Y73.

In yet further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.7 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, Gl 10, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, MI 11, N94, P146, Q41, R102, R27, R4, R56, Sl 1, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, Sl 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, I107, I5, I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115, V118, V32, V48, V87, W149, Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, F100, F28, F46, G110, G126, G52, G70, I107, I49, I5, I60, I89, L109, L114, L12, L38, L68, L82, L86, Ml 11, N59, N94, P83, R102, R33, R4, S112, S72, S76, T103, T116, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, and Y99.

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0. 8 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, Mill, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, Ml 11, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, I107, I5, I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115, V118, V32, V48, V87, W149, Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, F100, F28, F46, G110, G126, G52, G70, I107, I49, I5, I60, I89, L109, L114, L12, L38, L68, L82, L86, Ml 11, N59, N94, P83, R102, R33, R4, S112, S72, S76, T103, T116, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, Y99, A108, A122, A29, A55, C77, D10, D106, D45, D61, D62, D65, D85,

E47, E50, F100, F150, F28, F46, G110, G124, G126, G15, G36, I153, I194, I5, I60, 189, K3, K97, L105, L109, L114, Ll 19, L38, L42, L68, L84, L86, Ml, N59, P24, P30, P83, R101, R27, R4, R56, S112, S54, S76, T103, T116, T120, T127, T128, T13, T35, T64, V113, V17, V19, V32, V48, V87, Y129, Y73, and Y99.

The present invention also provides perhydrolase variants, wherein the perhydrolase variants exhibit greater perhydrolysis activity and decreased peracid hydrolysis activity as compared to wild-type perhydrolase. In some embodiments, the variant perhydrolases exhibit perhydrolysis activity ratio of at least about 1.2, and peracid hydrolysis activity ratio of about 0.8 or less, as compared to wild-type perhydrolase. In alternative embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A29, A44, A55, A71, A79, C7, D10, D106, D31, D85, E26, E47, F150, F154, F196, F28, G124, G126, G36, G43, I153, L109, L42, L53, L109, L42, L53, L109, L42, L53, L68, L82, L86, Ml 11, N69, P104, P148, P18, P63, P66, P83, Q117, Q40, R101, R67, S54, S121, S72, S76, T25, T64, V115, and V19.

In additional embodiments, the perhydrolase exhibits perhydrolysis activity ratio of at least about 1.2, a peracid hydrolysis activity ratio of about 0.8 or less, and a protein concentration ratio of at least 0.5, as compared to wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A29, A44, A71, A79, C7, D85, E26, E47, E51, F150, F154, F196, F28, G124, G126, G36, I153, L109, L12, L53, L68, L82, Mlll, N69, P104, P148, P18, P63, P66, P83, Q117, Q40, R101, R67, S121, S54, S72, S76, T25, T64, V125, and V19.

The present invention provides variant perhydrolases that exhibit an increase in expression of the perhydrolase variants, as compared to the expression of wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2, wherein at least one substitution is selected from the group consisting of A2, 15, C7, F8, Sll, L12, T13, W14, W16, V17, P18, V19, E20, G22, A23, P24, T25, A29, P30, V32, T35, G36, V37, A39, F46, E47, S54, A55, R56, T58, I60, D61, D62, P63, T64, P66, R67, L68, N69, G70, S72, Y73, L74, P75, S76, C77, L78, A79, T80, L82, P83, L84, L86, 189, T93, T96, K97, A98, Y99, F100, R101, R102, T103, P104, L105, D106, I107, A108, L109, G110, S112, VI 13, LI 14, VI 15, T116, Q117, VI 18, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P130, P132, K133, L135, V136, S138, P141, L142, A143, M145, H147, W149, F150, Q151, I153, G157, Q159, T161, T162, L164, A165, R166, V167, Y168, A170, L171, A172, M175, K176, P178, A182, G183, S184, V185, I186, T188, I194, F196, V191, N201, L208, A209, Q211, Q213, S214, L215, and L216.

The present invention also provides isolated proteins comprising homologs of M. smegmatis perhydrolase, wherein the homologs are proteins within the SGNH-hydrolase family of proteins. In alternative preferred embodiments, the isolated proteins have at least about 35% identity with the amino acid sequence of M. smegmatis perhydrolase, in which the protein comprises at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, Sl l, D192, and H195. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO : 2.

The present invention also provides isolated proteins having at least about 38% identity with the amino acid sequence of M. smegmatis perhydrolase, wherein the protein exhibits perhydrolysis activity. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO : 2.

The present invention also provides homologs of M. smegmatis perhydrolase, wherein the homologs are perhydrolases comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In preferred embodiments, the homologs exhibit perhydrolysis. In some particularly preferred embodiments, the homologs exhibit a perhydrolysis to hydrolysis ratio that is great than about 1. In still further embodiments, the homologs are immunologically cross-reactive with antibodies raised against M. smegmatis perhydrolase. In yet additional embodiments, antibodies raised against the homolog cross-react with M. smegmatis perhydrolase.

The present invention also provides isolated proteins having at least-about 35% identity with the amino acid sequence of at least one M. smegmatis perhydrolase homolog, wherein the proteins exhibit perhydrolysis activity.

In some particularly preferred embodiments, the present invention provides proteins having perhydrolase activity, wherein the proteins are in the form of a multimer in solution. In some more preferred embodiments, the protein is a perhydrolase that comprises a dimer. In alternative particularly preferred embodiments, the protein is a perhydrolase that comprises an octamer. In still further embodiments, the protein is in the form of a multimer in solution and the protein is selected from the group consisting of M. smegmatis perhydrolase, M. smegmatis perhydrolase homologs, and M. smegmatis perhydrolase variants. In yet further embodiments, the protein is selected from the group

consisting of modified serine hydrolases and modified cysteine hydrolases, wherein the modified serine hydrolases or modified cysteine hydrolases comprise increased perhydrolase activity as compared to unmodified serine hydrolases or unmodified cysteine hydrolases The present invention also provides proteins having perhydrolase activity, wherein the protein comprises at least one motif selected from the group consisting of GDSL- GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In some embodiments, the protein is obtained from a member of the Rhizobiales. In some preferred embodiments, the protein is obtained from a member of the genus Mycobacterium.

The present invention also provides isolated genes identified using at least one primer selected from the group consisting of SEQ ID NOS: 21-69.

The present invention also provides methods for identifying a perhydrolase, comprising the steps of : identifying source of the perhydrolase; analyzing the source to identify sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT; expressing the sequences identified in step b) to produce the perhydrolase; and testing the perhydrolase for perhydrolysis activity.

In some embodiments, the analyzing step is an amplification step wherein the primer sequences set forth in SEQ ID NOS: 21-69 are used to amplifying the sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL- ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In still further embodiments, the source is selected from the group consisting of environmental sources and metagenomic sources. The present invention also provides proteins identified using the methods set forth herein. The present invention further provides isolated nucleic acid sequences encoding the proteins identified using the methods set forth herein. In some particularly preferred embodiments, the proteins exhibit a perhydrolysis to hydrolysis

ratio that is greater than about 1. In still further embodiments, the proteins exhibit a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase. In yet additional embodiments, the proteins comprise at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, Sll, D192, and H195.

In further embodiments, the analyzing step comprises searching at least one amino acid database. In yet further embodiments, the analyzing step comprises searching at least one nucleic acid database to identify nucleic acid sequences encoding the amino acid sequences of the perhydrolase. In still further embodiments, the source is selected from the group consisting of environmental sources and metagenomic sources. The present invention further provides isolated nucleic acid sequences encoding the proteins identified using the methods set forth herein. In some particularly preferred embodiments, the proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than about 1. In still further embodiments, the proteins exhibit a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase. In yet additional embodiments, the proteins comprise at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, Sll, D192, and H195, as set forth in SEQ ID NO : 2.

The present invention also provides variant perhydrolases having altered substrate specificities as compared to wild-type M. smegmatis perhydrolase. In some embodiments, the variant perhydrolases have altered para nitrophenyl caproate (PNC) activity, as compared to wild-type M. smegmatis perhydrolase.

The present invention also provides variant perhydrolases having altered pI values as compared to wild-type M. smegmatis perhydrolase. In some embodiments, the variant perhydrolases comprise at least one positively charged mutation, while in alternative

embodiments, the variant perhydrolases comprise at least one negatively charged mutation.

The present invention also provides variant perhydrolases that have increased stability, as compared to wild-type M. smegmatis perhydrolase. In some preferred embodiments, the stability of the variant perhydrolase is selected from the group consisting of thermostability, enzymatic stability, and chemical stability.

The present invention also provides variant perhydrolases, wherein the variant perhydrolase exhibits at least one altered surface property. In some preferred embodiments, the variants comprise at least one mutation comprising at least one substitution at sites selected from the group consisting of the residues set forth in Table 15-1.

The present invention also provides perhydrolase variants having at least one improved property as compared to wild-type perhydrolase.

The present invention also provides expression vectors comprising a polynucleotide sequence encoding at least one perhydrolase variant. The present invention further provides host cells comprising at least one such expression vector. In some preferred embodiments, a host cell is selected from the group consisting of Bacillus sp. , Streptomyces sp. , Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by the host cells.

The present invention also provides compositions comprising at least a portion of at least one perhydrolase. In some preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO : 2. In further embodiments, the perhydrolase is encoded by a polynucleotide sequence comprises SEQ ID NO : 1. In additional embodiments, the sequence comprises at least a portion of SEQ ID NO : 1. In further embodiments, the present invention provides expression vectors comprising the polynucleotide sequence encoding at least a portion of at least one perhydrolase. The present invention also provides host comprising at least one expression vectors. In some

embodiments, the host cells are selected from the group consisting of Bacillus sp. , Streptomyces sp. , Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by these host cells.

The present invention also provides variant perhydrolases, wherein the perhydrolases comprise at least one substitution corresponding to the amino acid positions in SEQ ID NO : 2, and wherein the variant perhydrolase has better performance in at least one property, compared to wild-type M. smegmatis perhydrolase.

The present invention further provides isolated polynucleotides comprising a nucleotide sequence (i) having at least about 70% identity to SEQ ID NO : 1, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ID NO : 1, under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence set forth in SEQ ID NO : 1. In some embodiments, the present invention also provides vectors comprising these polynucleotide sequences. In additional embodiments, the present invention also provides host comprising at least one expression vectors. In some embodiments, the host cells are selected from the group consisting of Bacillus sp. , Streptomyces sp. , Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by these host cells.

The present invention also provides polynucleotides comprising a sequence complementary to at least a portion of the sequence set forth in SEQ ID NO : 1.

The present invention also provides methods of producing enzymes having perhydrolase activity, comprising: transforming a host cell with an expression vector comprising a polynucleotide having at least 70% sequence identity to SEQ ID NO: 1; cultivating the transformed host cell under conditions suitable for the host cell to produce the perhydrolase; and recovering the perhydrolase. In some preferred embodiments, the host cell is selected from the group consisting of Streptomyces, Pantoea, Escherichia, and Bacillus species.

The present invention also provides probes comprising a 4 to 150 polynucleotide sequence substantially identical to a corresponding fragment of SEQ ID NO : 1, wherein the probe is used to detect a nucleic acid sequence coding for an enzyme having perhydrolase activity.

The present invention also provides cleaning compositions comprising: a) at least 0.0001 weight percent of a perhydrolase that exhibits a perhydrolysis to hydrolysis ratio that is greater than 1; b) a molecule comprising an ester moiety; and c) optionally, an adjunct ingredient.

The present invention further provides cleaning compositions comprising: a) at least 0.0001 weight percent of a perhydrolase that exhibits a perhydrolysis to hydrolysis ratio that is greater than 1; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, the peroxygen source being selected from the group consisting of : a per-salt; an organic peroxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture, and mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient.

The present invention also provides cleaning compositions comprising: a) from about 0.0001 to about 1 weight percent of a variant perhydrolase having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in Msmegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, the peroxygen source being selected from the group consisting of : a per-salt; an organic peroxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture; and mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient. In some preferred embodiments, the cleaning compositions further comprise at least one adjunct ingredient. In some particularly

preferred embodiments, the adjunct ingredient is selected from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti- redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof.

In additional embodiments, the present invention provides cleaning compositions wherein: the perhydrolase exhibits a perhydrolysis to hydrolysis molar ratio that is greater than about 0.1 ; the per-salt is selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof; the carbohydrate is selected from the group consisting of mono- carbohydrates, di-carbohydrates, tri-carbohydrates, oligo-carbohydrates and mixtures thereof ; the carbohydrate oxidase is selected from the group consisting of aldose oxidase (IUPAC classification EC1. 1.3. 9), galactose oxidase (IUPAC classification EC1. 1.3. 9), cellobiose oxidase (IUPAC classification EC1. 1.3. 25), pyranose oxidase (IUPAC classification EC1. 1.3. 10), sorbose oxidase (IUPAC classification EC1. 1.3. 11) hexose oxidase (IUPAC classification EC1.1. 3.5). glucose oxidase (IUPAC classification EC1. 1.3. 4) and mixtures thereof; and the molecule comprising an ester moiety has the formula : R [ (ruz m (R) n] p (i) wherein Rl is a moiety selected from the group consisting of H, substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; (ii) each R2 is an alkoxylate moiety; (iii) R3 is an ester-forming moiety having the formula:

R4CO-wherein R4 is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl ; (iv) x is 1 when Rl is H; when Rl is not H, x is an integer that is equal to or less than the number of carbons in Rl ; (v) p is an integer that is equal to or less than x; (vi) m is an integer from 0 to 50; and (vii) n is at least 1 In alternative embodiments, the present invention provides cleaning compositions wherein: a) R is an C2-C32 substituted or unsubstituted alkyl or heteroalkyl moiety; b) each R2 is independently an ethoxylate or propoxylate moiety; and c) m is an integer from 1 to 12. In some embodiments, R3 is an ester-forming moiety having the formula: R4CO- wherein R4 is: a) a substituted or unsubstituted alkyl, alkenyl or alkynyl moiety comprising from 1 to 22 carbon atoms; or b) a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety comprising from 4 to 22 carbon atoms.

In still further embodiments of the cleaning compositions, the molecule comprising the ester moiety has the formula: R'0. [ (Rm (Rn] p wherein: a) Rl is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, the R'moiety that comprises an amine moiety being selected from the group consisting of substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; b) each R2 is an alkoxylate moiety; c) R3 is an ester-forming moiety having the formula: R4CO- wherein R4 may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; d) x is 1 when Rl is H; when Rl is not H, x is an integer that is equal to or less than the number of carbons in Rl ; e) p is an integer that is equal to or less than x; f) m is

an integer from 0 to 12; and g) n is at least 1.

In still further embodiments of the present cleaning compositions, the molecule comprising an ester moiety has a weight average molecular weight of less than 600,000 Daltons. In yet additional embodiments, an adjunct ingredient is selected. from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof.

The present invention further provides methods of cleaning comprising the steps of : a) contacting a surface and/or an article comprising a fabric with any of the cleaning compositions provided above and/or a composition comprising any of the cleaning compositions provided above; and b) optionally washing and/or rinsing the surface or material.

In alternative embodiments, the present invention provides methods of cleaning, the method comprising the steps of : a) contacting a surface and/or an article comprising a fabric with any suitable cleaning composition provided above and/or a composition comprising any suitable cleaning provided above; and b) optionally washing and/or rinsing the surface or material.

The present invention also provides bleaching compositions comprising at least one perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2..

In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase variant having at least one improved property as compared to wild-type perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase variant comprising at least one substitution corresponding to the amino acid positions in SEQ II) NO : 2, and wherein the variant perhydrolase has better performance in at least one property compared to wild-type M. smegmatis perhydrolase..

In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase that is at least approximately about 35% homologous to M. smegmatis

perhydrolase.. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2..

In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant having at least one improved property as compared to wild-type perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant comprising at least one substitution corresponding to the amino acid positions in SEQ ID NO : 2, and wherein the variant perhydrolase has better performance in at least one property compared to wild-type M. smegmatis perhydrolase.

In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase that is at least approximately about 35% homologous to M. smegmatis perhydrolase.. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

In some preferred embodiments, the perhydrolase is at least approximately 70% homologous to M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO : 2. In some embodiments, the present invention provides perhydrolases that cross react with antibody generated against M. smegmatis perhydrolase, particularly that comprising the amino acid sequence set forth in SEQ ID NO : 2. In further embodiments, the present invention provides perhydrolases that are structural homologs of the M. smegmatis perhydrolase, in which active site comprises sites homologous to Sl 1, D192, and H195 of the M. smegmatis perhydrolase. In yet additional embodiments, the present invention provides perhydrolases comprising one or more modifications at the following residues: Cys7, AsplO, Sert 1, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99,

Va1125, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thrl59, Thrl 86, Ilel 92, Ilel 94, and Phel96. However, it is not intended that the present invention be limited to perhydrolases with these modifications only at these residues, as perhydrolases with other modifications also find use with the present invention.

In some embodiments, at least one perhydrolase of the present invention is used in a cleaning process wherein an article to be cleaned is exposed to a sufficient amount of the at least one perhydrolase under conditions such that the perhydrolase cleans and/or bleaches, and/or decolorizes any/all stains present on the article (e. g., laundry and dish detergents). In some embodiments, the cleaning further comprises disinfecting. In some embodiments, the article cleaned, bleached and/or disinfected using at least one perhydrolase of the present invention comprises textiles and/or hard surfaces, while in other embodiments, the article is paper or pulp, and in still further embodiments, at least one perhydrolase is used as a personal care product to whiten or bleach hair, teeth, skin, etc. Thus, in some embodiments, the present invention provides compositions for use in various cleaning, bleaching, and/or disinfecting applications. Indeed, it is not intended that the present invention be limited to any particular application.

In some preferred embodiments, the perhydrolase comprises SEQ ID NO : 2. In some preferred alternative embodiments, the perhydrolase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the present invention provides enzymes with activities that result in high peracid/acid ratios. In alternative embodiments, the present invention provides the perhydrolase of Afycobacterium smegmatis, as well as sequence and/or structural homologs of this protein. In additional embodiments, the present invention provides enzymes that have been modified so as to express perhydrolase activity with a high perhydrolysis to hydrolase ratio either in addition to or instead of the enzyme's original activity. In additional embodiments, the present invention provides modified enzymes with altered substrate specificity, Km, kcat, perhydrolase activity, and/or peracid

degradation activity.

In additional embodiments, the present invention provides means to identify, produce, and characterize enzymes that comprise the perhydrolysis activity of the present invention. The present invention further provides methods and compositions comprising at least one perhydrolase for cleaning, disinfecting, bleaching, and other applications, including but not limited to paper and pulp bleaching, fabric and garment cleaning, hard surface cleaning, and personal care applications (e. g., oral care, hair care, and skin care).

In some preferred embodiments, the present invention provides methods and compositions for bleaching cotton and other fabrics. Indeed, the present invention finds use in the bleaching and cleaning of various textiles. It is not intended that the present invention be limited to any particular setting, application or use, as it is contemplated that it will find use in numerous areas where an enzymatic generation of peracids is desired over the use of preformed peracids or hydrogen peroxide or other bleaching chemicals, under conditions including but not limited to a wide range of pHs and temperatures. The present invention also finds use in applications where peracid hydrolysis is useful, such as in the clean up of peracids.

Furthermore, the present invention provides means to produce perhydrolase enzymes suitable for cleaning, disinfecting, bleaching, and other applications, including personal care.

DESCRIPTION OF THE FIGURES Figure 1 provides a phylogenetic tree of M. smegmatis perhydrolase and other related sequences.

Figure 2 provides an overview phylogenetic tree, showing the major branches of the bacteria and the origin of the active clones/sequences compared to M. smegmatis.

Figure 3 provides a schematic of four structural families of serine hydrolases, including perhydrolase (SGNH-hydrolase family), chymotrypsin, subtilisin, and alß

hydrolase.

Figure 4 provides a diagram of the structure of the perhydrolase fold.

Figure 5 provides a map of plasmid pET26-M4aE11.

Figure 6 provides a purification table showing the enzyme activity of the enzyme of the present invention through various steps in the purification process.

Figure 7 provides a graph which shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 40 minutes.

Figure 8 provides a graph showing the peracid production by 30 mM acetate equivalents and 29 mM hydrogen peroxide, tested at various pHs. These results show that using the perhydrolase composition of the present invention, there is peracid generation over a wide pH range. In contrast, with TAED and hydrogen peroxide, peracid generation is limited to alkaline conditions.

Figure 9 provides a graph showing the peracid production by 0.1 ppm perhydrolase enzyme in 30 mM ethyl acetate and 20 mM hydrogen peroxide at various temperatures. These results show that the perhydrolase of the present invention works at a wide range of temperatures, including low temperatures.

Figure 10 provides a graph showing the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 4, 10, and 30 minutes.

Figure 11 provides a graph showing the ratio of peracetic acid to acetic acid generated by various enzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10 minutes.

Figure 12 provides a map of plasmid pMSATNcoI.

Figure 13 provides a map of plasmid pMSATNcol-1.

Figure 14 provides a map of plasmid pAH505.

Figure 15 provides a map of plasmid pSFNASally.

Figure 16 provides a map of plasmid pCP606.

Figure 17 provides a map of plasmid pCP649.

Figure 18 provides a map of plasmid pSECGT-MSAT.

Figure 19 provides a map of plasmid pSEGT-phdA4.

Figure 20 provides a map of plasmid pMC355rbs.

Figure 21 provides a graph showing the degree of bleaching by three detergents tested alone and in comparison with the M. smegmatis perhydrolase of the present invention.

Figure 22 provides a graph showing the bleaching ability of the M. smegmatis perhydrolase tested on cotton.

Figure 23 provides a graph showing the bleaching ability of the M. smegmatis perhydrolase tested on linen.

DESCRIPTION OF THE INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. In particular, the present invention provides improved methods and compositions comprising perhydrolysis enzymes with high peracid/acid ratios for cleaning, bleaching, disinfecting and other applications. In some preferred embodiments, the present invention provides improved methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA

fields, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e. g., Sambrook et al.,"Molecular Cloning: A Laboratory Manual", Second Edition (Cold Spring Harbor), [1989] ); and Ausubel et al.,"Current Protocols in Molecular Biology" [1987] ). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

Definitions Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed. , John Wiley and Sons, NY (1994) ; and Hale and Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms"a","an,"and"the"include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5'to 3'orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not

limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein, the term"bleaching"refers to the treatment of a material (e. g., fabric, laundry, pulp, etc. ) or surface for a sufficient length of time and under appropriate pH and temperature conditions to effect a brightening (i. e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include but are not limited to CIOz, H202, peracids, NO2, etc.

As used herein, the term"disinfecting"refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present invention be limited to any particular surface, item, or contaminant (s) or microbes to be removed.

As used herein, the term"perhydrolase"refers to an enzyme that is capable of catalyzing a reaction that results in the formation of sufficiently high amounts of peracid suitable for applications such as cleaning, bleaching, and disinfecting. In particularly preferred embodiments, the perhydrolase enzymes of the present invention produce very high perhydrolysis to hydrolysis ratios. The high perhydrolysis to hydrolysis ratios of these distinct enzymes makes these enzymes suitable for use in a very wide variety of applications. In additional preferred embodiments, the perhydrolases of the present invention are characterized by having distinct tertiary structure and primary sequence. In

particularly preferred embodiments, the perhydrolases of the present invention comprises distinct primary and tertiary structures. In some particularly preferred embodiments, the perhydrolases of the present invention comprise distinct quaternary structure. In some preferred embodiments, the perhydrolase of the present invention is the M. smegmatis perhydrolase, while in alternative embodiments, the perhydrolase is a variant of this perhydrolase, while in still further embodiments, the perhydrolase is a homolog of this perhydrolase. In further preferred embodiments, a monomeric hydrolase is engineered to produce a multimeric enzyme that has better perhydrolase activity than the monomer.

However, it is not intended that the present invention be limited to this specific M. smegmatis perhydrolase, specific variants of this perhydrolase, nor specific homologs of this perhydrolase.

As used herein, the term"multimer"refers to two or more proteins or peptides that are covalently or non-covalently associated and exist as a complex in solution. A "dimer"is a multimer that contains two proteins or peptides; a"trimer"contains three proteins or peptides, etc. As used herein,"octamer"refers to a multimer of eight proteins or peptides.

As used herein, the phrase"perhydrolysis to hydrolysis ratio"is the ratio of the amount of enzymatically produced peracid to that of enzymatically produced acid by the perhydrolase, under defined conditions and within a defined time. In some preferred embodiments, the assays provided herein are used to determine the amounts of peracid and acid produced by the enzyme.

As used herein, "personal care products"means products used in the cleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth, including, but not limited to shampoos, body lotions, shower gels, topical moisturizers, toothpaste, and/or other topical cleansers. In some particularly preferred embodiments, these products are utilized on humans, while in other embodiments, these products find use with non-human animals (e. g. , in veterinary applications).

As used herein, "pharmaceutically-acceptable"means that drugs, medicaments and/or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and other animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.

As used herein, "cleaning compositions"and"cleaning formulations"refer to compositions that find use in the removal of undesired compounds from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes) etc. The term encompasses any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e. g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the perhydrolase and other enzyme (s) used in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.

The terms further refer to any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e. g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).

Indeed, the term"cleaning composition"as used herein, includes unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type;

machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners ; as well as cleaning auxiliaries such as bleach additives and"stain-stick"or pre-treat types.

As used herein, the terms"detergent composition"and"detergent formulation" are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In some preferred embodiments, the term is used in reference to laundering fabrics and/or garments (e. g.,"laundry detergents"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e. g.,"dishwashing detergents"). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to perhydrolase, the term encompasses detergents that contain surfactants, transferase (s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.

As used herein, "enhanced performance"in a detergent is defined as increasing cleaning of bleach-sensitive stains (e. g., grass, tea, wine, blood, dingy, etc. ), as determined by usual evaluation after a standard wash cycle. In particular embodiments, the perhydrolase of the present invention provides enhanced performance in the oxidation and removal of colored stains and soils. In further embodiments, the perhydrolase of the present invention provides enhanced performance in the removal and/or decolorization of stains. In yet additional embodiments, the perhydrolase of the present invention provides enhanced performance in the removal of lipid-based stains and soils. In still further embodiments, the perhydrolase of the present invention provides enhanced performance in removing soils and stains from dishes and other items.

As used herein the term"hard surface cleaning composition, "refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like. Such compositions are provided in any form, including but not limited to solids, liquids, emulsions, etc.

As used herein, "dishwashing composition"refers to all forms for compositions for cleaning dishes, including but not limited to granular and liquid forms.

As used herein, "fabric cleaning composition"refers to all forms of detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms.

As used herein,"textile"refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics.

The term encompasses yams made from natural, as well as synthetic (eg., manufactured) fibers.

As used herein, "textile materials"is a general term for fibers, yarn intermediates, yarn, fabrics, and products made from fabrics (e. g., garments and other articles).

As used herein, "fabric"encompasses any textile material. Thus, it is intended that the term encompass garments, as well as fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material.

As used herein, the term"compatible, "means that the cleaning composition materials do not reduce the enzymatic activity of the perhydrolase to such an extent that the perhydrolase is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.

As used herein, "effective amount of perhydrolase enzyme"refers to the quantity of perhydrolase enzyme necessary to achieve the enzymatic activity required in the specific application (e. g. , personal care product, cleaning composition, etc. ). Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the cleaning application, the

specific composition of the cleaning composition, and whether a liquid or dry (e. g., granular, bar) composition is required, and the like.

As used herein, "non-fabric cleaning compositions"encompass hard surface cleaning compositions, dishwashing compositions, personal care cleaning compositions (e. g., oral cleaning compositions, denture cleaning compositions, personal cleansing compositions, etc. ), and compositions suitable for use in the pulp and paper industry.

As used herein, "oral cleaning compositions"refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Oral care compositions that find use in conjunction with the perhydrolases of the present invention are well known in the art (See e. g., U. S. Patent Nos 5,601, 750, 6,379, 653, and 5,989, 526, all of which are incorporated herein by reference).

As used herein,"pulp treatment compositions"refers to the use of the present perhydrolase enzymes in compositions suitable for use in papermaking. It is intended that the term encompass compositions suitable for the treatment of any pulp material, including wood, as well as non-wood materials, such as"agricultural residues"and"fiber crops, "including but not limited to wheat straw, rice straw, corn stalks, bagasse (sugar cane), rye grass straw, seed flax straw, flax straw, kenaf, industrial hemp, sisal, textile flat straw, hesperaloe, etc. Thus, the present invention also encompasses the use of the perhydrolases of the present invention in pulp treatment methods.

As used herein, "oxidizing chemical"refers to a chemical that has the capability of bleaching pulp or any other material. The oxidizing chemical is present at an amount, pH and temperature suitable for bleaching. The term includes, but is not limited to hydrogen peroxide and peracids.

As used herein,"acyl"is the general name for organic acid groups, which are the residues of carboxylic acids after removal of the-OH group (e. g., ethanoyl chloride,

CH3CO-Cl, is the acyl chloride formed from ethanoic acid, CH3COO-H). The names of the individual acyl groups are formed by replacing the"-ic"of the acid by"-yl." As used herein, the term"acylation"refers to the chemical transformation which substitutes the acyl (RCO-) group into a molecule, generally for an active hydrogen of an - OH group.

As used herein, the term"transferase"refers to an enzyme that catalyzes the transfer of functional compounds to a range of substrates.

As used herein, "leaving group"refers to the nucleophile which is cleaved from the acyl donor upon substitution by another nucleophile.

As used herein, the term"enzymatic conversion"refers to the modification of a substrate to an intermediate or the modification of an intermediate to an end-product by contacting the substrate or intermediate with an enzyme. In some embodiments, contact is made by directly exposing the substrate or intermediate to the appropriate enzyme. In other embodiments, contacting comprises exposing the substrate or intermediate to an organism that expresses and/or excretes the enzyme, and/or metabolizes the desired substrate and/or intermediate to the desired intermediate and/or end-product, respectively.

As used herein, the phrase"detergent stability"refers to the stability of a detergent composition. In some embodiments, the stability is assessed during the use of the detergent, while in other embodiments, the term refers to the stability of a detergent composition during storage.

As used herein, the phrase, "stability to proteolysis"refers to the ability of a protein (e. g., an enzyme) to withstand proteolysis. It is not intended that the term be limited to the use of any particular protease to assess the stability of a protein.

As used herein, "oxidative stability"refers to the ability of a protein to function under oxidative conditions. In particular, the term refers to the ability of a protein to function in the presence of various concentrations of H202 and/or peracid. Stability under various oxidative conditions can be measured either by standard procedures known to

those in the art and/or by the methods described herein. A substantial change in oxidative stability is evidenced by at least about a 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity present in the absence of oxidative compounds.

As used herein, "pH stability"refers to the ability of a protein to function at a particular pH. In general, most enzymes have a finite pH range at which they will function. In addition to enzymes that function in mid-range pHs (i. e., around pH 7), there are enzymes that are capable of working under conditions with very high or very low pHs.

Stability at various pHs can be measured either by standard procedures known to those in the art and/or by the methods described herein. A substantial change in pH stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity at the enzyme's optimum pH. However, it is not intended that the present invention be limited to any pH stability level nor pH range.

As used herein, "thermal stability"refers to the ability of a protein to function at a particular temperature. In general, most enzymes have a finite range of temperatures at which they will function. In addition to enzymes that work in mid-range temperatures (e. g., room temperature), there are enzymes that are capable of working in very high or very low temperatures. Thermal stability can be measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the catalytic activity of a mutant when exposed to a different temperature (i. e. , higher or lower) than optimum temperature for enzymatic activity. However, it is not intended that the present invention be limited to any temperature stability level nor temperature range.

As used herein, the term"chemical stability"refers to the stability of a protein (e. g., an enzyme) towards chemicals that adversely affect its activity. In some

embodiments, such chemicals include, but are not limited to hydrogen peroxide, peracids, anionic detergents, cationic detergents, non-ionic detergents, chelats, etc. However, it is not intended that the present invention be limited to any particular chemical stability level nor range of chemical stability.

As used herein, the phrase"perhydrolase activity improvement"refers to the relative improvement of perhydrolase activity, in comparison with a standard enzyme. In some embodiments, the term refers to an improved rate of perhydrolysis product, while in other embodiments, the term encompasses perhydrolase compositions that produce less hydrolysis product. In additional embodiments, the term refers to perhydrolase compositions with altered substrate specificity.

As used herein, the phrase"alteration in substrate specificity"refers to changes in the substrate specificity of an enzyme. In some embodiments, a change in substrate specificity is defined as a difference between the Kcat/Km ratio observed with an enzyme compared to enzyme variants or other enzyme compositions. Enzyme substrate specificities vary, depending upon the substrate tested. The substrate specificity of an enzyme is determined by comparing the catalytic efficiencies it exhibits with different substrates. These determinations find particular use in assessing the efficiency of mutant enzymes, as it is generally desired to produce variant enzymes that exhibit greater ratios for particular substrates of interest. For example, the perhydrolase enzymes of the present invention are more efficient in producing peracid from an ester substrate than enzymes currently being used in cleaning, bleaching and disinfecting applications. Another example of the present invention is a perhydrolase with a lower activity on peracid degradation compared to the wild type. Another example of the present invention is a perhydrolase with higher activity on more hydrophobic acyl groups than acetic acid.

However, it is not intended that the present invention be limited to any particular substrate composition nor any specific substrate specificity.

As used herein,"surface property"is used in reference to an electrostatic charge, as well as properties such as the hydrophobicity and/or hydrophilicity exhibited by the surface of a protein.

As used herein, the phrase"is independently selected from the group consisting of ...."means that moieties or elements that are selected from the referenced Markush group can be the same, can be different or any mixture of elements as indicated in the following example: A molecule having 3 R groups wherein each R group is independently selected from the group consisting of A, B and C. Here the three R groups may be: AAA, BBB, CCC, AAB, AAC, BBA, BBC, CCA, CCB, or ABC.

In reference to chemical compositions, the term"substituted"as used herein, means that the organic composition or radical to which the term is applied is: (a) made unsaturated by the elimination of at least one element or radical; or (b) at least one hydrogen in the compound or radical is replaced with a moiety containing one or more (i) carbon, (ii) oxygen, (iii) sulfur, (iv) nitrogen or (v) halogen atoms; or (c) both (a) and (b).

Moieties which may replace hydrogen as described in (b) immediately above, that contain only carbon and hydrogen atoms, are hydrocarbon moieties including, but not limited to, alkyl, alkenyl, alkynyl, alkyldienyl, cycloalkyl, phenyl, alkyl phenyl, naphthyl, anthryl, phenanthryl, fluoryl, steroid groups, and combinations of these groups with each other and with polyvalent hydrocarbon groups such as alkylene, alkylidene and alkylidyne groups. Moieties containing oxygen atoms that may replace hydrogen as described in (b) immediately above include, but are not limited to, hydroxy, acyl or keto, ether, epoxy, carboxy, and ester containing groups. Moieties containing sulfur atoms that may replace hydrogen as described in (b) immediately above include, but are not limited to, the sulfur- containing acids and acid ester groups, thioether groups, mercapto groups and thioketo

groups. Moieties containing nitrogen atoms that may replace hydrogen as described in (b) immediately above include, but are not limited to, amino groups, the nitro group, azo groups, ammonium groups, amide groups, azido groups, isocyanate groups, cyano groups and nitrile groups. Moieties containing halogen atoms that may replace hydrogen as described in (b) immediately above include chloro, bromo, fluoro, iodo groups and any of the moieties previously described where a hydrogen or a pendant alkyl group is substituted by a halo group to form a stable substituted moiety.

It is understood that any of the above moieties (b) (i) through (b) (v) can be substituted into each other in either a monovalent substitution or by loss of hydrogen in a polyvalent substitution to form another monovalent moiety that can replace hydrogen in the organic compound or radical.

As used herein, the terms"purified"and"isolated"refer to the removal of. contaminants from a sample. For example, perhydrolases are purified by removal of contaminating proteins and other compounds within a solution or preparation that are not perhydrolases. In some embodiments, recombinant perhydrolases are expressed in bacterial or fungal host cells and these recombinant perhydrolases are purified by the removal of other host cell constituents; the percent of recombinant perhydrolase polypeptides is thereby increased in the sample.

As used herein, "protein of interest, "refers to a protein (e. g., an enzyme or "enzyme of interest") which is being analyzed, identified and/or modified. Naturally- occurring, as well as recombinant proteins find use in the present invention.

As used herein, "protein"refers to any composition comprised of amino acids and recognized as a protein by those of skill in the art. The terms"protein,""peptide"and polypeptide are used interchangeably herein. Wherein a peptide is a portion of a protein, those skilled in the art understand the use of the term in context.

As used herein, functionally and/or structurally similar proteins are considered to be"related proteins."In some embodiments, these proteins are derived from a different

genus and/or species, including differences between classes of organisms (e. g. , a bacterial protein and a fungal protein). In some embodiments, these proteins are derived from a different genus and/or species, including differences between classes of organisms (e. g. , a bacterial enzyme and a fungal enzyme). In additional embodiments, related proteins are provided from the same species. Indeed, it is not intended that the present invention be limited to related proteins from any particular source (s). In addition, the term"related proteins"encompasses tertiary structural homologs and primary sequence homologs (e. g., the perhydrolase of the present invention). In further embodiments, the term encompasses proteins that are immunologically cross-reactive. In most particularly preferred embodiments, the related proteins of the present invention very high ratios of perhydrolysis to hydrolysis.

As used herein, the term"derivative"refers to a protein which is derived from a protein by addition of one or more amino acids to either or both the C-and N-terminal end (s), substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, and/or deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, and/or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of a protein derivative is preferably achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative protein.

Related (and derivative) proteins comprise"variant proteins. "In some preferred embodiments, variant proteins differ from a parent protein and one another by a small number of amino acid residues. The number of differing amino acid residues may be one or more, preferably 1,2, 3,4, 5,10, 15,20, 30,40, 50, or more amino acid residues. In some preferred embodiments, the number of different amino acids between variants is between 1 and 10. In some particularly preferred embodiments, related proteins and particularly variant proteins comprise at least 35%, 40%, 45%, 50%, 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity.

Additionally, a related protein or a variant protein as used herein, refers to a protein that differs from another related protein or a parent protein in the number of prominent regions. For example, in some embodiments, variant proteins have 1,2, 3,4, 5, or 10 corresponding prominent regions that differ from the parent protein.

Several methods are known in the art that are suitable for generating variants of the perhydrolase enzymes of the present invention, including but not limited to site- saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.

In particularly preferred embodiments, homologous proteins are engineered to produce enzymes with the desired activity (ies). In some particularly preferred embodiments, the engineered proteins are included within the SGNH-hydrolase family of proteins. In some most preferred embodiments, the engineered proteins comprise at least one or a combination of the following conserved residues: L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, LI 14, L135, F180, G205. In alternative embodiments, these engineered proteins comprise the GDSL-GRTT and/or ARTT motifs.

In further embodiments, the enzymes are multimers, including but not limited to dimers, octamers, and tetramers. In yet additional preferred embodiments, the engineered proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than 1.

An amino acid residue of a perhydrolase is equivalent to a residue of M. smegmatis perhydrolase if it is either homologous (i. e. , having a corresponding position in either the primary and/or tertiary structure) or analogous to a specific residue or portion of that residue in M. smegmatis perhydrolase (i. e. , having the same or similar functional capacity to combine, react, and/or chemically interact).

In some embodiments, in order to establish homology to primary structure, the amino acid sequence of a perhydrolase is directly compared to the M. smegmatis

perhydrolase primary sequence and particularly to a set of residues known to be invariant in all perhydrolases for which sequence is known. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i. e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of M. smegmatis perhydrolase are defined. In preferred embodiments, alignment of conserved residues conserves 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues are also adequate to define equivalent residues. In preferred embodiments, conservation of the catalytic serine and histidine residues are maintained.

Conserved residues are used to define the corresponding equivalent amino acid residues of M. smegmatis perhydrolase in other perhydrolases (e. g. , perhydrolases from other Mycobacterium species, as well as any other organisms).

In some embodiments of the present invention, the DNA sequence encoding M. smegmatis perhydrolase is modified. In some embodiments, the following residues are modified: Cys7, AsplO, Serll, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thr159, Thrl86, Ilel92, Ilel94, and Phel96. However, it is not intended that the present invention be limited to sequence that are modified at these positions. Indeed, it is intended that the present invention encompass various modifications and combinations of modifications.

In additional embodiments, equivalent residues are defined by determining homology at the level of tertiary structure for a perhydrolase whose tertiary structure has been determined by x-ray crystallography. In this context, "equivalent residues"are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the carbonyl hydrolase and M. smegmatis perhydrolase (N on N, CA on CA, C on C, and O on O) are within 0. 13nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and

positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the perhydrolase in question to the M. smegmatis perhydrolase. As known in the art, the best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. Equivalent residues which are functionally and/or structurally analogous to a specific residue of M. smegmatis perhydrolase are defined as those amino acids of the perhydrolases that preferentially adopt a conformation such that they either alter, modify or modulate the protein structure, to effect changes in substrate binding and/or catalysis in a manner defined and attributed to a specific residue of the M. smegmatis perhydrolase. Further, they are those residues of the perhydrolase (in cases where a tertiary structure has been obtained by x- ray crystallography), which occupy an analogous position to the extent that although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of M. smegmatis perhydrolase. The coordinates of the three dimensional structure of M. smegmatis perhydrolase were determined and are set forth herein (See e. g., Example 14) and find use as outlined above to determine equivalent residues on the level of tertiary structure.

In some embodiments, some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. The perhydrolase mutants of the present invention include various mutants, including those encoded by nucleic acid that comprises a signal sequence. In some embodiments of perhydrolase mutants that are encoded by such a sequence are secreted by an expression host. In some further embodiments, the nucleic acid sequence comprises a homolog having a secretion signal.

Characterization of wild-type and mutant proteins is accomplished via any means suitable and is preferably based on the assessment of properties of interest. For example, pH and/or temperature, as well as detergent and/or oxidative stability is/are determined

in some embodiments of the present invention. Indeed, it is contemplated that enzymes having various degrees of stability in one or more of these characteristics (pH, temperature, proteolytic stability, detergent stability, and/or oxidative stability) will find use. In still other embodiments, perhydrolases with low peracid degradation activity are selected.

As used herein, "expression vector"refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification,"plasmid," "expression plasmid, "and"vector"are often used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.

In some preferred embodiments, the perhydrolase gene is ligated into an appropriate expression plasmid. The cloned perhydrolase gene is then used to transform or transfect a host cell in order to express the perhydrolase gene. This plasmid may replicate in hosts in the sense that it contains the well-known elements necessary for plasmid replication or the plasmid may be designed to integrate into the host chromosome. The necessary elements are provided for efficient gene expression (e. g., a promoter operably linked to the gene of interest). In some embodiments, these necessary elements are supplied as the gene's own homologous promoter if it is recognized, (i. e. , transcribed, by the host), a transcription terminator (a polyadenylation region for

eukaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the perhydrolase gene. In some embodiments, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid- infected host cells by growth in antimicrobial-containing media is also included.

The following cassette mutagenesis method may be used to facilitate the construction of the perhydrolase variants of the present invention, although other methods may be used.

First, as described herein, a naturally-occurring gene encoding the perhydrolase is obtained and sequenced in whole or in part. Then, the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded perhydrolase. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protein gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the perhydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region

which does not contain a site.

Once the naturally-occurring DNA and/or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

As used herein, "corresponding to, "refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.

As used herein, "corresponding region, "generally refers to an analogous position along related proteins or a parent protein.

The terms"nucleic acid molecule encoding,""nucleic acid sequence encoding," "DNA sequence encoding, "and"DNA encoding"refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

As used herein, the term"analogous sequence"refers to a sequence within a protein that provides similar function, tertiary structure, and/or conserved residues as the protein of interest (i. e., typically the original protein of interest). For example, in epitope regions that contain an alpha helix or a beta sheet structure, the replacement amino acids in the analogous sequence preferably maintain the same specific structure. The term also refers to nucleotide sequences, as well as amino acid sequences. In some embodiments, analogous sequences are developed such that the replacement amino acids result in a variant enzyme showing a similar or improved function. In some preferred embodiments, the tertiary structure and/or conserved residues of the amino acids in the protein of interest are located at or near the segment or fragment of interest. Thus, where the

segment or fragment of interest contains, for example, an alpha-helix or a beta-sheet structure, the replacement amino acids preferably maintain that specific structure.

As used herein, "homologous protein"refers to a protein (e. g. , perhydrolase) that has similar action and/or structure, as a protein of interest (e. g. , an perhydrolase from another source). It is not intended that homologs be necessarily related evolutionarily.

Thus, it is intended that the term encompass the same or similar enzyme (s) (i. e., in terms of structure and function) obtained from different species. In some preferred embodiments, it is desirable to identify a homolog that has a quaternary, tertiary and/or primary structure similar to the protein of interest, as replacement for the segment or fragment in the protein of interest with an analogous segment from the homolog will reduce the disruptiveness of the change. In some embodiments, homologous proteins have induce similar immunological response (s) as a protein of interest.

As used herein, "homologous genes"refers to at least a pair of genes from different species, which genes correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i. e., the development of new species) (e. g. , orthologous genes), as well as genes that have been separated by genetic duplication (e. g. , paralogous genes). These genes encode "homologous proteins." As used herein, "ortholog"and"orthologous genes"refer to genes in'different species that have evolved from a common ancestral gene (i. e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution.

Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.

As used herein,"paralog"and"paralogous genes"refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to

genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species.

As used herein,"wild-type"and"native"proteins are those found in nature. The terms"wild-type sequence, "and"wild-type gene"are used interchangeably herein, to refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project. The genes encoding the naturally-occurring protein may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protein of interest, preparing genomic libraries from organisms expressing the protein, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The term"recombinant DNA molecule"as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.

The term"recombinant oligonucleotide"refers to an oligonucleotide created using molecular biological manipulations, including but not limited to, the ligation of two or more oligonucleotide sequences generated by restriction enzyme digestion of a polynucleotide sequence, the synthesis of oligonucleotides (e. g., the synthesis of primers or oligonucleotides) and the like.

The degree of homology between sequences may be determined using any suitable method known in the art (See e. g., Smith and Waterman, Adv. Appl. Math. , 2: 482 [1981] ; Needleman and Wunsch, J. Mol. Biol., 48 : 443 [1970]; Pearson and Lipman, Proc. Natl.

Acad. Sci. USA 85: 2444 [1988] ; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., Nucl. Acid Res. , 12: 387-395 [1984]).

For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol., 35: 351-360 [1987] ). The method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5: 151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al., (Altschul et al., J. Mol. Biol. , 215: 403-410, [1990]; and Karlin et al., Proc. Natl.

Acad. Sci. USA 90: 5873-5787 [1993] ). One particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol.,, 266 : 460-480 [1996]). parameters"W,""T,"and"X"determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89 : 10915 [1989]) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

As used herein, "percent (%) nucleic acid sequence identity"is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of the sequence.

As used herein, the term"hybridization"refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.

As used herein, the phrase"hybridization conditions"refers to the conditions under which hybridization reactions are conducted. These conditions are typically classified by degree of"stringency"of the conditions under which hybridization is measured. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example,"maximum stringency"typically occurs at about Tm-5°C (5° below the Tm of the probe);"high

stringency"at about 5-10° below the Tm;"intermediate stringency"at about 10-20° below the Tm of the probe; and"low stringency"at about 20-25° below the Tm. Alternatively, or in addition, hybridization conditions can be based upon the salt or ionic strength conditions of hybridization and/or one or more stringency washes. For example, 6xSSC = very low stringency; 3xSSC = low to medium stringency ; IxSSC = medium stringency; and 0. 5xSSC = high stringency. Functionally, maximum stringency conditions may be used to identify nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe.

For applications requiring high selectivity, it is typically desireable to use relatively stringent conditions to form the hybrids (e. g., relatively low salt and/or high temperature conditions are used).

The phrases"substantially similar and"substantially identical"in the context of at least two nucleic acids or polypeptides typically means that a polynucleotide or polypeptide comprises a sequence that has at least about 40% identity, more preferable at least about 50% identity, yet more preferably at least about 60% identity, preferably at least about 75% identity, more preferably at least about 80% identity, yet more preferably at least about 90%, still more preferably about 95%, most preferably about 97% identity, sometimes as much as about 98% and about 99% sequence identity, compared to the reference (i. e., wild-type) sequence. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See e. g., Altschul, et al., J. Mol. Biol. 215: 403-410 [1990]; Henikoff et al., Proc. Natl. Acad. Sci.

USA 89: 10915 [1989]; Karin et al., Proc. Natl. Acad. Sci USA 90: 5873 [1993]; and Higgins et al., Gene 73: 237-244 [1988]). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85: 2444-2448 [1988]). One indication that two polypeptides are substantially identical is

that the first polypeptide is immunologically cross-reactive with the second polypeptide.

Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e. g., within a range of medium to high stringency).

As used herein, "equivalent residues"refers to proteins that share particular amino acid residues. For example, equivalent resides may be identified by determining homology at the level of tertiary structure for a protein (e. g. , perhydrolase) whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the protein having putative equivalent residues and the protein of interest (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non- hydrogen protein atoms of the proteins analyzed. The preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art of crystallography and protein characterization/analysis.

As used herein, the terms"hybrid perhydrolases"and"fusion perhydrolases"refer to proteins that are engineered from at least two different or"parental"proteins. In preferred embodiments, these parental proteins are homologs of one another. For example, in some embodiments, a preferred hybrid perhydrolase or fusion protein contains the N-terminus of a protein and the C-terminus of a homolog of the protein. In some preferred embodiment, the two terminal ends are combined to correspond to the full-length active protein.

The term"regulatory element"as used herein refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals and termination signals.

As used herein, "host cells"are generally prokaryotic or eukaryotic hosts which are transformed or transfected with vectors constructed using recombinant DNA techniques known in the art. Transformed host cells are capable of either replicating vectors encoding the protein variants or expressing the desired protein variant. In the case of vectors which encode the pre-or prepro-form of the protein variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.

The term"introduced"in the context of inserting a nucleic acid sequence into a cell, means transformation, transduction or transfection. Means of transformation include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA and the like as known in the art. (See, Chang and Cohen, Mol. Gen. Genet. , 168: 111-115 [1979]; Smith et al., Appl. Env. Microbiol. , 51: 634 [1986]; and the review article by Ferrari et al., in Harwood, Lu ils Plenum Publishing Corporation, pp. 57-72 [1989]).

The term"promoter/enhancer"denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (for example, the long terminal repeats of retroviruses contain both promoter and enhancer functions). The enhancer/promoter may be"endogenous"or"exogenous"or"heterologous."An endogenous enhancer/promoter is one which is naturally linked with a given gene in the genome. An exogenous (heterologous) enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i. e. , molecular biological techniques).

The presence of"splicing signals"on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal

of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.7-16. 8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

The term"stable transfection"or"stably transfected"refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term"stable transfectant"refers to a cell which has stably integrated foreign or exogenous DNA into the genomic DNA of the transfected cell.

The terms"selectable marker"or"selectable gene product"as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.

As used herein, the terms"amplification"and"gene amplification"refer to a process by which specific DNA sequences are disproportionately replicated such that the amplified gene becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e. g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i. e., input) sequences encoding this gene product, or both. Selection of cells by growth in the presence of a drug (e. g., an inhibitor of an inhibitable enzyme) may result in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i. e., input) sequences encoding this gene product, or both.

"Amplification"is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i. e. , replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i. e. , synthesis of the proper polynucleotide sequence) and nucleotide (ribo-or deoxyribo-) specificity. Template

specificity is frequently described in terms of"target"specificity. Target sequences are "targets"in the sense that they are sought to be sorted out from other nucleic acid.

Amplification techniques have been designed primarily for this sorting out.

As used herein, the term"co-amplification"refers to the introduction into a single cell of an amplifiable marker in conjunction with other gene sequences (i. e., comprising one or more non-selectable genes such as those contained within an expression vector) and the application of appropriate selective pressure such that the cell amplifies both the amplifiable marker and the other, non-selectable gene sequences. The amplifiable marker may be physically linked to the other gene sequences or alternatively two separate pieces of DNA, one containing the amplifiable marker and the other containing the non- selectable marker, may be introduced into the same cell.

As used herein, the terms"amplifiable marker, ""amplifiable gene,"and "amplification vector"refer to a marker, gene or a vector encoding a gene which permits the amplification of that gene under appropriate growth conditions.

As used herein, the term"amplifiable nucleic acid"refers to nucleic acids which may be amplified by any amplification method. It is contemplated that"amplifiable' nucleic acid"will usually comprise"sample template." As used herein, the term"sample template"refers to nucleic acid originating from a sample which is analyzed for the presence of"target" (defined below). In contrast, "background template"is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

"Template specificity"is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will

process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qp replicase, MDV-1 RNA is the specific template for the replicase (See e. g. , Kacian et al., Proc. Natl. Acad. Sci. USA 69: 3038 [1972J). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (See, Chamberlin et al., Nature 228 : 227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (See, Wu and Wallace, Genomics 4 : 560 [1989] ). Finally, Taq and Pfu polymerasesj by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences.

As used herein, the term"primer"refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i. e. , in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term"probe"refers to an oligonucleotide (i. e. , a sequence of

nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded.

Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any"reporter molecule, "so that is detectable in any detection system, including, but not limited to enzyme (e. g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the term"target, "when used in reference to amplification methods (e. g., the polymerase chain reaction), refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the"target"is sought to be sorted out from other nucleic acid sequences. A"segment"is defined as a region of nucleic acid within the target sequence.

As used herein, the term"polymerase chain reaction" ("PCR") refers to the methods of U. S. Patent Nos. 4,683, 195,4, 683,202, and 4,965, 188, hereby incorporated by reference, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.

The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i. e. , denaturation, annealing and extension

constitute one"cycle" ; there can be numerous"cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the"polymerase chain reaction" (hereinafter"PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be"PCR amplified".

As used herein, the term"amplification reagents"refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc. ), needed for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e. g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

As used herein, the terms"PCR product, ""PCR fragment,"and"amplification product"refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

As used herein, the terms"restriction endonucleases"and"restriction enzymes"

refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

The Present Invention In some most particularly preferred embodiments, the present invention finds use in the enzymatic generation of peracids from ester substrates and hydrogen peroxide. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Importantly, the present invention provides means for effective cleaning, bleaching, and disinfecting over broad pH and temperature ranges. In some embodiments, the pH range utilized in this generation is 4-12. In alternative embodiments, the temperature range utilized is between 5° and 90°C. The present invention provides advantages over the presently used systems (See e. g., EP Appln. 87- 304933.9) in that bleaching is possible at the optimum pH of peracid oxidation, as well as providing bleaching at neutral pH, acidic pHs, and at low temperatures. While the present invention is described herein most fully in regard to laundry and fabric care, it is not intended that the present invention be limited to these applications. Indeed, the present invention finds use in various settings, particularly those in which bleaching by peracids and/or hydrogen peroxide are desired, including but not limited to laundry, fabric treatment, pulp and paper processing, personal care applications, disinfection and cleaning of hard surfaces. For example, it is contemplated that the compositions of the present invention will find use in bleaching of pulp, including use in methods such as those set forth in U. S. Patent Nos. 6,569, 286, 5,785, 812,6, 165,318, and 4,400, 237, all of which are herein incorporated by reference.

Historically, sodium perborate, and more recently, sodium percarbonate, have been used as bleaching compounds, particularly in European laundry detergents. This

compound decomposes rapidly in aqueous solution to yield hydrogen peroxide (H202), which is the active bleaching species. As sodium perborate is more active at temperatures above 80°C, and less active in the temperature range of 40-60°C (i. e., wash temperatures that have become most commonly preferred as of the 1950s), bleaching activators have been incorporated into laundry detergents that contain sodium perborate. Indeed, most laundry detergents contain bleaching activators. These activators are compounds with O- or N-bounded acetyl groups that are able to react with the strongly nucleophilic hydroperoxy anion to yield peroxyacetic acid. Since the reacting species is hydroperoxy anion, alkaline pHs are essential for the efficient conversion of these activators to peracids. The peroxyacetic acid is decomposed in weakly basic media to form singlet oxygen (See, Hofmann et al., J. Prakt. Chem. , 334: 293-297 [1992]).

Hydrogen peroxide is a particularly effective bleach at high temperatures (e. g., >40°C) and pH (>10), conditions that are typically used in washing fabrics in some settings. However, as indicated above, cold water washing is becoming more commonly used and results in less effective bleaching by H202 than use of hot water. To overcome this low temperature disadvantage, detergent formulations typically include bleach boosters, such as TAED (N, N, N'N'-tetraacetylethylenediamine), NOBS (nonanoyloxybenzene sulfonate), etc. These boosters combine with H202 to form peracetic acid, a peracid species that is more effective than H202 alone. Although it helps the bleaching capability of detergent, the TAED reaction is only approximately 50% efficient, as only two out of the four acetyl groups in TAED are converted to peracids.

Additionally, conversion of TAED into peracetic acid by hydrogen peroxide is efficient only at alkaline pHs and high temperatures. Thus, the TAED reaction is not optimized for use in all bleaching applications (e. g. , those involving neutral or acidic pHs, and cold water). The present invention provides means to overcome the disadvantages of TAED use. For example, the present invention finds use in cold water applications, as well as those involving neutral or acidic pH levels. Furthermore, the present invention provides

means for peracid generation from hydrogen peroxide, with a high perhydrolysis to hydrolysis ratio. The present invention further provides advantages over compositions that contain enzymes such as esterases and lipases) which have very low perhydrolysis to hydrolysis ratios.

In addition to its applications in detergents, the present invention provides methods and compositions for the use of peracids in textile bleaching and in various other applications. In some embodiments, the present invention provides one-step methods for textile processing applications, including but not limited to one-step desizing, scouring and bleaching processes (See e. g., EP WO 03002810, EP 1255888, WO 0164993, and US 20020007516, all of which are hereby incorporated by reference). As described in greater detail herein, in some embodiments, bleaching involves processing textile material before it is dyed and/or after it is incorporated into textile goods. However, it is not intended that the present invention be limited to any particular regimen of use nor any particular textile material.

Furthermore, the peracetic technology of the present invention finds use as an effective bactericide (See, Baldry, J. Appl. Bacteriol. , 54: 417-423 [1983] ). Thus, the present invention provides compositions and methods for the sterilization/disinfection of various objects, including but not limited to medical devices, medical equipment, industrial equipment, and fermenters, as well as any additional object that needs to be sterilized or disinfected. As discussed in greater detail below, during the development of the present invention, the enzyme of the present invention was used in a standard cell kill experiment to demonstrate this suitability. In additional embodiments, the present invention provides compositions and methods suitable for use in biofilm control, such as in cooling towers.

Also as described in more detail in the Examples below, the present invention provides many advantages for cleaning and/or sterilization of a wide range of objects, including but not limited to clothing, fabrics, medical devices, etc. In addition, the

present invention provides compositions that are effective in cleaning, bleaching, and disinfecting, over a range of wash temperatures and pHs. In additional embodiments, the present invention finds use in degradation of peracids through the perhydrolase peracid degradation activity. In some preferred embodiments, this activity is used in peracid waste clean up applications.

Furthermore, the perhydrolase enzymes of the present invention are active on various acyl donor substrates, as well as being active at low substrate concentrations, and provide means for efficient perhydrolysis due to the high peracid: acid ratio. Indeed, it has been recognized that higher perhydrolysis to hydrolysis ratios are preferred for bleaching applications (See e. g. , U. S. Patent No. 5,352, 594,5, 108,457, 5,030, 240,3974, 082, and 5,296, 616, all of which are herein incorporated by reference). In preferred embodiments, the perhydrolase enzymes of the present invention provide perhydrolysis to hydrolysis ratios that are greater than 1. In particularly preferred embodiments, the perhydrolase enzymes provide a perhydrolysis to hydrolysis ratio greater than 1 and are find use in bleaching.

In addition, it has been shown to be active in commonly used detergent formulations (e. g., Ariel Futur, WOB, etc. ). Thus, the present invention provides many advantages in various cleaning settings.

As indicated above, key components to peracid production by enzymatic perhydrolysis are enzyme, ester substrate, and hydrogen peroxide. Hydrogen peroxide can be either added directly in batch, or generated continuously" situ."Current washing powders use batch additions of H202, in the form of percarbonate or perborate salts that spontaneously decompose to H202. The perhydrolase enzymes of the present invention find use in the same washing powder batch method as the H202 source.

However, these enzymes also find use with any other suitable source of H202, including that generated by chemical, electro-chemical, and/or enzymatic means. Examples of chemical sources are the percarbonates and perborates mentioned above, while an

example of an electrochemical source is a fuel cell fed oxygen and hydrogen gas, and an enzymatic example includes production of H202 from the reaction of glucose with glucose oxidase. The following equation provides an example of a coupled system that finds use with the present invention. Glucose oxidase Glucose + HzO------------------gluconic acid + H202 + Perhydrolase H202 + ester substrate----------------------------------------+ alcohol + peracid It is not intended that the present invention be limited to any specific enzyme, as any enzyme that generates H202 with a suitable substrate finds use in the methods of the present invention. For example, lactate oxidases from Lactobacillus species which are known to create H202 from lactic acid and oxygen find use with the present invention.

Indeed, one advantage of the methods of the present invention is that the generation of acid (e. g., gluconic acid in the above example) reduces the pH of a basic solution to the pH range in which the peracid is most effective in bleaching (i. e. , at or below the pKa).

Other enzymes (e. g., alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase, etc. ) that can generate hydrogen peroxide also find use with ester substrates in combination with the perhydrolase enzymes of the present invention to generate peracids. In some preferred embodiments, the ester substrates are selected from one or more of the following acids: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, as described herein, the present

invention provides definite advantages over the currently used methods and compositions for detergent formulation and use, as well as various other applications.

DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

Cloning and Characterization of M. smegmatis Perhydrolase The cloning of the M. smegmatis perhydrolase (i. e., referred to herein as the"phf gene, which encodes the"Phd"protein; this perhydrolase gene is sometimes herein referred to as the"act"gene and the protein is sometimes referred to as the"Act"protein) of the present invention was based on peptide sequence data from the acyltransferase purified from Mycobacterium parafortuitum (previously known as Corynebacterium oxydans) and published information regarding the 7-aminocephalosporanic acid (7-ACA) arylesterase gene of Agrobacterium radiobacter (Sakai et al., J. Ferment. Bioengineer., 85 : 138-143 [1998] ). Two peptide sequences from purified M. parafortuitum acyltransferase were found to be similar to internal N-and C-terminal regions of the A. radiobacter 7-ACA-arylesterase (47% and 42% identity respectively).

A set of PCR primers was designed based on the amino acid sequence of these internal peptides (designated"AtintF"and"AtintR"). Another set of primers was developed based on the 5'and 3'ends ("ATNcoI"and"ATBamHl") of the A. radiobacter 7-ACA DNA sequence. A single product of the expected size was amplified from M. parafortuitum chromosomal DNA using both sets of primers. The full length product, amplified by the ATNcoI/ATBamHl primer pair, was cloned into pET16b and

transformed into BL21 cells (Novagen, Madison,-WI). This clone had a sequence identical to that of the A. radiobacter 7-ACA gene. As it was determined that purified M. parafortuitum perhydrolase was not the 7-ACA acyl esterase, it was concluded that this was not the gene encoding the perhydrolase of the present invention.

Thus, efforts were further focused on M. smegmatis for cloning and expression of the perhydrolase of the present invention. To identify the M. parafortuitum gene based on enzyme activity screening, a plasmid library of M. parafortuitum DNA in M. smegmatis was constructed using a plasmid with a promoter to drive expression of cloned genes. Surprisingly, M. smegmatis itself was found to be positive for perhydrolase and acyltransferase activity. Thus, in some instances herein, the perhydrolase is referred to as "ACT" (or"Act").-A protein BLAST search of the M. smegmatis unfinished genome using the sequence of the A. radiobacter 7-ACA identified a 2 kb contig containing an ORF (open reading frame) that encoded a hypothetical protein that was similar but not identical to the 7-ACA protein. Based on this sequence, primers were designed and used to amplify the gene from M. smegmatis (ATCC 10143). By adding an E. coli ribosome binding site upstream of the start codon, a clone that expressed active enzyme was obtained. The vector used was either pCR2. 1TOPO or pBluntIITOPO (Invitrogen, Carlsbad, CA), in E. coli Top 10 cells. The gene was expressed constitutively from the plasmid-encoded lac promoter. This enzyme carried out the same reactions as the originally described M. parafortuitum acyltransferase.

During the characterization of the perhydrolase of the present invention, standard protein BLAST searches identified a few proteins (<20) with sequence similarity of 30- 80%. This group included the 7-ACA arylesterases from A. radiobacter and other organisms, which have 43% identity with M. smegmatis perhydrolase. All of the identified homologs with at least 40% similarity have a GDS motif very near the N- terminal end. All of the proteins also contain most of the conserved residues which could place them within the suggested GDSL family of lipolytic enzymes (See e. g., Upton and

Buckley, Trends Biochem. Sci. , 20: 178 [1995] ). However, enzymes mentioned in this paper do not appear on homology searches with the perhydrolase protein. Indeed these proteins have less than 20% similarity with the perhydrolase and its homologs, suggesting that the acyltransferase-related (and perhydrolase of the present invention) enzymes form a subfamily.

The natural function of the enzyme of the present invention and the closely related proteins, apart from the 7-ACA arylesterase, have not been biochemically determined. M. smegmatis appears to be the only organism with the acyltransferase/perhydrolase in an operon with a putative penicillin binding protein (PBP). While it is not intended that the present invention be limited to any particular mechanism, this suggests that the enzyme may be involved in cell wall synthesis/structure or modification of molecules taken up from the environment. There are no homologues of the perhydrolase of the present invention that have been identified in M. tuberculosis or M. leprae to date. However, some organisms were determined to have multiple homologues (e. g., S. meliloti).

During the development of the present invention, various mutations were made in the M. smegmatis perhydrolase in order to assess its activity. This enzyme contains two cysteine residues, which were hypothesized as potentially forming disulfide bonds, both of which were changed to alanine, in order to determine whether or not the C residues had any effect on the activity of the enzyme. Activity assay results obtained using the transesterification (in aqueous solution) assay described herein indicated that C7A, as well as C77A, and a double mutant (C7A and C77A) were of the same size and specific activity.

Many enzymes have the amino acid serine as part of their active site and are therefore referred to, among other designations, as"serine hydrolases. "The active site may consist of a catalytic triad of S (serine), D (aspartic acid) and H (histidine).

Examples of such enzymes include, but are not limited to subtilisin (D32-H64-S215), chymotrypsin (H57-D 102-S 195) and lipases in the alpha/beta hydrolase family (e. g.,

S126-D176-H206). A typical motif for lipases is the GDSL motif (Upton and Buckley, supra [1995] ) in which the S is the active site serine. Since the perhydrolase of the present invention was determined to have a GDSL (amino acids 9-12) motif, the Sl 11 was mutated to an A, in order to confirm the involvement of this S in the active site. As indicated in the Examples, the activity assay results indicated that Sl lA had only 1% of the activity of the wild-type enzyme. Deletion of the C-terminal 25 amino acids also resulted in abrogation of the activity, suggesting that these amino acids either contained a residue involved directly in the active site, and/or that the structure of the protein was affected such that the active site was no longer able to catalyze the reactions. In addition, the predicted active site residues, D192 and H195 were mutated to A. Neither mutant had activity, confirming that the active site residues of the perhydrolase of the present invention consist of Sll, D192 and H195. However, it is not intended that the present invention be limited to any particular mechanism, nor is the present invention limited to mutation (s) at any particular active site residues.

Cloning of M. parafortuitum Perhydrolase There were some differences between the N-terminal peptide sequence obtained from the M. parafortuitum enzyme and the N-terminal sequence of M. smegmatis perhydrolase. However, there was a sequence in the C-terminal region of the M. smegmatis perhydrolase identical to the C-terminal peptide sequence of the M. parafortuitum enzyme. Two primers were designed to amplify a partial sequence of the M. parafortuitum perhydrolase gene; the sequence of the reverse primer was identical to the sequence of the corresponding region in M smegmatis perhydrolase gene, and the sequence of the forward primer was based on M. smegmatis codon usage. The forward primer, MP5 : 5'- ATGGGTACCCGACGAATTCTGTCCTTCGGTGATTCCCTGACCT-3' (SEQ ID NO: 11) and the reverse primer MPC-intR 5'-

GATTCCGTCGACGCCGTCGGTGCTGATCACCGAACCCGCGTCGAAGAACGG- 3' (SEQ ID NO: 12). The partial gene was amplified from the chromosome of M. parafortuitum and cloned into pCR2. lTOPO (Invitrogen, Carlsbad, CA). Sequence analysis showed that the enzyme is very similar, but not identical to the M. smegmatis perhydrolase (77% identity). Based on the molecular weights of the monomers of the perhydrolases determined by SDS-PAGE (MP AT: 26 kDa, MSAT: 24 kDa, MP cloned AT :-18 kDa), the clone from primers made to the internal fragment was determined to be missing approximately 70 amino acids (-8 kDa). The remaining sequence at the 5'- end of the M. parafortuitum gene can be obtained by any of several methods suitable and familiar to those skilled in the art of molecular biology, including, but not limited to, inverse PCR, probing of plasmid/cosmid libraries of M. parafortuitum chromosomal DNA, sequencing of the gene directly from chromosomal DNA (e. g., as performed by Fidelity Systems, Bethesda Maryland).

Expression of the M. smegmatis Perhydrolase The perhydrolase is an intracellular protein in its native host. Production of the perhydrolase in non-native hosts may also be done intracellularly. However, in some embodiments, a signal sequence is added to the perhydrolase, which facilitates expression of the perhydrolase by secretion into the periplasm (i. e., in Gram-negative organisms, such as E. coli), or into the extracellular space (i. e., in Gram-positive organisms, such as Bacillus and Actinomycetes), or eukaryotic hosts (e. g., Trichoderma, Aspergillus, Saccharomyces, and Pichia). Of course, these are just a few examples of possible prokaryotic and eukaryotic hosts. It is not intended that the present invention be limited to these specific hosts, as various other organisms find use as expression hosts in the present invention.

A variety of commercially available expression systems, including but not limited to pBAD, plac, T7, find use in the expression of the perhydrolase in Gram-negative hosts

(e. g., E. coli). In some embodiments, the same types of promoters find use in another Gram-negative host, Pantoea citrea.

To test expression in E. coli two strategies were used: 1) adding an RBS (ribosome binding site) to the 5'end of the phd gene and cloning the gene into pCRBLUNTIITOPO (Invitrogen), thus allowing expression directly from the pLac promoter available in that vector; and 2) cloning the pAd gene under control of the T7 promoter in the plasmid pET16b (Novagen). In the latter system, expression of the gene is inducible by addition of IPTG to the growing culture and use of a specific host cell (e. g. , BL21 (, % DE3) pLysS (Novagen) ) that contains the kDE3 lysogen encoding the T7 RNA polymerase. The first strategy produces a plasmid capable of allowing expression of the perhydrolase protein in other Gram-negative hosts (e. g. , P. citrea) ; To express protein in E. coli or P. citrea using the first strategy, cultures were grown from single, purified colonies at 37°C overnight in L broth plus the appropriate antibiotic (example, kanamycin 50 ug/ml). Expression of the protein was determined by the pNB assay (See, Example 1) after lysis of the cells.

Expression of the perhydrolase using the T7 expression system requires induction of the culture with the addition of IPTG (e. g. , 100 mmole IPTG added at an ODsso of 0.4). Overnight cultures, inoculated from a single colony, are used to inoculate the expression culture of the desired volume (25 mls to several liters) at an ODsso of 0.1. The expression culture was then grown at the desired temperature (e. g., 25°C, 30°C, 37°C) until an ODsso of 0.4 was reached, after which IPTG was added. Expression was allowed to continue for 3 hours to overnight. Protein expression was monitored by pNB activity assay as described in Example 1. Usually, expression from the T7 system gives a high titer of protein, sufficient for further analysis such as crystallography.

Bacillus species are well-known as suitable hosts for expression of extracellular proteins (e. g., proteases). Intracellular expression of proteins is less well known.

Expression of the perhydrolase protein intracellularly in Bacillus subtilis can be done

using a variety of promoters, including, but not limited to pVeg, pSPAC, pAprE, or pAmyE in the absence of a signal sequence on the 5'end of the gene. In some embodiments, expression is achieved from a replicating plasmid (high or low copy number), while in alternative embodiments, expression is achieved by integrating the desired construct into the chromosome. Integration can be done at any locus, including but not limited to the aprE, amyE, or pps locus. In some embodiments, the perhydrolase is expressed from one or more copies of the integrated construct. In alternative embodiments, multiple integrated copies are obtained by the integration of a construct capable of amplification (e. g., linked to an antibiotic cassette and flanked by direct repeat sequences), or by ligation of multiple copies and subsequent integration into the chromosome. In some embodiments, expression of the perhydrolase with either the replicating plasmid or the integrated construct is monitored using the pNB activity assay (described herein) in an appropriate culture.

As with Bacillus, in some embodiments, expression of the perhydrolase in the Gram-positive host Streptomyces is done using a replicating plasmid, while in other embodiments, expression of the perhydrolase is accomplished via integration of the vector into the Streptomyces chromosome. Any promoter capable of being recognized in Streptomyces finds use in driving transcription of the perhydrolase gene (e. g. , glucose isomerase promoter, A4 promoter). Replicating plasmids, either shuttle vectors or Streptomyces only, also find use in the present invention for expression (e. g, pSECGT).

Structure of M. smegmatis Perhydrolase The crystal structure of the M. smegmatis perhydrolase was determined to 2.2 Angstroms. The structure confirmed findings with gel filtration sizing columns, that indicated this enzyme is an octamer. The structure of the monomer places the enzyme in the class known as SGNH-hydrolases (See e. g., Molgaard et al., Structure 8: 373-383 [2000] ). The active site residues were identified as Sl l-D192-H195, based on

homology, confirming the identification of the catalytic triad based on loss of activity in the Sl 1A, D192A, and H I 95A mutations described above. Figure 3 provides schematics showing the structure of the M. smegmatis perhydrolase, as well as other serine hydrolases. As indicated, this enzyme has a different structure than the enzymes shown here (chymotrypsin, subtilisin, and a/ (3 hydrolase). Indeed, the structural analysis of the perhydrolases of the present invention indicates that this group of enzymes has a different form and active site than do these other enzymes. A schematic diagram of the structure of the monomer is illustrated in Figure 4. The structures of four other enzymes in the SGNH-hydrolase family have been solved, namely Aspergillus aculeatus rhamnogalucturonan acetylesterase (RGAE), Bos taurus platelet activating factor (PAF- AH (lb) a), Streptomyces scabies esterase (SsEst) and the thioesterase/Protease I/Phospholipase Li (TAP or Tes) from E. coli. Very little sequence or functional homology is present in these enzymes. Basically, the sequence identity is reserved for the residues involved in the active site and those defining the family. While the overall folding of the enzymes is similar (See e. g., Molgaard et al., supra [2000], for overlaying of structures), there are structural differences. For example, there is a loop covering the active site in SsEst, compared to RGAE and TAP which have active sites that are surface- exposed. The M. smegmatis perhydrolase has an active site that is somewhat buried. The binding residues ofthe M. smegmatis perhydrolase were identified as Cys7, Asp 10, Serl 1, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Va1125, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thrl59, Thrl86, Ilel92, Ilel94, and Phel96. These sites were derived from direct observation and by modeling studies to model substrate binding to the enzyme, using methods known in the art.

As indicated above, the M. smegmatis perhydrolase was found to be an octamer in the crystalline state. However, it is contemplated to be either a hexamer or octamer in solution. The octamer is seen to be a tetramer of dimers, two molecules are much more

closely and extensively interacting and these are termed the"act transferase"dimers.

Several of the conserved sites are found along this dimer interface. For example, residues Trp 14, Arg 27, Arg 56, His 81 and Pro 83, were found to be conserved in natural isolates that have perhydrolase activity and are contemplated to be critical in forming the interface. In addition one other residue, Glu 51, which is conserved in all but one of the natural isolates (and in that case it is a homologous enzyme) was identified.

One additional feature of interest in that in the natural isolates showing perhydrolase activity, all share an insertion of residues 69-81. This region forms a loop that is at the dimer interface. Without this loop, it is believed that much of the dimer interface would be lost and it is likely that dimers and subsequent aggregation would not occur. Thus, there is a correlation of the insertion with the structural aggregation particularly dimer formations and the appearance of perhydrolase activity. However, it is not intended that the present invention be limited to any particular mechanisms.

Key residues were found to be associated with desired activity in selected homologs. Indeed, there are several conserved residues that are contemplated to have importance for acyltransferase activity. These include Leu 6, Trp 14, Arg 27, Trp 34, Asp 62, Leu74, Leu 78 His 81, Pro83, Met 90, Lys 97, and Leu 114.

In additional analyses, the association of the perhydrolase with carbamate was investigated. The native octamer was determined in space group P4 with unit cell dimensions : a= 98. 184 b= 98.184 and c= 230.119 a=90. 00 p=90. 00 y=90. 00, this crystal diffracted to about 2.0 A. The carbamate-inhibited crystal grew in the space group PI with unit cell dimensions a=67. 754, b=80. 096, and c=85. 974 a=104. 10°, P=l 12. 10', and y=97. 40' and these crystals diffract to a resolution exceeding 1.0 A.

The carbamate was bound in a manner to exploit the interactions between the keto oxygen of the carbamate and residues forming the oxyanion hole, the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobic side chain extends along the

hydrophobic surface of the binding site out into the surface opening between pairs of dimers in the octamer structure. The carbamate moiety direction highlights the pivotal role of the S54V mutation. The hydrophobic moiety passes adjacent to the side chain of ser 54. Mutating the serine side to valine increased the hydrophobicity, and also served as-a gatekeeper to prevent hydrophilic nucleophiles (e. g., water) for competing with desired deacylating nucleophiles. The residues surrounding the carbamate moiety on the same and neighboring molecules forming the extended entry are expected to influence the selection of the optimal de-acylating nucleophile. The structure showed that each monomer was inhibited with carbamate covalently attached. Thus, all octamer active sites were found to be active and functional. The side chain of carbamate resembles the leaving groups of the substrates tested. Thus, the carbamate moiety indicates the access direction for substrate.

M. smegmatis Perhydrolase is an SGNH-Hydrolase The perhydrolase of the present invention has certain components that indicate it is in the SGNH-hydrolase family of enzymes. This family is defined by having the four conserved amino acids SGN and H in four blocks, similar to the blocks that describe the lipolytic family of enzymes (See, Upton and Buckley, supra). In the case of the M. smegmatis perhydrolase, these correspond to 511, G52, N94 and H195 which correspond to Blocks I II, III and V according to Upton and Buckley (Upton and Buckley, supra) and Molgaard et al. (Molgaard et al., supra). These amino acids are also conserved within the closest sequence homologs of the perhydrolase.

As indicated herein, the sequences were aligned using the Alignment program in Vector NTi (Informax, Invitrogen) In the following alignment providing a comparison of homolog sequences, the double underline indicates the residues involved in the active site. AR: Agrobacterium rhizogenes Q9KWA6; RR: Rhizobium rhizogenes NF006; SM: Sinorhizobium meliloti RSM02162; MS: Mycobacterium smegmatis Act; MP:

Mycobacterium parafortuitum Phd partial sequence; PD: Prosthecobacter dejongeii RVM04532. The amino acids within the blocks defining the SGNH-hydrolase family are indicated in bold letters. Block I Block II GDS G AR (1)----------MAESRSILCFGDSLTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED--PN RR (1)----------MAESRSILCFGDSLTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED-PN RM (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSP TLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDD-PN SM (1)-----------MVEKRSVLCFGDSLTWGWIPVKESSP TLRYPYEORWTGAMAARLGDGYHIIEEGLSARTTSLDD-PN MS (1)-------------MAKRILCFGDSLTWGWVPVEDGAP TERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDD-PT MP-------------GTRRILSFGDSLTWGWIPVEEGVP TERFPRDVRWTGVLADLLGDRYEVIEEGLSARTTTAED-PA PD l)--------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTGVLAKALGA GFRVIEEGQNGRTTVHED--PL Block III GxND AR (67) DPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGKLAGOVLTSAGG IGTPYPAPKLLIVSPPPLAP RR (67) DPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGKLAGQVLTSAGG IGTPYPAPKLLIVSPPPLAP RM (78) DARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGG VGTPYPAPKVLVVAPPPLAP SM (67) DARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGG VGTPYPAPKVLWAPPPLAP MS (65) DPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIA. GTNDTKAYFRRTPLDIALGMStILVTQVLTSAGGtTGTTYPAPKVLVVSPPPLAP MP (65) DPRLNGSQYLPSCLASHLPLDLVILMLGTNDTKANFGRTPFDIATGMGVLATQVLTSAGG VGTSYPAPQVLIVAPPPLGE PD (65) NICRKGKDYLPACLESHKPLDLVILMLGTNDLKSTFNVPPGEIAAGAGVLGRMILAGDAG P-ENRPPQLLLMCPPKVRDL Block V DGIHF AR (147) MPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVRTDGC=GIaFSAETNITL GHAIAAKVEAIFSQEAKNAA (SEQ ID NO : 14) RR (147) MPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCaGIEFSAETNITL GHAIAAKVEAIFSQEAKNAA (SEQ ID NO : 15) RM (158) MPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGI=GIaLSAETNIRL GHAIADKVAALF--------(SEQ XD NO : 16) SM (147) MPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIpGI$LSAETNIRL GHAIADKVAALF-------- (SEQ ID NO : 17) MS (145) MPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVaGIEFTEANNRDL GVALAEQVRSLL--------(SEQ ID N0 : 18) MP 145) LPHPWFDLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDGVBGI---------- -------------------- (SEQ ID NO : 19) PD (144) SAMPDLDAKIPHGAARSAEFPRHYKAQAVALKCEYFNSQEIVETSPVDGIkiLEASEHLK LGEALAEKVKVLLG------- (SEQ'ID NO : 20) The primers used to identify homologs for each of the Blocks indicated above are provided below : Block I (forward 5'-3) le : acggtcctgtgctttggngaytcnyt (SEQ ID NO : 21) If : acggtcctgtgctttggngayagyyt (SEQ ID N0 : 22)

lg : gcggtcctgttctwnggngaytcnyt (SEQ ID NO : 23) lh : gcggtcctgttctwnggngayagyyt (SEQ ID NO : 24) li : gctcgaaccgtcctctgttttggngaytcnyt (SEQ ID NO : 25) li : gctcgaaccgtcctctgttttggngayagyyt (SEQ H) NO : 26) lk : gctcgaaccgtcctctgtttnggngaytc (SEQ ID NO : 27) 11 : gctcgaaccgtcctctgttttggngaytcnytn (SEQIDNO : 28) Im : gctcgaaccgtcctctgttttggngaytcnytg (SEQIDNO : 29) 1A : gccaagcgaattctgtgtttcggngaytcnyt (SEQIDNO : 30) 1B : gccaagcgaattctgtgtttcggngayagyyt (SEQIDNO : 31) Block III (reverse 5'-3) 3c : attccgcgcttcagrtcrttnvtncc (SEQ ID NO : 32) 3d : attccgcgcttcagrtcrttnwgncc (SEQ ID NO : 33) 3e : attccgcgcttcagrtcrttnscncc (SEQ ID NO : 34) 3f : attccgcgcttcagrterttnrancc (SEQ ID NO : 35) 3k : attccgcgcttcagrtcrttnrtncc (SEQ ID NO : 36) 31 : attccgcgcttcagrtcrttnytncc (SEQ ID NO : 37) 3m : attccgcgcttcagrtcrttnsgncc (SEQ ID NO : 38) 3n : attccgcgcttcagrtcrttnwcncc (SEQ ID NO : 39) 3o : attccgcgcttcagrtcrttnyancc (SEQ ID NO : 40) 3p : attccgcgcttgrsrtcrttnrtncc (SEQ ID NO : 41) 3q : attccgcgcttgrsrtcrttnytncc (SEQ ID NO : 42) 3r : attccgcgcttgrsrtcrttnsgncc (SEQ ID NO : 43) 3s : attccgcgcttgrsrtcrttnwcnnn (SEQ ID NO : 44) 3t : attccgcgcttgrsrtcrttnyancc (SEQ ID NO : 45) 3A : gcgccggaagtaggccttggtrtcrttnvtncc (SEQ ID NO : 46) 3B : gcgccggaagtaggccttggtrtcrttnwgncc (SEQ ID NO : 47) 3C : gcgccggaagtaggccttggtrtcrttnscncc (SEQ ID NO : 48) 3D : gcgccggaagtaggccttggtrtcrttnrancc (SEQ ID NO : 49) Block III (forward 5'-3) 3g : cggaattatcatgctgggnabnaayga (SEQ ID NO : 50) 3h : cggaattateatgctgggncwnaayga (SEQ ID NO : 51) 3i : cggaattatcatgctgggngsnaayga (SEQ ID NO : 52) 3j : cggaattatcatgctgggntynaayga (SEQ ID NO : 53) 3u : ccggaattatcatgctnggnabnaayga (SEQ ID NO : 54) 3v : ccggaattatcatgctnggncwnaayga (SEQ ID NO : 55) 3w : ccggaattatcatgctnggngsnaayga (SEQ ID NO : 56) 3x : ccggaattatcatgctnggntynaayga (SEQ ID NO : 57)

Block V (reverse 5'-3) 5c : acccttagcgtttggrtgnrtnccrtc (SEQ ID NO : 58) 5d : atccttagcgtttggrtgnavnccrtc (SEQ ID NO : 59) 5e : aatcttagccgtgrrrtgnrtnccrtc (SEQ ID N0 : 60) 5f : aatcttagccgtgrrrtgnrcnccrtc (SEQ ID N0 : 61) 5g : aatcttagccgtgrrrtgntmccrtc (SEQ ID NO : 62) 5h : ccgctggtcctcatctggrtgnrtnccrtc (SEQ ID NO : 63) 5i : ccgctggtcctcatctggrtgnrcnccrtc (SEQ ID NO : 64) 5j : ccgctggtcctcatctggrtgntmccrtc (SEQ ID NO : 65) 5k : ccgctggtcctcatcraartgnrtncc (SEQ ID NO : 66) 5A : cgattgttcgcctcgtgtgaartgnrtnccrtc (SEQ ID NO : 67) 5B : cgattgttcgcctcgtgtgaartgnrcnccrtc (SEQ ID NO : 68) 5C : cgattgttcgcctcgtgtgaartgntmccrtc (SEQ ID NO : 69) As described in greater detail herein, the sequence and structure results are supported by the activity data that indicate the perhydrolase enzymes of the present invention differ from lipolytic enzymes known in the art.

Identification of Homologs As well known in the art, proteins with a desired activity may be identified in several ways, including but not limited to: 1) searching available databases for proteins with sequence homology (30-100%) ; 2) screening environmental isolates for the desired activity; and 3) examining type strains from ATCC of the genus identified to have activities (e. g., Mycobacterium and Corynebacterium, as described herein in particular embodiments).

By doing a standard protein-protein BLAST search, several homologs were identified from fully or partially sequenced genomes. From the known gene sequence, several homologs were amplified by PCR from the chromosome of the parent organism

and cloned into a pET expression vector, essentially as described for the cloning of pAd from M. smegmatis into pET16b. Homologues identified by this BLAST search included: Agrobacterium rhizogenes Q9KWA6, A. rhizogenes Q9KWB1 A. tumefaciens Q8UFG4, A. tumefaciens Q8UACO (now AgrL, identical to 7-ACA arylesterase), A. tumefaciens Q9ZI09, A. tumefaciens (radiobacter) ACA, Prosthecobacter. dejongeii RVM04532, Rhizobium. loti Q98MY5, R. meliloti Q92XZ1, R. meliloti Q9EV56, R. rhizogenes NF006, R. rhizogenes NF00602875, R. solanacerarum Q8XQI0, Sinorhizobium meliloti RSM02162, S. meliloti RSM05666, Mesorhizobium loti RMLO00301, A. rhizogenes Q9KWA6, and A. rhizogenes Q9KVWB 1.

Based on these results, a homology tree of proteins with sequence homology (20- 80%) to M. smegmatis perhydrolase was generated. As shown in Figure 2, an enzyme in the family of lipolytic enzymes described by Upton and Buckley (supra) is that of V mimics. This phylogenetic tree was generated using the alignment program in Vector NTi (Informax, Invitrogen). The green arrow indicates M. smegmatis perhydrolase, the red arrow indicates A. radiobacter 7-ACA arylesterase, the blue arrow indicates E. coli TAP, and the black arrow indicates A. aculeatus RGAE.

As further indicated in Figure 2, the perhydrolase is not closely related to this enzyme. The perhydrolase and its closest relatives, Prosthecobacter dejongeii RVM04532, R. rhizogenes NF006, A. rhizogenes Q9KWA6, R. meliloti Q92XZ1, S. meliloti RSM02162, A. rhizogenes Q9KWB1 and R. rhizogenes NF00602875 come off their own branch (i. e. , a branch that is different from the 7-ACA arylesterase-like proteins and the RGAE/TAP-like proteins). However, it is contemplated that some additional, more distantly related homologs will find use in the present invention due to perhydrolase activity or will serve as a suitable backbone for modification to the desired perhydrolase activity.

In addition to the sequence and homology analysis, environmental isolates were grown on a rich medium (N-MISO: g/1 : glucose 10 g, yeast extract 10 g, KNO3 1.5,

KH2P04 3.4 g, NaH2PO4. H20 3.4 g, Salt Solution C 10 ml [Salt Solution C: g/l : MgS047H20 25, FeS047H20 2.8, MnS04H20 1.7, NaCl 0.6, NaMoS04. 2H20, ZnS04. 7H20 0.06, in O. 1N HC1]), assayed and those positive for the transesterification reaction were purified as described in the Examples. This is one of the screening methods that can be used to identify perhydrolase These data show that the present invention finds use in identification of additional enzymes with the desired perhydrolase activity.

Additional Investigations of Homologues In addition to the above analyses, an enzyme library of novel"GDSL-type" esterases which are homologous to the prototype M. smegmatis perhydrolase was created.

In order to identify new"GDSL"-type esterases, a sequence homology based screening procedure was established and used to screen libraries set up from complex metagenomic DNA (at BRAIN).

An enzyme library comprising 19"GDSL"-type esterases (See, below) was developed. The sequences in this library were:

S248_M2bBll (AminoAcid) MFALCTAASAAPDRTVVFFGDSLTAGYGLDDPQTQSYPARIQEKVDAAGLRWK VVNAGLSGETSAGGLRRVDWVLGQHIDAFVLALGANDGLRGIDPQVTRANLQEII NRVRSRWPRAAIVIAGMKMPQSMGQDYAANFDRIFPGLAARNSATLIPFLLEGV AAHPSLNQGDGIHPTAAGDALVAGTVWTYLLPILRSAH (SEQ ID NO : 71) S248_M40cD4 (DNA) ATGCGCTTTGCTAAGCTCACTGCCGTCATCTTTGCCCTGATAGTCTTGCACAG CCCCCTTGCCGCCGCCGCGCCGCCCACCGTGATGGTGTTTGGCGACAGTCTGA CCGCCGGGTTGGGATTGCCGGCCGATGCTGCATTTCCGGCGCAGCTCCAGGC AAAGCTGCACGATATGGGTATCCTGCAGAAATCGCCGCGCGCGCCACCTCGG GGCAAACGACGGCCGGCGGGTTGGCGAGCCTTGCGGATGCGCTGGCCGCAA AGCCGGATTTGGTGATCCTCGAACTCGGCGCCAATGACATGCTGCGCGCGGT CGATCCGGCCAGCGTGCGCGCCAATCTCGATGCAATGATGACGAAAATCCAG GCGAGCGGCGCTAAACTGCTGCTGACCGGAATGCAGGCGGCGCCCAATTGGG GCGAGGACTATAAGCACGATTTCGACCGCCTTTATCCCGAGCTTGCGAAGGC GCACGGGGTGACGCTTTATCCATTCTTTCTTGATGGGGTGGCGCTGGACCCGG CGCTGAACCAGGCGGATGGAATGCACCCGAACGCCAAGGGGGTCGCCGTGA TCGTCGACCGTATCGCGCCCGTCGTCGCCAAGATGCTGAGAGGCCAGTCATA A (SEQ ID NO : 72) S248_M40cD4 (AminoAcid) MRFAKLTAVIFALIVLHSPLAAAAPPTVMVFGDSLTAGLGLPADAAFPAQLQAKL HDMGIPAEIAARATSGQTTAGGLASLADALAAKPDLVILELGANDMLRAVDPAS VRANLDAMMTKIQASGAKLLLTGMQAAPNWGEDYKHDFDRLYPELAKAHGVT LYPFFLDGVALDPALNQADGMHPNAKGVAVIVDRIAPWAKMLRGQS (SEQ ID N0 : 73) S24S_M44aA5 (DNA) ATGATCGCATGGCTTACCGGATGCGGCAGCGCAAAGACGCAACCGCAGCCCG CAAGTTCCATCCCGCCATCCAGTATTCCAGCAACCGCAAAACCTGCGACAAC GGATATCAGACCGATCATCGTTGCTTTCGGCGACAGCCTGACTGCAGGATAC GGCGTCAGTAGTGAACAAAGCTATCCGGCCAATCTTCAACGCGATCTGGATG CGCGTGGATATCATGCCCACGTCATCAACGAAGGCATCAGCGGCAACACATC GAAAGACGGCGTTCTCAGGGCCCAGGCGATTGCGGCACTCCATCCGGCTGTC GTCATCGTTGCCTTCGGCGGCAACGACGGTCTGCGTGGCCTCCCCATCGGAG ACACGGAAATGAATCTGGCAACGATCATCTCAACCATGCAGCATGCCCATGC CAAGGTAATTTTAGGCGGAATTACTTTGCCTCCCAACTATGGCAGCGAATAC

ATCGCCAAATTCAATGCGATCTATAAAAAGCAGGCAGCCGCGTATCATGTGC CCCTGCTGCCCTTCATGCTGAAGGGGGTGTATGGCGTGCCCGGTTCCATGCAG AGCGACGGCATCCATCCGACCGCCAAGGGCTGCCAGCAAGTGGCCAGAAACT TCCTGCCCTTGTTATTGCCGCTCCTGCACAAATCAGGGAAGAAATCCATGGAG TCGAAAGCATTGTCTCGACGTCATTAA (SEQ ID NO : 74) S248_M44aA5 (Amino Acid) MIAWLTGCGSAKTQPQPASSIPPSSIPATAKPATTDIRPIIVAFGDSLTAGYGVSSEQ SYPANLQRDLDARGYHAHVINEGISGNTSKDGVLRAQAIAALHPAWIVAFGGN DGLRGLPIGDTEMNLATIISTMQHAHAKVILGGITLPPNYGSEYIAKFNAIYKKQA AAYHVPLLPFMLKGVYGVPGSMQSDGIHPTAKGCQQVARNFLPLLLPLLHKSGK KSMESKALSRRH (SEQ ID NO : 75) S261_M2aA12 (DNA) ATGAAAAACATCCTTGCATTTGGCGACAGTCTGACCTGGGGTTTTGTGGCCGG ACAGGATGCGCGCCATCCGTTTGAAACCCGCTGGCCAAACGCATTGGCGGCC GGCCTTGGGGGCAAAGCCCGCGTAATTGAAGAGGGTCAGAACGGCCGCACT ACGGTGTTCGACGATGCCGCCACCTTCGAATCTCGAAATGGCTCGGTGGCATT GCCGCTGCTACTGATCAGCCACCAGCCGTTGGACCTGGTAATCATCATGCTCG GCACCAATGACATCAAGTTTGCCGCCCGCTGCCGCGCCTTTGATGCTTCAATG GGCATGGAACGGCTGATCCAGATCGTCAGAAGTGCCAACTACATGAAGGGCT ACAAGATACCTGAAATCCTCATCATATCGCCGCCCAGCCTCGTGCCGACGCA GGATGAATGGTTCAACGACCTCTGGGGCCATGCCATCGCCGAGTCAAAACTC TTCGCCAAGCACTACAAGCGCGTGGCCGAAGAACTGAAAGTGCATTTCTTTG ATGCAGGCACGGTGGCCGTCGCCGACAAGACCGACGGCGGACATCTCGATGC TGTGAATACTAAAGCCATTGGCGTCGCATTGGTGCCGGTGGTGAAATCAATA CTCGCTCTCTAA (SEQ ID NO : 76) S261M2aA12 (Amino Acid) MKNILAFGDSLTWGFVAGQDARHPFETRWPNALAAGLGGKARVIEEGQNGRTT VFDDAATFESRNGSVALPLLLISHQPLDLVIIMLGTNDIKFAARCRAFDASMGMER LIQIVRSANYMKGYKIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAKHYKRVA EELKVHFFDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL (SEQ ID NO : 77)

S279_M70aE8 (DNA) ATGCCGAAAATAGCCAAACTCGCGCCGTCGGATGTGATCGTAGCTTTCGGCG ACAGTCTGACGTTCGGCACCGGCGCAACGGAAGCGGAGAGTTATCCCATCGT GCTCGCACAATTGATCGGTCGCACCGTGGTGCGCGCGGGTGTGCCGGGTGAG GTAACCGAAGGCGGGCTTGCGCGCCTGACCGACGTTATCGAAGAACACAAGC CGAAGCTGATTATTGTTTGCCTGGGCGGCAACGACATGCTGCGCAAGGTCCA GGAAGACCAGACCCGCGCCAATTTGCGCGCCATTATTAAAACCATCAAGGCG CAAGGCATCGCCGTGGTACTGGTCGGTGTGCCGAAGCCCGCGCTGGTGACCA GTGCGCCGCCGTTCTACGAGGAGATCGCCAAAGAGTTCGGTATCCCTTACGA AGGCAAGATTGTTACCGACGTGTTGTACCAACGCGATCAGAAATCCGATTCC ATACATCCCAATGCCAAAGGCTATCGGCGCATGGCCGAAGCGATAGCCACGC TGCTGAAAAAATCCGGAGCCATTTAA (SEQ ID NO : 78) S279 : M70aE8 (Amino Acid) MPKIAKLAPSDVIVAFGDSLTFGTGATEAESYPIVLAQLIGRTWRAGVPGEVTEG GLARLTDVIEEHKPKLIIVCLGGNDMLRKVQEDQTRANLRAIIKTIKAQGIAWLV GVPKPALVTSAPPFYEEIAKEFGIPYEGKIVTDVLYQRDQKSDSIHPNAKGYRRMA EAIATLLKKSGAI (SEQ ID N0 : 79) S279_M75bA2 (DNA) ATGGAACGGACCGGCCGCGCTGGCGATCGGTGTCGGCGTGGGGCTGGCGAGC CTGAGCCCGGTCGCGCTGGCGACGCCGCCGCGGGGCACCGTGCCGGTGTTCA CCCGATCGGGGACAGCCTGACGGACGAGTATTTTGAGCCGTTCTTCCAGTGG GGGTTCTGCGGGAAGTCGTGGGCCGAGATTTTGGTGGAGACGGGGCGGGCGA GCATGGGCCCGACGGCGCAGCAGGCGGGGATCAGCGAGCCGGAGGGATGGT CGGATCCGCGGAACACGGGGTATCAGCACAACTGGGCGCGGTACTCGTGGAG CTCCTCAGACGCGCTGACCGAGGAGTCGCCGGGGGCGACGCTGAGCGTGCTG CTTGGGGCGGAGTACGCGGTGGTGTTCATTGGGACCAACGACTTCAATCCGT CGTGGCCGGCGTATCAGAGCGTGTATCTGAGCCAGTGGAGCGACGAGCAGAT CGACACGTACGTGAACGGGGTGGTGCAGAACATCGCGCAGATGGTGGACTCG CTGAAGTCGGTCGGGGCGAAGGTGGTGCTTGCGCCGCCGGTGGATTTTCAGT TCGCGGGGTTCCTGCGGAACTCATGCCCGGATCCGATGCTGCGCGAGCAGGC GGGTATTCTGACACGGAAGTGCCACGACCGGGTGCGGTCGATGGCGCGGCAG AAGCACGTGGTGTTCGTGGACATGTGGCGGCTGAACCGCGATTTGTTCGGCA ACGGGTTCGCGATCAGCTACGGCCTTCGGAACACGGTGCGCGTGGGGGACTC GGAGATCGGGCTGCAACTGGCCGGGCTGACGGGATCGGCGGGGCTGGTTCCG GACGGGATCCATCCGCAGCGGGTGGTGCAGGGGATCTGGGCGAATGCGTTCA

TCGTGGGTCTGAACGCGCATGGGGCGAACATCGCGCCCATCGGCGAGGCGGA GATGTGCGCGATGGGGGGGGTCGTGTACGGGGGAACGGACACGCTGGCGAA CTTCCTGCCGCCGGTCGCGGGCTACGTGGAGGACTTCCGCAACGCGGGGGAC TTCGTGTGCACGGCGGACTTCAACCATGACCTTGGCGTGACGCCGACGGACA TCTTCGCGTTCATCAACGCGTGGTTCATGAATGATCCCTCGGCGCGGATGAGC AACCCGGAGCACACGCAGATCGAGGACATCTTCGTGTTTCTGAATCTGTGGC TGGTGGGGTGCTAA (SEQ ID NO : 80) S279 M75bA2 (Amino Acid) MERTGRAGDRCRRGAGEPEPGRAGDAAAGHRAGVHPIGDSLTDEYFEPFFQWG FCGKSWAEILVETGRASMGPTAQQAGISEPEGWSDPRNTGYQHNWARYSWSSS DALTEESPGATLSVLLGAEYAWFIGTNDFNPSWPAYQSVYLSQWSDEQIDTYVN GWQNIAQMVDSLKSVGAKWLAPPVDFQFAGFLRNSCPDPMLREQAGILTRKC HDRVRSMARQKHVVFVDMWRLNRDLFGNGFAISYGLRNTVRVGDSEIGLQLAG LTGSAGLVPDGIHPQRWQGIWANAFIVGLNAHGANIAPIGEAEMCAMGGWYG GTDTLANFLPPVAGYVEDFRNAGDFVCTADFNHDLGVTPTDIFAFINAWFMNDP SARMSNPEHTQIEDIFVFLNLWLVGC (SEQ ID NO : 81) M091_M4aEl l (DNA) ATGAAGACCATTCTCGCCTATGGCGACAGCCTGACCTATGGGGCCAACCCGA TCCCGGGCGGGCCGCGGCATGCCTATGAGGATCGCTGGCCCACGGCGCTGGA GCAGGGGCTGGGCGGCAAGGCGCGGGTGATTGCCGAGGGGCTGGGTGGTCG CACCACGGTGCATGACGACTGGTTTGCGAATGCGGACAGGAACGGTGCGCGG GTGCTGCCGACGCTGCTCGAGAGCCATTCGCCGCTCGACCTGATCGTCATCAT GCTCGGCACCAACGACATCAAGCCGCATCACGGGCGGACGGCCGGCGAGGC CGGGCGGGGCATGGCGCGGCTGGTGCAGATCATCCGCGGGCACTATGCCGGC CGCATGCAGGACGAGCCGCAGATCATCCTCGTGTCGCCGCCGCCGATCATCC TCGGCGACTGGGCGGACATGATGGACCATTTCGGCCCGCACGAAGCGATCGC CACCTCGGTGGATTTCGCTCGCGAGTACAAGAAGCGGGCCGACGAGCAGAAG GTGCATTTCTTCGACGCCGGCACGGTGGCGACGACCAGCAAGGCCGATGGCA TCCACCTCGACCCGGCCAATACGCGCGCCATCGGGGCAGGGCTGGTGCCGCT GGTGAAGCAGGTGCTCGGCCTGTAA (SEQ ID N0 : 82) M091_M4aEll (AminoAcid) MKTILAYGDSLTYGANPIPGGPRHAYEDRWPTALEQGLGGKARVIAEGLGGRTT VHDDWFANADRNGARVLPTLLESHSPLDLIVIMLGTNDIKPHHGRTAGEAGRGM

GCGAGTACCGAGCTCGGGAGCGTCACCGGGTTTGTCGCGGGCTCGGCCACCG CAGGCCGGGCGGATGCCAACTATTGGGCGGTCACGGTCGGCCTGCGGATGCC GTTGTAG (SEQ ID NO : 84) Est105 (Amino Acid) MRTLHRSLLASAAALFLAASGNATAQFSNVYFFGDSLTDAGSFKPVLPPGTGLFT TNPGPVWPQVFGANYGVAVTPANQGGTDYAQGGARVTSLPGVPTSQPTGSAVPI ATQISQFLGSGPADPNAFYSVWGGANDIFFQLGLAQAGMATPAQVQSAVGLAAV QLAQATAALNASGARFITVINVPDIGKTPFGVGSGQGAQITALSSFFNSTLFGALD ATGIQTMRVNGFAVLNEWADPAAYGFANASTPACGATPSLVCTSANFVTPLAA QTFLFADGVHPTTAGHALIAQAVQAMITGPQQMAALGDAPLAVEQANFRALDN RMWSSLNAPRSPGKLQGWAAYDYSHTDLQAGPTNGSGHMNTVAVGVDMKVS DHMLAGAMFGYTNTKGDFGGPGGGYTLKQPVGTAYAGYGVGPWYVGATLGT GGLDYSDVTRAIPLGLAVRTESAEARGYEFTGRILGGYWFTMRDLMHGPYARLA WTKAWKRFSEESTDSTALNYDRQERKQLLWSLGWQLAGNVGSIRPYARATWE IDSKDQDRSVGASSVTLGGFYSVPVAKPDNSYALFSLGASTELGSVTGFVAGSAT AGRADANYWAVTVGLRMPL (SEQ ID NO : 85) Estll4 (DNA) ATGGGGCGATCGAGAGTTCTGAAGGCTGTTTTCCTGGTGGCGTGCCTTGTGGG TCGGCTCGCGGCGCATGCCGAGGCGTCGCCCATCGTGGTCTACGGCGATAGC CTCTCTGACAACGGCAATCTGTTTGCGCTCACCGGCGGTGTCGCGCCGCCCTC GCCGCCGTACTTCAACGGACGGTTTTCTAATGGCCCGGTGGCCGTGGAGTATC TCGCGGCCGCGCTGGGATCTCCGCTGATCGATTTCGCGGTCGGCGGGGCGAC GACCGGCCTCGGCGTCAACGGCGATCCCGGTGGTTCGCCGACGAGTCTCGGC GCGGCGGGATTGCCGGGGCTTCAGACGACATTCGCCGCCACGCAAGGCACGC TGGGTCCGTACGTTGGTGGTCTCTTCGTGGTGTGGGCGGGTCCGAACGACTTC TTGTCGCCCTCGCCGCTTGACACGAACGCTTTTCAGATTGCGAACCGGGCCGT GTCCAACATCCTCGGCGTGGTGGCATCACTTCAGGCACTCGGCGTCGAGCGC ATCCTCGTCCCCGGCATGCCCGATCTCGGTCTGACGCCCGCTCTTCAGCCCAT CGCAGGCGCAGCCACCGCGTTCACCGATTTGTTCAACTCGATGCTGCGCGCG GGCTTGCCGAACGACGTGCTGTACCTGGACACGGCGACAATCTTCCGATCGA TCGTGGCAGACCCTGGGGCCTACGGCTTGACCAACGTGACCACGCCGTGCCT GATTGGTGCGACCGTCTGCGCGAATCCGGATCAGTACCTGTTCTGGGATGGT ATTCATCCTACGACGGCGGGGCACGCGATCTTGGGCAATGCCCTCGTCGCCC AGGCAGTCCCCGAGCCCGCGACCATGGTGCTCGTGCTGACGGGTCTGTCCAT GCACGTGATTGCGCGCCGGCGGCGGGCGTAA (SEQ ID NO : 86)

Estll4 (AminoAcid) MGRSRVLKAVFLVACLVGRLAAHAEASPIVVYGDSLSDNGNLFALTGGVAPPSP PYFNGRFSNGPVAVEYLAAALGSPLIDFAVGGATTGLGVNGDPGGSPTSLGAAGL PGLQTTFAATQGTLGPYVGGLFWWAGPNDFLSPSPLDTNAFQIANRAVSNILGV VASLQALGVERILVPGMPDLGLTPALQPIAGAATAFTDLFNSMLRAGLPNDVLYL DTATIFRSIVADPGAYGLTNVTTPCLIGATVCANPDQYLFWDGIHPTTAGHAILGN ALVAQAVPEPATMVLVLTGLSMHVIARRRRA (SEQ ID NO : 87) Sinorhizobium meliloti Smel (SMal993) (DNA) ATGACAATCAACAGCCATTCATGGAGGACGTTAATGGTGGAAAAGCGCTCAG TACTGTGCTTTGGGGATTCGCTGACATGGGGCTGGATTCCGGTGAAGGGATC CTCACCGACCTTGCGCTATCCCTATGAACAACGGTGGACCGGCGCAATGGCC GCGAGGCTTGGCGACGGTTACCACATCATCGAAGAGGGGCTGAGCGCCCGCA CCACCAGCCTCGACGACCCCAACGACGCGCGGCTCAACGGCAGCACCTACCT GCCCATGGCACTCGCCAGCCACCTCCCACTCGACCTCGTCATCATCATGCTGG GCACGAACGACACGAAATCCTATTTCCACCGCACGCCTTACGAGATCGCCAA CGGCATGGGCAAGCTAGTCGGCCAGGTGCTGACCTGCGCCGGTGGCGTCGGC ACGCCATATCCCGCGCCGAAGGTGCTTGTCGTCGCTCCGCCGCCGCTCGCGCC GATGCCCGACCCGTGGTTCGAAGGCATGTTCGGCGGCGGCTACGAGAAGTCG AAGGAACTCTCCGGCCTCTACAAGGCGCTTGCCGATTTCATGAAGGTCGAGT TTTTCGCCGCCGGTGATTGCATTTCCACCGATGGGATCGACGGCATTCACCTC TCGGCGGAAACCAACATCAGACTCGGGCACGCGATCGCGGACAAAGTTGCG GCGTTGTTC (SEQ ID NO : 88) Sinorhizobium meliloti SmeI (SMal993) (Amino Acid) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKGSSPTLRYPYEQRWTGAMAA RLGDGYHIIEEGLSARTTSLDDPNDARLNGSTYLPMALASHLPLDLVIIMLGTNDT KSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLWAPPPLAPMPDPWF EGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLG HAIADKVAALF (SEQ ID NO : 89) Sinorhizobium meliloti SmeII (Q92XZ1) (DNA) ATGGAGGAGACAGTGGCACGGACCGTTCTATGCTTCGGAGATTCCAACACTC ACGGCCAGGTACCTGGCCGCGGACCGCTTGATCGCTACCGACGCGAACAGCG CTGGGGCGGTGTTCTGCAAGGCCTGCTCGGCCCGAACTGGCAGGTTATCGAA GAAGGCCTGAGCGGACGCACGACCGTGCATGACGATCCGATCGAAGGTTCGC TCAAGAACGGCCGGACCTATCTGCGCCCCTGTCTGCAGAGCCATGCACCACT

CGACCTTATCATCATTATGCTCGGCACCAATGACCTGAAGCGGCGCTTCAACA TGCCACCGTCCGAGGTCGCAATGGGCATCGGCTGTCTCGTGCACGATATCCG AGAACTCTCGCCCGGCCGGACCGGCAACGATCCCGAAATCATGATCGTCGCC CCGCCGCCGATGCTGGAAGATCTCAAGGAATGGGAGTCGATTTTCTCAGGCG CACAGGAAAAATCTCGCAAGCTGGCGCTGGAGTTCGAGATAATGGCGGATTC TCTGGAGGCGCATTTCTTCGACGCCGGTACGGTCTGCCAGTGTTCGCCGGCCG ATGGCTTCCACATCGACGAGGATGCCCACCGCCTGCTCGGCGAGGCTCTCGC CCAGGAAGTGCTGGCGATCGGGTGGCCCGATGCGTAA (SEQ ID NO : 90) Sinorhizobium meliloti SmelI (Q92XZ1) (Amino Acid) MEETVARTVLCFGDSNTHGQVPGRGPLDRYRREQRWGGVLQGLLGPNWQVIEE GLSGRTTVHDDPIEGSLKNGRTYLRPCLQSHAPLDLIIIMLGTNDLKRRFNMPPSE VAMGIGCLVHDIRELSPGRTGNDPEIMIVAPPPMLEDLKEWESIFSGAQEKSRKLA LEFEIMADSLEAHFFDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO : 91) Sinorhizobium meliloti SmeIII (Q9EV56) (DNA) ATGAAGACAGTCCTTTGCTACGGTGACAGTCTGACCTGGGGATACGATGCAA CCGGTTCCGGCCGGCATGCGCTGGAGGACCGTTGGCCGAGCGTGCTGCAGAA GGCGCTCGGTTCGGACGCGCATGTCATCGCCGAAGGGCTGAACGGGCGGACG ACCGCCTATGACGACCATCTCGCCGATTGCGACCGGAACGGCGCGCGTGTCC TCCCGACGGTCCTGCACACCCACGCGCCACTCGATCTCATCGTGTTCATGCTC GGCTCGAACGACATGAAGCCGATCATTCACGGCACCGCTTTCGGCGCGGTGA AGGGCATCGAGCGCCTCGTCAATCTGGTGCGCAGGCACGACTGGCCGACGGA AACGGAGGAGGGGCCCGAGATTCTCATCGTCTCGCCGCCGCCGCTCTGCGAG ACGGCCAACAGCGCCTTTGCCGCCATGTTCGCGGGCGGGGTCGAGCAATCCG CAATGCTGGCGCCGCTTTATCGCGATCTCGCCGACGAGCTCGACTGCGGCTTC TTCGACGGCGGATCGGTGGCCAGGACGACGCCGATCGACGGTGTCCACCTCG ACGCGGAGAACACCCGGGCGGTCGGCAGAGGGTTGGAGCCTGTCGTGCGGA TGATGCTCGGGCTTTAA (SEQ ID NO : 92) Sinorhizobium meliloti SmeIII (Q9EV56) (Amino Acid) MKTVLCYGDSLTWGYDATGSGRHALEDRWPSVLQKALGSDAHVIAEGLNGRTT AYDDHLADCDRNGARVLPTVLHTHAPLDLIVFMLGSNDMKPIIHGTAFGAVKGIE RLVNLVRRHDWPTETEEGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAPLY RDLADELDCGFFDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRMMLGL (SEQ ID NO : 93)

Agrobacterium tumefaciens Atu III (AAD02335) (DNA) ATGGTGAAGTCGGTCCTCTGCTTTGGCGATTCCCTCACCTGGGGATCAAATGC GGAAACGGGTGGCCGGCACAGCCATGACGATCTTTGGCCGAGCGTCTTGCAG AAGGCGCTCGGTCCTGACGTGCATGTGATTCACGAAGGTCTGGGTGGTCGCA CCACCGCCTATGACGACAACACCGCCGATTGCGACCGCAACGGCGCGCGGGT TCTTCCGACGTTGTTGCACAGCCATGCGCCGCTGGATCTGGTGATTGTCATGC TCGGGACCAACGACCTGAAGCCGTCAATCCATGGATCGGCGATCGTTGCCAT GAAGGGTGTCGAAAGGCTGGTGAAGCTCACGCGCAACCACATCTGGCAGGTG CCGGACTGGGAGGCGCCTGACGTGCTGATCGTCGCACCGCCGCAGCTGTGTG AAACGGCCAATCCGTTCATGGGCGCGATCTTTCGTGATGCGATCGATGAATC GGCGATGCTGGCGTCCGTTTACCGGGACCTTGCCGACGAGCTTGATTGCGGCT TTTTCGATGCGGGTTCCGTCGCCCGAACGACGCCGGTGGATGGCGTTCATCTC GATGCTGAAAATACGCGGGCCATCGGGCGGGGGCTGGAGCCCGTCGTTCGCA TGATGCTCGGACTTTAA (SEQ ID N0 : 94) Agrobacterium tumefaciens Atu in (AAD02335) (Amino Acid) MVKSVLCFGDSLTWGSNAETGGRHSHDDLWPSVLQKALGPDVHVIHEGLGGRT TAYDDNTADCDRNGARVLPTLLHSHAPLDLVIVMLGTNDLKPSIHGSAIVAMKG VERLVKLTRNHIWQVPDWEAPDVLIVAPPQLCETANPFMGAIFRDAIDESAMLAS VYRDLADELDCGFFDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL (SEQ ID NO : 95) Mesorhizobium loti MloI (Q98MY5) (DNA) ATGAAGACGGTGCTTTGCTACGGCGACTCGCTGACCTGGGGCTACAATGCCG AAGGCGGCCGCCATGCGCTGGAAGACCGCTGGCCGAGCGTGCTGCAAGCAG CGTTAGGCGCCGGCGTGCAAGTGATTGCCGATGGCCTCAACGGCCGCACCAC GGCCTTCGACGATCATCTGGCCGGTGCTGATCGCAACGGCGCCAGGCTGCTG CCGACGGTCCTGACGACGCACGCGCCGATCGACCTGATCATCTTCATGCTCG GCGCCAACGACATGAAGCCTTGGATCCACGGCAATCCGGTCGCAGCCAAGCA AGGCATCCAGCGGTTGATCGACATCGTGCGTGGTCACGACTACCCGTTCGAC TGGCCGGCGCCGCAGATCCTGATCGTCGCGCCGCCTGTAGTCAGCCGCACCG AAAATGCCGACTTCAAGGAAATGTTCGCCGGTGGCGATGACGCCTCGAAGTT TTTGGCACCGCAATATGCCGCGCTCGCCGACGAAGCCGGCTGTGGCTTCTTCG ACGCCGGCAGCGTGGCCCAAACCACACCGCTCGATGGCGTTCACCTCGATGC CGAAAACACGCGAGAAATCGGCAAGGCGCTGACGCCGATCGTGCGCGTCAT GCTGGAATTGTAA (SEQ ID NO : 96)

Mesorhizobium loti Mlo I (Q98MY5) (Amino Acid) MKTVLCYGDSLTWGYNAEGGRHALEDRWPSVLQAALGAGVQVIADGLNGRTT AFDDHLAGADRNGARLLPTVLTTHAPIDLIIFMLGANDMKPWIHGNPVAAKQGIQ RLIDIVRGHDYPFDWPAPQILIVAPPWSRTENADFKEMFAGGDDASKFLAPQYA ALADEAGCGFFDAGSVAQTTPLDGVHLDAENTREIGKALTPIVRVMLEL (SEQ ID NO : 97) Moraxella bovis Mbo (AAK53448) (DNA) ATGAAAAAATCCGCCTTTGCCAAATACTCAGCACTTGCCCTAATGGTTGGGAT GTGCCTGCACACCGCTTACGCCAAGGAGTTTAGCCAAGTCATCATTTTTGGGG ACAGCTTGTCCGATACAGGTCGCCTAAAAGATATGGTCGCCCGAAAAGATGG CACCCTTGGCAACACCTTACAGCCATCTTTTACCACCAACCCCGACCCTGTAT GGTCAAGCTTATTTGCCCAAAGTTATGGCAAAACCGCCAGTCCCAACACGCC TGACAATCCCACTGGCACTAACTATGCCGTGGGCGGAGCTCGCTCTGGCTCG GAGGTCAATTGGAATGGTTTTGTGAATGTACCCTCCACCAAAACGCAAATCA CCGACCATTTGACCGCCACAGGTGGCAAAGCCGACCCTAATACCCTGTATGC CATTTGGATTGGCTCTAATGACTTAATTTCAGCTTCTCAAGCCACCACAACAG CCGAAGCCCAAAACGCCATTAAAGGTGCGGTAACTCGCACCGTGATAGACAT CGAAACACTCAATCAAGCAGGGGCGACAACCATTTTGGTGCCAAATGTGCCT GATTTGAGCCTCACGCCCCGAGCCATCTATGGCGAAAGCCTCATGGCAGGCG TGCAAGACAAAGCCAAACTCGCCTCAAGTCTGTATAATAGCGGTCTGTTTGA AGCATTAAATCAATCCACCGCCAACATCATCCCTGCCAACACCTTTGCCCTAC TCCAAGAAGCGACCACAAATAAAGAAGCCTTTGGTTTTAAAAACACGCAAGG CGTGGCGTGTCAAATGCCCGCTCGTACCACAGGGGCGGATGATGTGGCTTCT ACTTCCTTGGCATGTACCAAAGCCAATCTTATAGAAAACGGGGCAAATGACA CCTACGCCTTTGCCGATGACATTCACCCATCGGGACGCACGCACCGCATTTTG GCACAGTATTACCGTTCTATCATGGACGCCCCTACTCACATGGGTAAACTCTC AGGCGAGCTTGTCAAAACAGGTTCAGCCCACGACCGTCATGTTTACCGTCAG CTTGACAGGCTTAGTGGCTCACAGCACAGCATTTGGGCAAACGTCTATGCCA GCGACCGTACCGACCCCACCACCCAAATCGGCTTGGACGTGGCAGGTTCATC AAGCCATACAGGGGCGTATCTGAGCCACCAAAACCAAGATTATGTGCTGGAT GACACCCTATCATCAGATGTCAAAACCATTGGCATGGGGCTGTATCATCGCC ATGACATCGGCAATGTCCGTCTAAAAGGCGTGGCAGGTATCGACCGACTTAG CGTGGATACGCACCGCCATATCGACTGGGAGGGGACAAGCCGTTCGCACACC GCAGATACCACCGCCAGACGTTTTCATGCAGGGCTACAAGCCAGCTATGGCA TAGACATGGGCAAAGCCACCGTGCGTCCGCTTATCGGCGTACATGCCCAAAA AGTCAAAGTAAATGACATGACCGAGAGCGAATCAACTTTATCCACCGCCATG

CGTTTTGGCGAGCAAGAACAAAAGTCCCTACAAGGCGAGATTGGCGTCGATG TGGCTTATCCGATTAGCCCTGCTTTGACTCTGACGGGCGGTATCGCTCACGCT CATGAGTTTAACGATGATGAACGCACCATTAATGCCACTTTAACCTCCATTCG TGAATACACGAAGGGCTTTAATACAAGCGTTAGCACCGACAAATCTCACGCC ACCACCGCTCATCTGGGCGTACAAGGGCAACTTGGCAAGGCAAATATTCATG CAGGCGTTCACGCCACCCACCAAGACAGCGATACAGACGTGGGTGGTTCGCT TGGGGTTCGCTTGATGTTTTAA (SEQ ID NO : 98) Moraxella bovis Mbo (AAK53448) (Amino Acid) MKKSAFAKYSALALMVGMCLHTAYAKEFSQVIIFGDSLSDTGRLKDMVARKDG TLGNTLQPSFTTNPDPVWSSLFAQSYGKTASPNTPDNPTGTNYAVGGARSGSEVN WNGFVNVPSTKTQITDHLTATGGKADPNTLYAIWIGSNDLISASQATTTAEAQNA IKGAVTRTVIDIETLNQAGATTILVPNVPDLSLTPRAIYGESLMAGVQDKAKLASS LYNSGLFEALNQSTANIIPANTFALLQEATTNKEAFGFKNTQGVACQMPARTTGA DDVASTSLACTKANLIENGANDTYAFADDIHPSGRTHRILAQYYRSIMDAPTHMG KLSGELVKTGSAHDRHVYRQLDRLSGSQHSIWANVYASDRTDPTTQIGLDVAGS SSHTGAYLSHQNQDYVLDDTLSSDVKTIGMGLYHRHDIGNVRLKGVAGIDRLSV DTHRHIDWEGTSRSHTADTTARRFHAGLQASYGIDMGKATVRPLIGVHAQKVKV NDMTESESTLSTAMRFGEQEQKSLQGEIGVDVAYPISPALTLTGGIAHAHEFNDD ERTINATLTSIREYTKGFNTSVSTDKSHATTAHLGVQGQLGKANIHAGVHATHQD SDTDVGGSLGVRLMF (SEQ ID NO : 99) Chromobacterium violaceum Cvi (Q7NRP5) (DNA) ATGCGCTCTATCGTCTGCAAAATGCTGTTCCCTTTGTTGCTGCTGTGGCAGCT GCCCGCCCTGGCCGCCACCGTGCTGGTGTTCGGCGACAGCCTGTCCGCCGGC TACGGCCTGGCCCCGGGCCAGGGATGGGCGGCGCTGCTGGCGCGCGACCTCT CGCCCCGGCACAAGGTGGTCAACGCCAGCGTGTCCGGCGAAACCAGCGCCGG CGGCCTGTCCAGGCTGCCCGACGCGCTCGCCCGCCACCAGCCCGACGTGCTG GTGCTGGAACTCGGCGCCAACGATGGCCTGCGCGGCCTGCCGATGGCTGACA TGAGGCGCAACCTGCAGCGGATGATAGACCTGGCCCAGGCGCGCAAGGCCA AGGTGCTGCTGGTGGGCATGGCGCTGCCACCCAACTATGGCCCCCGCTACGG CGCCGAGTTCCGCGCCGTTTATGACGATTTGGCCCGCCGCAACCGCCTGGCCT ACGTGCCGCTGCTGGTCGAGGGCTTCGCCGGCGACCTCGGCGCCTTCCAGCC CGACGGCCTGCATCCCCGCGCGGAGAAGCAGGCCACCATGATGCGCACGGTC AAGGCAAAACTGCCAGTGAAATAA (SEQ ID NO : 100) Chromobacterium violaceum Cvi (Q7NRP5) (Amino Acid) MRSIVCKMLFPLLLLWQLPALAATVLVFGDSLSAGYGLAPGQGWAALLARDLSP RHKVVNASVSGETSAGGLSRLPDALARHQPDVLVLELGANDGLRGLPMADMRR

NLQRMIDLAQARKAKVLLVGMALPPNYGPRYGAEFRAVYDDLARRNRLAYVPL LVEGFAGDLGAFQPDGLHPRAEKQATMMRTVKAKLPVK (SEQIDNO : 101) Vibrio vulnificus Vvu (AA007232) (DNA) ATGTTTTTCCTTTCTAGCGTCGCACACGCAACCGAGAAAGTGTTAATTCTTGG CGACAGCCTAAGTGCAGGATACAACATGTCTGCAGAGCAGGCTTGGCCTAAT TTGTTACCAGAAGCATTGAATACATACGGAAAAAACGTAGAAGTGATCAACG CCAGTATCTCTGGAGACACAACCGGCAATGGACTATCTCGTCTGCCTGAGTTG TTAAAAACGCACTCACCAGACTGGGTGCTTATTGAGTTGGGTGCCAATGATG GCTTGCGAGGTTTCCCGCATAAAGTGATCTCTTCAAACCTTTCGCGAATGATT CAACTCAGTAAAGCCTCAGACGCTAAAGTCGCATTGATGCAAATTCGTGTAC CGCCTAACTATGGCAAGCGCTACACCGATGCATTTGTCGAACTCTACCCTACG CTTGCTGAACATCACCAAGTCCCGTTGCTCCCCTTTTTCTTAGAGGAAGTGAT CGTGAAACCGGAATGGATGATGCCTGATGGCTTACACCCAATGCCCGAAGCT CAGCCTTGGATCGCTCAATTTGTTGCAAAAACGTTTTACAAACATCTCTAA (SEQ ID NO : 102) Vibrio vulnificus Vvu (AA007232) (Amino Acid) MFFLSSVAHATEKVLILGDSLSAGYNMSAEQAWPNLLPEALNTYGKNVEVINASI SGDTTGNGLSRLPELLKTHSPDWVLELGANDGLRGFPHKVISSNLSRMIQLSKAS DAKVALMQIRVPPNYGKRYTDAFVELYPTLAEHHQVPLLPFFLEEVIVKPEWMM PDGLHPMPEAQPWIAQFVAKTFYKHL (SEQ ID N0 : 103) Ralstonia eutropha Reu (ZP00166901) (DNA) ATGCCATTGACCGCGCCGTCTGAAGTCGATCCGCTGCAAATCCTGGTCTATGC CGATTCGCTTTCGTGGGGCATCGTGCCCGGCACCCGCCGGCGGCTTCCCTTCC CGGTTCGCTGGCCAGGCCGGCTCGAACTCGGCCTGAACGCCGACGGCGGCGC CCCGGTCCGCATCATCGAGGACTGCCTGAACGGCCGGCGCACCGTCTGGGAC GACCCATTCAAACCGGGCCGCAACGGCTTGCAAGGGCTGGCGCAGCGCATCG AGATCCATTCCCCGGTGGCGCTCGTGGTTTTGATGCTGGGCAACAACGATTTC CAGTCCATGCATCCGCACAACGCCTGGCATGCGGCACAGGGCGTCGGCGCGC TGGTCCACGCCATCCGGACGGCGCCGATCGAACCGGGAATGCCGGTGCCGCC GATCCTGGTGGTGGTGCCGCCGCCGATCCGCACGCCCTGCGGGCCGCTCGCG CCCAAGTTCGCCGGCGGCGAACACAAGTGGGCAGGCCTGCCCGAGGCGCTGC GCGAACTGTGCGCCACTGTCGACTGCTCGCTGTTCGATGCGGGTACCGTGATC CAGAGCAGTGCCGTCGACGGCGTACACCTTGACGCCGATGCCCATGTCGCCC

TGGGCGATGCCCTGCAACCGGTCGTTCGTGCGCTGCTCGCCGAATCCTCGGG ACATCCCTCCTAA (SEQ ID NO : 104) Ralstonia eutropha Reu (ZP00166901) (Amino Acid) MPLTAPSEVDPLQILVYADSLSWGIVPGTRRRLPFPVRWPGRLELGLNADGGAPV RIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALVVLMLGNNDFQSMHP HNAWHAAQGVGALVHAIRTAPIEPGMPVPPILVWPPPIRTPCGPLAPKFAGGEH KWAGLPEALRELCATVDCSLFDAGTVIQSSAVDGVHLDADAHVALGDALQPW RALLAESSGHPS (SEQ ID NO : 105) Salmonella typhimurium Stm (AAC38796) (DNA) ATGACCCAAAAGCGTACCCTGCTAAAATACGGCATACTCTCGCTGGCGCTGG CCGCGCCATTATCTGCCTGTGCGTTTGACTCTCTTACGGTGATTGGCGATAGC CTTAGCGATACCGGTAATAACGGTCGCTGGACCTGGGATAGTGGTCAAAATA AGCTCTACGACGAACAGTTGGCCGAACGATATGGGCTGGAATTAAGCCCTTC CAGCAATGGCGGCTCTAATTATGCCGCCGGCGGCGCGACGGCGACCCCGGAA TTAAACCCGCAGGATAATACCGCGGATCAGGTACGGCAGTGGCTTGCCAAAA CGGGGGGAAAAGCCGACCACAACGGTTTGTATATTCACTGGGTCGGCGGAAA CGATCTGGCGGCGGCCATCGCGCAACCAACCATGGCACAGCAAATAGCCGGT AATAGCGCCACTAGCGCGGCGGCGCAGGTAGGGCTGTTACTGGATGCCGGCG CCGGGCTGGTCGTGGTGCCAAACGTACCGGATATTAGTGCGACGCCAATGCT TCTGGAGGCGGTAATCACCGCTGGGCTGGGCGCAGCGGCGCCCCCGGCGCTA AAAGCGGCGTTAGATGCGCTGGCGGAGGGCGCTACGCCCGATTTCGCCAGTC GGCAACAGGCGATCCGCAAGGCGCTGCTGGCGGCGGCTGCAACGGTAAGCA GCAATCCATTTATTCAGCAACTGCTCGTTGAACAACTGCTGGCGGGCTATGAA GCGGCGGCAGGGCAGGCGTCAGCTCTGACCGATTATTATAATCAGATGGAAG AGAAGGGGCTGGAGCAACACGGCGGCAATATAGCCCGTGCCGATATCAACG GCCTCTTTAAGGAAATTCTTGCCAACCCGCAGGCGTTTGGTCTGACAAATACC GTAGGTATGGCCTGCCCGCCTGGCGTATCCGCTTCGGCGTGCTCCTCGGCAAT GCCTGGATTTAATGCGTCGCAGGACTATGTGTTTGCCGATCATTTACATCCCG GTCCGCAGGTCCATACCATTATTGCGCAATATATTCAGTCGATCATTGCCGCG CCGGTACAGGCGACATACCTGAACCAAAGCGTTCAGTCGATGGCGCAAGGCA GTCGTACCACGCTTGACAGCCGTTATCAGCAGCTTCGCCAGGGGGAAAATCC TGTTGGTTCGCTGGGCATGTTCGGCGGATACAGCGGGGGATATCAACGTTAT GATAATAATGAGGCCGACGGGAACGGTAATCATAATAATCTGACGGTTGGCG TCGATTATCAGCTTAACGAGCAGGTTCTGCTGGGAGGGCTGATAGCCGGTTCT CTGGATAAGCAACATCCTGACGATAATTATCGTTATGATGCCCGCGGTTTTCA

GGCCGCCGTATTCAGCCATTTACGCGCCGGTCAGGCGTGGCTGGATAGCGAT TTACACTTTCTGTCCGCTAAATTCAGTAACATTCAGCGCAGTATAACGCTCGG TGCGCTAAGACGGGTGGAAGAGGGCGAAACCAACGGTCGGCTGTCGGGCGC GAGCTTAACCAGCGGTTATGATTTTGTCATGGTGCCGTGGTTAACGACCGGAC CGATGCTGCAATATGCATGGGATTACAGCCACGTTAATGGTTATAGCGAGAA GCTCAATACCAGTACATCAATGCGTTTTGGTGACCAAAACGCCCATTCGCAG GTGGGTAGCGCGGGTTGGCGTCTGGATCTTCGCCACAGCATCATTCACTCCTG GGCGCAGATTAATTATCGCCGTCAGTTTGGCGATGATACGTATGTGGCGAAC GGCGGCCTTAAATCGACCGCGCTGACGTTTAGCCGCGACGGAAAAACGCAGG ATAAAAACTGGGTTGATATCGCGATTGGCGCAGATTTTCCGCTGTCGGCAAC GGTGTCCGCTTTCGCCGGGCTGTCGCAAACGGCAGGGTTAAGCGATGGCAAT CAAACCCGTTATAACGTTGGGTTTAGCGCCCGATTTTAA (SEQ ID NO : 106) Salmonella typhimurium Stm (AAC38796) (Amino Acid) MTQKRTLLKYGILSLALAAPLSACAFDSLTVIGDSLSDTGNNGRWTWDSGQNKL YDEQLAERYGLELSPSSNGGSNYAAGGATATPELNPQDNTADQVRQWLAKTGG KADHNGLYIHWVGGNDLAAAIAQPTMAQQIAGNSATSAAAQVGLLLDAGAGLV VVPNVPDISATPMLLEAVITAGLGAAAPPALKAALDALAEGATPDFASRQQAIRK ALLAAAATVSSNPFIQQLLVEQLLAGYEAAAGQASALTDYYNQMEEKGLEQHG GNIARADINGLFKEILANPQAFGLTNTVGMACPPGVSASACSSAMPGFNASQDYV FADHLHPGPQVHTIIAQYIQSIIAAPVQATYLNQSVQSMAQGSRTTLDSRYQQLRQ GENPVGSLGMFGGYSGGYQRYDNNEADGNGNHNNLTVGVDYQLNEQVLLGGLI AGSLDKQHPDDNYRYDARGFQAAVFSHLRAGQAWLDSDLHFLSAKFSNIQRSIT LGALRRVEEGETNGRLSGASLTSGYDFVMVPWLTTGPMLQYAWDYSHVNGYSE KLNTSTSMRFGDQNAHSQVGSAGWRLDLRHSIIHSWAQINYRRQFGDDTYVAN GGLKSTALTFSRDGKTQDKNWVDIAIGADFPLSATVSAFAGLSQTAGLSDGNQTR YNVGFSARF (SEQ ID NO : 107) In total, nine of the new"GDSL"-type esterases were identified in 6 metagenomic libraries and BRAIN's esterase/lipase library. Eight of these genes were heterologously expressed in E. coli and the resulting enzymes analyzed for activity in the assays described herein. The characterization of these enzymes for perhydrolase activity revealed that one displayed the desired activity. A second one was predicted to show this activity due to the presence of amino acids conserved among this group of enzymes.

Comparison of the sequences of enzymes for which the presence or absence of the desired perhydrolase activity was determined led to the identification of 19 amino acid positions which were conserved among the enzymes which displayed the desired perhydrolase activity. Thus, it is contemplated that these conserved amino acids are essential for the perhydrolase reaction and/or is a structural feature of perhydrolase enzymes.

One of the identified structural motifs ("G/ARTT") conserved among esterases with the desired perhydrolase activity was used to design degenerate primers which provided the means to focus the screening on true perhydrolases among"GDSL"-type esterases. Indeed, the use of these"G/ARTT"primers led to the identification of enzymes with the desired perhydrolase activity from the metagenome. However, it is not intended that the use of the metagenome be limited to any particular assay method.

Indeed, it is contemplated that the metagenome be searched by assaying for a particular enzyme activity or activities desired (e. g., perhydrolysis and/or acyltransferase (cofactor dependent or independent) activity). In addition, screening using poly and/or monoclonal anti-sera directed against a protein of interest finds use in the present invention. In additional embodiments, the metagenome is searched using degenerate primer sets based on the sequence of the protein of interest.

In addition, the knowledge of the structure/function relationship of perhydrolases allowed searching for these enzymes in genome sequences of cultivable microorganisms.

Of 16"GDSL"-type esterases identified in different bacterial isolates, the corresponding genes of 10 enzymes were amplified and heterologously expressed in E. coli. The resulting enzyme samples of seven clones were analyzed using the assays described herein. Of five samples characterized to date, 4 enzymes indeed showed the desired activity and all results confirmed the proposed relationship between primary structural determinants and the function of perhydrolases. Thus, an enzyme library of 19"GDSL"- type esterases comprising at least 6 perhydrolases with the desired perhydrolase activity

was set up. The identified correlation between the structure and function of perhydrolases provides a definition of the sequence space used by enzymes with the desired perhydrolase activity.

Comparisons were made of protein sequences of enzymes for which the absence or presence of the desired perhydrolase activity. This revealed a correlation between the presence of certain amino acids and the capability to perform perhydrolase reactions.

This knowledge was used to identify enzymes containing these conserved amino acids in sequenced genomes from cultivable microorganisms. The following enzymes were identified and experiments to amplify the genes from the genomic DNA of the corresponding strains using specific primers were performed.

Table 1."GDSL"-type Esterases with a"GRTT"-Motif From Bacterial Isolates Isolate Protein Acronym Amplicon Expression Identifier Vector Sinorhizobium Smal993 Sme I yes pLO_SmeI meliloti Sinorhizobium Q92XZ1 Sme II yes pET26SmeII meliloti Sinorhizobium Q9EV56 Sme III yes pET26SmeIII meliloti Agrobacterium Q9KWB1 Arh I no rhizogenes Agrobacterium Q9KWA6 ArhII no rhizogenes

Agrobacterium AAD02335 Atu III yes pET26_AtuIII tumefaciens Mesorhizobium loti Q98MY5 Mlo I yes pET26_Mlo Mesorhizobium loti ZP 00197751 Mlo II no Ralstonia Q8XQI0 Rso no solanacearum Ralstonia eutropha ZP_00166901 Reu yes n. d.

Moraxella bovis AAK53448 Mbo yes pET26 Mbo Burkholderia ZP_00216984 Bce no - cepacia Chromobacterium Q7NRP5 Cvi yes pET26 Cvi violaceum Pirellula NP865746 Psp n. d. n. d.

Vibrio vulnificus AA007232 Vvu yes pET26 Vvu Salmonella AAC38796 Sty yes pET26 Sty typhimurium In the cases of A. rliizogenes, M. loti (enzyme II), R. solanacearum and B. cepacia no amplicon could be generated. It was thought that this was probably due to genetic differences between the strains used in this investigation and those used for the sequencing of the genes deposited in the public domain databases. One reason might be that the corresponding genes are located on plasmids which are not present in the strains used in this investigation. However, it is not intended that the present invention be limited to any particular mechanism or theory.

The amplicons from all other strains were sequenced. In many cases there were differences between the sequence from the databases and the sequence determined during the development of the present invention. By sequencing two clones from independent amplifications, mutations introduced by the polymerase could be nearly excluded. The sequences of the genes and the deduced amino acid sequences of"GDSL"-type esterases with a"GRTT"-motif or variations from bacterial isolates are provided below: SMal 993_Sinorhizobium meliloti (Sme I) (SEQ ID NOS : 88 and 89) Q92XZ1 Sinorhizobium meliloti (Sme II) (SEQ ID NOS : 90 and 91) Q9EV56 Sinorhizobium meliloti (Sme HI) (SEQ ID NOS : 92 and 93) AAD02335 Agrobacterium tumefaciens (Atu IIn (SEQ ID NOS : 94 and 95) Q98MY5 Mesorhizobium loti (Mlo 1) (SEQ ID NOS : 96 and 97) ZP 00166901 Ralstonia eutropha (Reu) (SEQ ID NOS : 104 and 105) AAK53448 Moraxella bovis (Mbo) (SEQ ID NOS : 98 and 99) Q7NRP5 Chromobacterium violaceum (Cvi) (SEQ ID NOS : 100 and 101) AA007232 Vibrio vulnificus (Vvu) (SEQ ID NOS : 102 and 103) AAC38796 Salmonella typhimurium (Stm) (SEQ ID NOS : 106 and 107) Q9SWB1_Agrobacteriutn rhizogenes (Arh I) MICHKGGEEMRSVLCYGDSNTHGQIPGGSPLDRYGPNERWPGVLRRELGSQWY VIEEGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHAILDLVIIMLGTNDLKARFGQ PPSEVAMGIGCLVYDIRELAPGPGGKPPEIMWAPPPMLDDIKEWEPIFSGAQEKS RRLALEFEIIADSLEVHFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGW R (SEQ ID NO : 108) ATGATTTGCCATAAAGGTGGGGAGGAAATGCGGTCAGTCTTATGCTACGGCG ACTCGAATACGCACGGCCAGATTCCGGGGGGCTCACCGCTCGACCGATACGG GCCGAACGAGCGCTGGCCTGGCGTTTTGAGACGGGAGCTTGGAAGCCAGTGG TATGTGATCGAGGAGGGCCTGAGTGGCCGCACGACGGTTCGCGACGATCCGA TCGAGGGCACGATGAAAAACGGCCGGACCTACCTGCGTCCGTGCCTCATGAG CCACGCGATCCTCGATCTCGTGATTATCATGCTCGGGACGAACGACCTGAAA GCGCGCTTCGGTCAACCGCCATCGGAAGTGGCGATGGGGATCGGCTGCCTCG TCTACGATATCAGGGAGCTGGCGCCCGGACCGGGCGGCAAGCCCCCCGAAAT CATGGTGGTTGCTCCGCCGCCGATGCTGGACGATATCAAGGAATGGGAACCC

ATATTTTCCGGCGCCCAGGAGAAATCCCGGCGTCTCGCGCTTGAGTTTGAAAT TATTGCTGATTCGCTTGAAGTACACTTCTTTGACGCCGCGACCGTCGCATCGT GTGATCCTTGCGATGGTTTTCACATCAACCGGGAAGCGCATGAAGCCTTGGG AACAGCGCTTGCCAGGGAAGTGGAGGCGATCGGTTGGAGATGATGA (SEQ ID NO : 109) Q9KWA6 Agrobacterium rhizogenes (Arh IPJ MAESRSILCFGDSLTWGWIPVPESSPTLRYPFEQRWTGAMAAALGDGYSIIEEGLS ARTTSVEDPNDPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANG MGKLAGQVLTSAGGIGTPYPAPKLLIVSPPPLAPMPDPWFEGMFGGGYEKSLELA KQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEA KNAAA (SEQ ID NO : 110) ATGGCAGAGAGCCGCTCAATATTATGTTTTGGGGATTCACTCACATGGGGTTG GATTCCGGTACCGGAGTCGTCGCCGACGCTCAGATATCCCTTTGAGCAGCGCT GGACCGGTGCAATGGCTGCGGCACTCGGTGACGGCTATTCAATCATCGAGGA AGGCCTTTCCGCCCGCACGACCAGCGTCGAGGATCCGAACGATCCCAGGCTG AACGGCAGCGCCTACCTGCCGATGGCGCTCGCCAGCCATCTGCCGCTCGATC TCGTCATCATCCTTCTCGGCACCAACGACACCAAGTCCTATTTCCGCCGCACG CCCTATGAGATCGCCAACGGCATGGGCAAGCTTGCCGGACAGGTTCTGACCT CGGCCGGCGGGATCGGCACGCCCTACCCTGCCCCGAAGCTTCTGATCGTTTC GCCGCCGCCGCTCGCTCCCATGCCTGACCCGTGGTTCGAAGGCATGTTCGGTG GCGGTTACGAAAAGTCGCTCGAACTCGCAAAGCAGTACAAGGCGCTCGCCAA CTTCCTGAAGGTCGACTTCCTCGACGCCGGCGAGTTTGTAAAGACCGACGGC TGCGATGGAATCCATTTCTCCGCCGAGACGAACATCACGCTCGGCCATGCGA TCGCGGCGAAGGTCGAAGCGATTTTCTCACAAGAGGCGAAGAACGCTGCGGC TTAG (SEQ ID NO : 111) ZP 00197751 Mesofhizobium loti (Mlo I) MKTILCYGDSLTWGYDAVGPSRHAYEDRWPSVLQGRLGSSARVIAEGLCGRTTA FDDWVAGADRNGARILPTLLATHSPLDLVIVMLGTNDMKSFVCGRAIGAKQGME RIVQIIRGQPYSFNYKVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAPLYAAL AQQTGCAFFDAGTVARTTPLDGIHLDAENTRAIGAGLEPWRQALGL (SEQ ID NO : 112) ATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATGATGCCGT CGGACCCATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATG ATGCCGTCGGACCCTCACGGCATGCTTATGAGGATCGATGGCCCTCCGTACTG

CAAGGCCGCCTCGGTAGCAGTGCGCGGGTGATCGCCGAGGGGCTTTGCGGCC GCACAACTGCGTTTGACGACTGGGTCGCTGGTGCGGACCGGAACGGTGCGCG CATCCTGCCGACGCTTCTTGCGACCCATTCACCGCTTGACCTCGTTATCGTCA TGCTCGGGACGAACGACATGAAATCGTTCGTTTGCGGGCGCGCTATCGGCGC CAAGCAGGGGATGGAGCGGATCGTCCAGATCATCCGCGGGCAGCCTTATTCC TTCAATTATAAGGTACCGTCGATTCTTCTCGTGGCGCCGCCGCCGCTGTGCGC TACCGAAAACAGCGATTTCGCGGAAATTTTTGAAGGTGGCATGGCTGAATCG CAAAAGCTCGCGCCGCTTTATGCCGCGCTGGCCCAGCAAACCGGATGCGCCT TCTTCGATGCAGGCACTGTGGCCCGCACGACACCGCTCGACGGTATTCACCTC GATGCTGAAAACACGCGCGCCATTGGTGCCGGCCTGGAGCCGGTGGTCCGCC AAGCGCTTGGATTGTGA (SEQ ID NO : 113) Q8XQI0 Ralstonia solanacearum (so) MQQILLYSDSLSWGIIPGTRRRLPFAARWAGVMEHALQAQGHAVRIVEDCLNGR TTVLDDPARPGRNGLQGLAQRIEAHAPLALVILMLGTNDFQAIFRHTAQDAAQG VAQLVRAIRQAPIEPGMPVPPVLIWPPAITAPAGAMADKFADAQPKCAGLAQAY RATAQTLGCHVFDANSVTPASRVDGIHLDADQHAQLGRAMAQWGTLLAQ (SEQ ID NO : 114) ATGCAACAGATCCTGCTCTATTCCGACTCGCTCTCCTGGGGCATCATCCCCGG CACCCGCCGGCGCCTGCCGTTCGCCGCCCGCTGGGCCGGGGTCATGGAACAC GCGCTGCAGGCGCAAGGGCACGCCGTGCGCATCGTCGAAGACTGCCTCAATG GACGCACCACGGTGCTCGACGATCCCGCGCGGCCGGGGCGCAACGGACTGCA GGGGCTCGCGCAGCGGATCGAAGCGCACGCCCCGCTTGCCCTGGTCATCCTG ATGCTCGGCACCAACGACTTCCAGGCGATCTTCCGGCACACCGCCCAGGACG CGGCGCAAGGCGTGGCGCAGCTGGTGCGGGCCATCCGCCAGGCGCCGATCGA ACCCGGCATGCCGGTGCCGCCCGTGCTGATCGTGGTGCCGCCGGCCATCACC GCGCCGGCCGGGGCGATGGCCGACAAGTTTGCCGACGCGCAGCCCAAGTGCG CCGGCCTTGCGCAGGCCTATCGGGCAACGGCGCAAACGCTAGGCTGCCACGT CTTCGATGCGAACAGCGTCACGCCGGCCAGCCGCGTGGACGGCATCCACCTC GATGCCGACCAGCATGCGCAGCTGGGCCGGGCGATGGCGCAGGTCGTCGGG ACGCTGCTTGCGCAATAA (SEQ ID NO : 115) ZP 00216984 Burkholderia cepacia (Bce) ATGACGATGACGCAGAAAACCGTGCTCTGCTACGGCGATTCGAACACGCATG GCACACGCCCGATGACGCATGCTGGCGGACTGGGGCGGTTTGCACGCGAAGA ACGCTGGACCGGCGTGCTGGCGCAAACGCTCGGTGCGAGCTGGCGGGTCATT GAAGAAGGGTTGCCCGCGCGTACGACCGTGCATGACGATCCGATCGAAGGCC

GGCACAAGAATGGTTTGTCGTATCTGCGCGCGTGCGTCGAAAGCCACTTGCC CGTCGATGTCGTCGTGCTGATGCTCGGGACCAACGATCTGAAGACACGCTTCT CGGTCACGCCCGCCGACATCGCGACATCGGTCGGCGTATTGCTTGCCAAGAT CGCTGCGTGCGGCGCCGGTCCGTCCGGTGCGTCACCGAAGCTCGTGCTGATG GCGCCTGCGCCGATCGTCGAGGTCGGATTCCTCGGCGAGATCTTTGCGGGCG GCGCAGCGAAGTCGCGGCAGCTCGCGAAGCGGTACGAACAGGTGGCAAGCG ATGCCGGTGCGCACTTTCTCGATGCCGGCGCGATCGTCGAGGTGAGCCCGGT GGATGGCGTTCACTTCGCGGCCGATCAGCATCGTGTGCTCGGGCAGCGGGTC GCTGCCCTTCTGCAGCAGATTGCGTAA (SEQ ID NO : 116) MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFAREERWTGVLAQTLGASWRVI EEGLPARTTVHDDPIEGRHKNGLSYLRACVESHLPVDVWLMLGTNDLKTRFSV TPADIATSVGVLLAKIAACGAGPSGASPKLVLMAPAPIVEVGFLGEIFAGGAAKSR QLAKRYEQVASDAGAHFLDAGAIVEVSPVDGVHFAADQHRVLGQRVAALLQQI A (SEQ ID NO : 117) NP 865746 Pirellula sp (Psp) MHSILIYGDSLSWGIIPGTRRRFAFHQRWPGVMEIELRQTGIDARVIEDCLNGRRT VLEDPIKPGRNGLDGLQQRIEINSPLSLWLFLGTNDFQSVHEFHAEQSAQGLALL VDAIRRSPFEPGMPTPKILLVAPPTVHHPKLDMAAKFQNAETKSTGLADAIRKVS TEHSCEFFDAATVTTTSVVDGVHLDQEQHQALGTALASTIAEILADC (SEQ ID NO : 118) ATGCATTCAATCCTCATCTATGGCGATTCTCTCAGTTGGGGAATCATTCCCGG CACGCGTCGTCGCTTCGCGTTCCATCAGCGTTGGCCGGGCGTCATGGAGATTG AACTGCGACAAACTGGAATCGATGCCCGCGTCATCGAAGACTGCCTCAATGG CCGACGAACCGTCTTGGAAGATCCAATCAAACCCGGACGCAATGGCCTGGAT GGTTTGCAGCAACGGATCGAAATCAATTCACCTCTGTCACTGGTCGTGCTCTT TCTGGGGACCAACGATTTCCAGTCCGTCCACGAATTCCATGCCGAGCAATCG GCACAAGGACTCGCACTGCTTGTCGACGCCATTCGTCGCTCCCCTTTCGAACC AGGAATGCCGACACCGAAAATCCTGCTTGTCGCACCACCGACGGTTCACCAC CCGAAACTTGATATGGCGGCGAAGTTCCAAAACGCGGAAACGAAATCGACG GGACTCGCAGATGCGATTCGCAAGGTCTCAACAGAACACTCCTGCGAATTCT TCGATGCGGCCACGGTCACCACAACAAGTGTCGTCGACGGAGTCCATCTCGA TCAAGAACAACATCAAGCACTCGGTACCGCACTGGCATCGACAATCGCTGAA ATACTAGCAGACTGTTGA (SEQ ID NO : 119)

As indicated above, the above sequences are the protein sequences and the coding sequences of"GDSL-type"esterases with a"GRTT"-motif or similar motifs from different bacterial isolates. The DNA sequences represent the target-DNA from which specific primers were deduced. All amplicons were ligated as NdelXhoI-fragments to pET26 thereby eliminating thepelB-leader sequence of this vector. All of the"GDSL- type"esterases from these isolates were expressed in E. coli Rosetta (DE3) at 28°C. The expression was induced by addition of 100 pM IPTG at an O. D. sso = 1 and the cells were harvested 20 h after induction. Only the cells expressing the enzymes from M. bovis and S. typhimurium were collected 4 h after induction, since previous experiments had shown that the highest activity could be obtained at this point of time. Table 2 summarizes the expression experiments.

Table 2: Expression and Characterization of"GDSL"-type Esterases From Bacterial Isolates for Perhydrolase Activity Strain Enzyme Expression Solubility3 Activity Perhydrolase GRTT Level2 4 Activity-Motif S. meliloti Sme I +++ ++ 5770,0 yes ARTT S. meliloti Sme II +++ +++ 85, 0 yes GRTT S. meliloti Sme in +++ ++ 746, 5 n. d. GRTT A. tumefaciens Atu III n. d5. n. d. n. d. n. d. GRTT M. loti Mlo I +++ ++ 1187,3 yes GRTT M. bovis1 Mbo + n. d. 25,2 yes ARTT C. violaceum Cvi + + 2422,7 n. d. GETS Vvulnificus Vvu n. d. n. d. n. d. n. d. GDTT R. eutropha Reu n. d. n. d. n. d. n. d. GRRT S. typhimurium'Sty + n. d. 17,2 no SRTT 1 outer membrane localized autotransporter protein

2 expression level: + moderate overexpression; ++ strong overexpression; +++ very strong overexpression as judged from SDS-PAGE-analysis as judged by SDS-PAGE-analysis 6 towards p-nitrophenyl butyrate<BR> <BR> <BR> not determined With the exception of the enzyme from S. typhimurium, all other enzymes tested showed the desired perhydrolase activity, confirming the correlation between the presence of certain conserved amino acids an the capability to perform perhydrolase reactions.

Although the enzyme from S. typhimurium contains the GRTT-motif, it is different from the other enzymes by the location of this motif downstream from block V. In all other enzymes, this motif is located between block I and III, indicating that it might have a different function in the enzyme from S. typhimurium. Thus, the absence of perhydrolase activity in the enzyme from S. typhimurium also supports the identified structure/function-relationship of the perhydrolases provided by the present invention.

Screening of New'4GDSL-type"Esterases in Metagenome Libraries i) Library S279 The full-length sequence of the gene from clone M75bA2 was completed, as provided below.

In the sequence ofS279M75bA2 provided above (DNA, SEQ ID NO : 80 ; and amino acid sequence, SEQ ID NO : 81), the coding sequence running from position 104 through 1312 is shown on a grey background. Conserved structural motifs are shown underlined and in bold.

The derived amino acid sequence showed the highest homology to a hypothetical protein (Y17D7A. 2) from Caenorhabditis elegans (BlastP2; swisspir), although with a

very high E-value of 2.5 (i. e. , indicating a non-reliable hit). The fact that no esterase is among the homologous proteins identified by the BlastP2-analysis indicates that this enzyme is a rather unusual"GDSL-type"esterase. Furthermore, the enzyme is characterized by unusually long peptides between the N-terminus and the"GDSL"-motif and the"DXXH"-motif of block V (containing the active site aspartic acid and histidine) and the C-terminus. The very C-terminal sequence shows similarity to a membrane lipoprotein lipid attachment site. A corresponding signal sequence of lipoproteins was not identified. The gene encoding M75bA5 was amplified but no further efforts were taken for this enzyme since it did not have the conserved amino acids typical of the perhydrolase of the present invention. ii) Library S248 The clone carrying the sequence-tag SP7_3j5h which could have been part of a gene encoding a"GDSL"-type esterase was identified (M31bAl 1), and the sequence was elongated. This facilitated the determination that this sequence did not encode a"GDSL- type"esterase, because block V could not be identified. The generation of this amplicon can be explained by an"unspecific"hybridization of primer 5h with the first mismatches at nucleotides 10,14 and 15 from the 3'-terminus of the primer. The sequence showed the highest homology to a hypothetical protein (K03E5. 5) from Caenorhabditis elegans with an E-value of 1.6, indicating a non-reliable hit. The sequence-tag from clone S248 lbAl l is provided below.

In the above sequence-tag of the clone S248 M31bAl 1, the primers 3j and 5h are indicated. Hybridization between primer and template is indicated by arrows, mismatches by open circles. Putative conserved structural motifs are indicated in bold and underlined.

Several further sequence-tags were generated using different primer pairs of the primers 2 and 5 but none turned out to encode a"GDSL"-type esterases. The screening of this library was completed. iii) Library M091 The elongation of the amplicon SP3_lj5h, which was identified in the insert-DNA of clone M24dG12 proved that the corresponding sequence does not encode a"GDSL"- type esterase. Whereas the sequence encoding a putative block V (DGTHP ; SEQ ID NO : 124) was found, the corresponding sequence encoding block I was missing. The amplicon was generated due to an"unspecific"hybridization of primer lj with the first mismatches at positions 5,10, 11 and 12 from the 3'-terminus of the primer. The sequence-tag of clone M091 M24dG12 s shown below:

Sequence-tag of the clone M091_M24dG12. The primers lj and 5h are indicated in the above sequence-tag of the clone M091 M24dG12. Hybridization between primer and template is indicated by arrows, mismatches by open circles. Putative conserved structural motifs are depicted in bold and underlined.

A further sequence-tag (SPl2b5h) was generated using the primer pair 2b/5h. A BlastX-analysis of the sequence from this tag yielded the highest homology to an arylesterase from Agrobacterium tumefaciens, with 70% identity. The single clone carrying the corresponding gene was identified (M4aEl 11) and the full length sequence determined to be as shown below:

In the above sequence, the conserved structural motifs are shown in bold and underlined. The BlastP-analysis with the deduced full length amino acid sequence identified the same hit with a identity of 48%. The primary structure of this enzyme showed the"GRTT"-motif proving the usefulness of the primers directed towards block 2 for the identification of"GRTT"-esterases. The gene was amplified to introduce unique restriction enzyme recognition sites and the absence of second site mutations was confirmed by sequencing. The gene was ligated to pET26 and was expressed in E. coli Rosetta (DE3). The vector map is provided in Figure 5. Expression and control strains were cultivated in LB in the presence of kanamycin (25 pg/ml), chloramphenicol (12.5 g/ml), and 1% glucose. At an ODsso of 1, expression was induced by addition of 100 uM IPTG. Samples were taken at 2,4, and 20 hours after induction. Cells were separated from the culture supernatant by centrifugation and after resuspending in sample buffer, they wee incubated for 10 minutes at 90°C. An amount of cells representing an ODsso of 0.1 was applied to a 4-12% acryl amide gradient gel, which was stained with Coomassie Brilliant Blue R250.

Strong overexpression of the gene was detected already 2 h after induction with 100 FM IPTG, as determined by SDS-PAGE analysis of crude cell extracts from E. coli Rosetta (DE3) pET26M4aEl 1. The amount of protein representing M4aEl 1 (calculated size 23.2 kDa) increased further over time.

Esterase activity of crude cell extracts from strains expressing the"GDSL"-type esterase M4aEl 1 was determined. An amount of cells corresponding to an O. D. sso = 2 were resuspended in 200 u. l of5mM Tris/HCl pH 8.0, and lysed by ultrasonication.

Then, 20 ul of each sample were used to determine the esterase activity towards p- nitrophenyl butyrate in a total volume of 200 p1. The activity was corrected for the background activity of the control strain. The activity towards p-nitrophenylbutyrate reached about 125 nmol/ml x min 20 h after induction.

In addition, SDS-PAGE analysis of the soluble and insoluble fraction of crude cell extracts from E. coli Rosetta (DE3) pET26M4aEl 1 was conducted. Cells from a culture induced with 100 uM IPTG and harvested 4 h and 20h after induction were lysed by ultrasonication and separated into soluble and insoluble fraction by centrifugation.

Sample buffer was added and directly comparable amounts of soluble and insoluble fractions were applied to a 4-12% acryl amide gradient gel, which was stained with Coomassie Brilliant Blue R250. The results of this analysis of the solubility revealed that M4aEl 1 is partially (estimated 80%) soluble. The screening of the library M091 was completed.

Thus, in total nine different"GDSL"-type esterases were identified in 6 different large insert metagenomic libraries and the esterases/lipases BRAIN's library comprising more than 4.3 Gbp. Eight of these genes were heterologously expressed in E. coli. The resulting enzyme samples of seven clones were characterized for the desired perhydrolase activity. Two of the enzymes displayed this activity. Table 3 summarizes the screening, expression and characterization of the metagenomic"GDSL"-type esterases.

Table 3: Expression and Characterization of Metagenomic"GDSL"-Type Esterases GDSL-type Homologyl Expression2 Solubility3 Activity4 Perhydrolase Esterase Level Activity S248_M2bB11 12.9% ++ + 136 - S248_M40cD4 14. 8% +++ ++ 50 S248 M44aA5 12.4% +++ ++ 75-/+ S261M2aA12 36.9% ++ ++ 72 +7 S279_M70aE8 11. 9% +++ + 167 S279_M75bA2 5.7% n. d5. ii. d. n. d. n. d.5 M091_M4aE11 33. 9% ++ 125 n. d.

Est105 4.3% +++--n. d.

Est114 7. 8% n. d. n. d. 13 t identity to the prototype enzyme from M. smegmatis calculated with the dialign algorithm (Morgenstern et al., 1996) 2 expression level: + moderate overexpression ; ++ strong overexpression ; +++ very <BR> <BR> strong overexpression as judged from SDS-PAGE-analysis<BR> <BR> <BR> as judged by SDS-PAGE-analysis 4 towards p-nitrophenyl butyrate; given as nmol/ (ml x min) not determined 7perhydrolysis activity 2x background<BR> <BR> <BR> perhydrolase activity more than 2x background Engineering of the Perhydrolase Based on the structure of the perhydrolase, residues which may alter substrate specificity (e. g., Km, kcat, Vmax, chain length, etc. ) and or the multimeric nature of the protein were identified. However, it is not intended that the present invention be limited to any particular residues. Nonetheless, site saturation libraries of residues D 10, L12, T13, W14, W16, S54, A55, N94, K97, Y99, P146, W149, F150, I194, F196, are constructed, as well as combinatorial libraries of residues: E51A, Y73A, H81D, T127Q and single mutations of the active site residues D192A, H195A and a site saturation

library of the conserved D95. Methods for production of such libraries are known to those skilled in the art and include commercially available kits as the Stratagene Quikchange Site-directed mutagenesis kit and/or Quikchange Multi-Site-directed mutagenesis kit.

Perhydrolase Activity The use of enzymes obtained from microorganisms is long-standing. Indeed there are numerous biocatalysts known in the art. For example, U. S. Patent No. 5,240, 835 (herein incorporated by reference) provides a description of the transacylase activity of obtained from C. oxydans and its production. In addition, U. S. Patent No. 3,823, 070 (herein incorporated by reference) provides a description of a Corynebacterium that produces certain fatty acids from an n-paraffin. U. S. Patent No. 4,594, 324 (herein incorporated by reference) provides a description of a Methylcoccus capsulatus that oxidizes alkenes. Additional biocatalysts are known in the art (See e. g, U. S. Patent Nos.

4,008, 125 and 4,415, 657; both of which are herein incorporated by reference). EP 0 280 232 describes the use of a C. oxydans enzyme in a reaction between a diol and an ester of acetic acid to produce monoacetate. Additional references describe the use of a C. oxydans enzyme to make chiral hydroxycarboxylic acid from a prochiral diol. Additional details regarding the activity of the C. oxydans transacylase as well as the culture of C. oxydans, preparation and purification of the enzyme are provided by U. S. Patent No.

5,240, 835 (incorporated by reference, as indicated above). Thus, the transesterification capabilities of this enzyme, using mostly acetic acid esters were known. However, the determination that this enzyme could carry out perhydrolysis reaction was quite unexpected. It was even more surprising that these enzymes exhibit very high efficiencies in perhydrolysis reactions. For example, in the presence of tributyrin and water, the enzyme acts to produce butyric acid, while in the presence of tributyrin, water and hydrogen peroxide, the enzyme acts to produce mostly peracetic acid and very little

butyric acid. This high perhydrolysis to hydrolysis ratio is a unique property exhibited by the perhydrolase class of enzymes of the present invention and is a unique characteristic that is not exhibited by previously described lipases, cutinases, nor esterases.

The perhydrolase of the present invention is active over a wide pH and temperature range and accepts a wide range of substrates for acyl transfer. Acceptors include water (hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classical acyl transfer). For perhydrolysis measurements, enzyme is incubated in a buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide.

Typical substrates used to measure perhydrolysis include esters such as ethyl acetate, triacetin, tributyrin, ethoxylated neodol acetate esters, and others. In addition, the wild type enzyme hydrolyzes nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. Peracid and acetic acid can be measured by the assays described herein. Nitrophenylester hydrolysis is also described.

Although the primary example used during the development of the present invention is the M. smegmatis perhydrolase, any perhydrolase obtained from any source which converts the ester into mostly peracids in the presence of hydrogen peroxide finds use in the present invention. hrtrstes In some preferred embodiments of the present invention, esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are utilized with the perhydrolase enzymes of the present invention. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. In additional embodiments, triacetin, tributyrin, neodol esters, and/or ethoxylated neodol esters serve as acyl donors for peracid formation.

Cleaning and Detergent Formulations The detergent compositions of the present invention are provided in any suitable form, including for example, as a liquid diluent, in granules, in emulsions, in gels, and pastes. When a solid detergent composition is employed, the detergent is preferably formulated as granules. Preferably, the granules are formulated to additionally contain a protecting agent (See e. g., U. S. Appln. Ser. No. 07/642,669 filed January 17,1991, incorporated herein by reference). Likewise, in some embodiments, the granules are formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium (See e. g., U. S. Patent No. 5,254, 283, incorporated herein by reference in its entirety). In addition, the perhydrolase enzymes of the present invention find use in formulations in which substrate and enzyme are present in the same granule. Thus, in some embodiments, the efficacy of the enzyme is increased by the provision of high local concentrations of enzyme and substrate (See e. g. , U. S. Patent Application Publication US2003/0191033, herein incorporated by reference).

Many of the protein variants of the present invention are useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the protein mutants of the invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents (See e. g., U. S. Patent Nos 4,404, 128 and 4,261, 868). A suitable detergent formulation is that described in U. S.

Patent No. 5,204, 015 (previously incorporated by reference). Those in the art are familiar with the different formulations which find use as cleaning compositions. As indicated above, in some preferred embodiments, the detergent compositions of the present invention employ a surface active agent (i. e. , surfactant) including anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions. Some surfactants suitable for use in the present invention are described in British Patent Application No. 2 094 826 A, incorporated herein by reference. In some embodiments,

mixtures surfactants are used in the present invention.

Suitable anionic surfactants for use in the detergent composition of the present invention include linear or branched alkylbenzene sulfonates ; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates ; alkane sulfonates and the like. Suitable counter ions for anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants that find use in the present invention include quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule.

Nonionic surfactants that find use in the present invention generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylen oxide adduct thereof, fatty acid glycerine monoesters, and the like.

In some preferred embodiments, the surfactant or surfactant mixture included in the detergent compositions of the present invention is provided in an amount from about 1 weight percent to about 95 weight percent of the total detergent composition and preferably from about 5 weight percent to about 45 weight percent of the total detergent composition. In various embodiments, numerous other components are included in the compositions of the present invention. Many of these are described below. It is not intended that the present invention be limited to these specific examples. Indeed, it is contemplated that additional compounds will find use in the present invention. The descriptions below merely illustrate some optional components.

Proteins, particularly the perhydrolase of the present invention can be formulated into known powdered and liquid detergents having pH between 3 and 12.0, at levels of about. 001 to about 5% (preferably 0.1% to 0.5%) by weight. In some embodiments,

these detergent cleaning compositions further include other enzymes such as proteases, amylases, mannanases, peroxidases, oxido reductases, cellulases, lipases, cutinases, pectinases, pectin lyases, xylanases, and/or endoglycosidases, as well as builders and stabilizers.

In addition to typical cleaning compositions, it is readily understood that perhydrolase variants of the present invention find use in any purpose that the native or wild-type enzyme is used. Thus, such variants can be used, for example, in bar and liquid soap applications, dishcare formulations, surface cleaning applications, contact lens cleaning solutions or products, , waste treatment, textile applications, pulp-bleaching, disinfectants, skin care, oral care, hair care, etc. Indeed, it is not intended that any variants of the perhydrolase of the present invention be limited to any particular use. For example, the variant perhydrolases of the present invention may comprise, in addition to decreased allergenicity, enhanced performance in a detergent composition (as compared to the wild-type or unmodified perhydrolase).

The addition of proteins to conventional cleaning compositions does not create any special use limitations. In other words, any temperature and pH suitable for the detergent are also suitable for the present compositions, as long as the pH is within the range in which the enzyme (s) is/are active, and the temperature is below the described protein's denaturing temperature. In addition, proteins of the invention find use in cleaning, bleaching, and disinfecting compositions without detergents, again either alone or in combination with a source of hydrogen peroxide, an ester substrate (e. g., either added or inherent in the system utilized, such as with stains that contain esters, pulp that contains esters etc), other enzymes, surfactants, builders, stabilizers, etc. Indeed it is not intended that the present invention be limited to any particular formulation or application.

Substrates

In some preferred embodiments of the present invention, esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are utilized with the perhydrolase enzymes in the detergent formulations of the present invention. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, in some preferred embodiments, detergents comprising at least one perhydrolase, at least one hydrogen peroxide source, and at least one ester acid are provided.

Hydrolases In addition to the perhydrolase described herein, various hydrolases find use in the present invention, including but not limited to carboxylate ester hydrolase, thioester hydrolase, phosphate monoester hydrolase, and phosphate diester hydrolase which act on ester bonds; a thioether hydrolase which acts on ether bonds; and a-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase, acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptide hydrolase which act on peptide bonds, all these enzymes having high perhydrolysis to hydrolysis ratios (e. g. , >1). Preferable among them are carboxylate ester hydrolase, and peptidyl-peptide hydrolase. Suitable hydrolases include: (1) proteases belonging to the peptidyl-peptide hydrolase class (e. g. , pepsin, pepsin B, rennin, trypsin, chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein, gastrisin, cathepsin D, bromelin, keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase, carboxypeptidase A and B, and aminopeptidase); (2) carboxylate ester hydrolase including carboxyl esterase, lipase, pectin esterase, and chlorophyllase; and (3) enzymes having high perhydrolysis to hydrolysis ratios. Especially effective among them are lipases, as well as esterases that

exhibit high perhydrolysis to hydrolysis ratios, as well as protein engineered esterases, cutinases, and lipases, using the primary, secondary, tertiary, and/or quaternary structural features of the perhydrolases of the present invention.

The hydrolase is incorporated into the detergent composition as much as required according to the purpose. It should preferably be incorporated in an amount of 0. 0001 to 5 weight percent, and more preferably 0.02 to 3 weight percent, . This enzyme should be used in the form of granules made of crude enzyme alone or in combination with other enzymes and/or components in the detergent composition. Granules of crude enzyme are used in such an amount that the purified enzyme is 0.001 to 50 weight percent in the granules. The granules are used in an amount of 0.002 to 20 and preferably 0.1 to 10 weight percent. In some embodiments, the granules are formulated so as to contain an enzyme protecting agent and a dissolution retardant material (i. e., material that regulates the dissolution of granules during use).

Cationic Surfactants and Long-Cham Fatty Acid Salts Such cationic surfactants and long-chain fatty acid salts include saturated or fatty acid salts, alkyl or alkenyl ether carboxylic acid salts, a-sulfofatty acid salts or esters, amino acid-type surfactants, phosphate ester surfactants, quaternary ammonium salts including those having 3 to 4 alkyl substituents and up to 1 phenyl substituted alkyl substituents. Suitable cationic surfactants and long-chain fatty acid salts include those disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. The composition may contain from about 1 to about 20 weight percent of such cationic surfactants and long-chain fatty acid salts.

Biiilders In some embodiments of the present invention, the composition contains from about 0 to about 50 weight percent of one or more builder components selected from the

group consisting of alkali metal salts and alkanolamine salts of the following compounds: phosphates, phosphonates, phosphonocarboxylates, salts of amino acids, aminopolyacetates high molecular electrolytes, non-dissociating polymers, salts of dicarboxylic acids, and aluminosilicate salts. Examples of suitable divalent sequestering agents are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

In additional embodiments, compositions of the present invention contain from about 1 to about 50 weight percent, preferably from about 5 to about 30 weight percent, based on the composition of one or more alkali metal salts of the following compounds as the alkalis or inorganic electrolytes: silicates, carbonates and sulfates as well as organic alkalis such as triethanolamine, diethanolamine, monoethanolamine and triisopropanolamine.

Anti-Re (1eynRition Agents In yet additional embodiments of the present invention, the compositions contain from about 0.1 to about 5 weight percent of one or more of the following compounds as antiredeposition agents: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose. In some preferred embodiments, a combination of carboxymethyl-cellulose and/or polyethylene glycol are utilized with the composition of the present invention as useful dirt removing compositions.

Ripiaching Agents The use of the perhydrolases of the present invention in combination with additional bleaching agent (s) such as sodium percarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxide adduct and/or a photo-sensitive bleaching dye such as zinc or aluminum salt of sulfonate phthalocyanine further improves the detergent effects. In additional embodiments, the perhydrolases of

the present invention are used in combination with bleach boosters (e. g., TAED and/or NOBS).

Bluing Agents and Fluorescent Dyes In some embodiments of the present invention, bluing agents and fluorescent dyes are incorporated in the composition. Examples of suitable bluing agents and fluorescent dyes are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

Casing Inhibitors In some embodiments of the present invention in which the composition is powdered or solid, caking inhibitors are incorporated in the composition. Examples of suitable caking inhibitors include p-toluenesulfonic acid salts, xylenesulfonic acid salts, acetic acid salts, sulfosuccinic acid salts, talc, finely pulverized silica, clay, calcium silicate (e. g., Micro-Cell by Johns Manville Co. ), calcium carbonate and magnesium oxide.

A nti mounts The antioxidants include, for example, tert-butyl-hydroxytoluene, 4,4'- butylidenebis (6-tert-butyl-3-methylphenol), 2,2'-butylidenebis (6-tert-butyl-4- methylphenol), monostyrenated cresol, distyrenated cresol, monostyrenated phenol, distyrenated phenol and 1, 1-bis (4-hydroxy-phenyl) cyclohexane.

Sollzhr ers In some embodiments, the compositions of the present invention also include solubilizers, including but not limited to lower alcohols (e. g. , ethanol, benzenesulfonate salts, and lower alkylbenzenesulfonate salts such as p-toluenesulfonate salts), glycols

such as propylene glycol, acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acid amides, benzoate salts and urea.

In some embodiments, the detergent composition of the present invention are used in a broad pH range of from acidic to alkaline pH. In a preferred embodiment, the detergent composition of the present invention is used in mildly acidic, neutral or alkaline detergent wash media having a pH of from above 4 to no more than about 12.

In addition to the ingredients described above, perfumes, buffers, preservatives, dyes and the like also find use with the present invention. These components are provided in concentrations and forms known to those in the art.

In some embodiments, the powdered detergent bases of the present invention are prepared by any known preparation methods including a spray-drying method and a granulation method. The detergent base obtained particularly by the spray-drying method and/or spray-drying granulation method are preferred. The detergent base obtained by the spray-drying method is not restricted with respect to preparation conditions. The detergent base obtained by the spray-drying method is hollow granules which are obtained by spraying an aqueous slurry of heat-resistant ingredients, such as surface active agents and builders, into a hot space. After the spray-drying, perfumes, enzymes, bleaching agents, inorganic alkaline builders may be added. With a highly dense, granular detergent base obtained such as by the spray-drying-granulation method, various ingredients may also be added after the preparation of the base.

When the detergent base is a liquid, it may be either a homogeneous solution or an inhomogeneous dispersion.

The detergent compositions of this invention may be incubated with fabric, for example soiled fabrics, in industrial and household uses at temperatures, reaction times and liquor ratios conventionally employed in these environments. The incubation conditions (i. e. , the conditions effective for treating materials with detergent compositions according to the present invention), are readily ascertainable by those of

skill in the art. Accordingly, the appropriate conditions effective for treatment with the present detergents correspond to those using similar detergent compositions which include wild-type perhydrolase.

As indicated above, detergents according to the present invention may additionally be formulated as a pre-wash in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements softening, depilling, pilling prevention, surface fiber removal or cleaning. When the detergent composition is a pre-soak (e. g., pre-wash or pre-treatment) composition, either as a liquid, spray, gel or paste composition, the perhydrolase enzyme is generally employed from about 0. 00001% to about 5% weight percent based on the total weight of the pre-soak or pre-treatment composition. In such compositions, a surfactant may optionally be employed and when employed, is generally present at a concentration of from about 0.0005 to about 1 weight percent based on the total weight of the pre-soak. The remainder of the composition comprises conventional components used in the pre-soak (e. g. , diluent, buffers, other enzymes (proteases), etc. ) at their conventional concentrations.

Cleaning Compositions Comprising Perhydrolase The cleaning compositions of the present invention may be advantageously employed for example, in laundry applications, hard surface cleaning, automatic dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin. However, due to the unique advantages of increased effectiveness in lower temperature solutions and the superior color-safety profile, the enzymes of the present invention are ideally suited for laundry applications such as the bleaching of fabrics.

Furthermore, the enzymes of the present invention find use in both granular and liquid compositions.

The enzymes of the present invention also find use in cleaning additive products.

Cleaning additive products including the enzymes of the present invention are ideally

suited for inclusion in wash processes where additional bleaching effectiveness is desired.

Such instances include, but are not limited to low temperature solution cleaning applications. The additive product may be, in its simplest form, one or more of the enzymes of the present invention. Such additive may be packaged in dosage form for addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired. Such single dosage form may comprise a pill, tablet, gelcap or other single dosage unit such as pre-measured powders or liquids. A filler or carrier material may be included to increase the volume of such composition. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Filler or carrier materials for liquid compositions may be water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to; methanol, ethanol, propanol and isopropanol. The compositions may contain from about 5% to about 90% of such materials. Acidic fillers can be used to reduce pH.

Alternatively, the cleaning additive may include activated peroxygen source defined below or the adjunct ingredients as defined below.

The cleaning compositions and cleaning additives of the present invention require an effective amount of the enzymes provided by the present invention. The required level of enzyme may be achieved by the addition of one or more species of the M. smegmatis perhydrolase, variants, homologues, and/or other enzymes or enzyme fragments having the activity of the enzymes of the present invention. Typically, the cleaning compositions of the present invention comprise at least 0.0001 weight percent, from about 0.0001 to about 1, from about 0. 001 to about 0.5, or even from about 0. 01 to about 0.1 weight percent of at least one enzyme of the present invention.

In some embodiments, the cleaning compositions of the present invention comprise a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, said peroxygen source being selected from the group

consisting of : (i) from about 0.01 to about 50, from about 0.1 to about 20, or even from about 1 to 10 weight percent of a per-salt, an organic peroxyacid, urea hydrogen peroxide and mixtures thereof; (ii) from about 0. 01 to about 50, from about 0.1 to about 20, or even from about 1 to 10 weight percent of a carbohydrate and from about 0.0001 to about 1, from about 0.001 to about 0.5, from about 0.01 to about 0.1 weight percent carbohydrate oxidase; and (iii) mixtures thereof.

Suitable per-salts include those selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof.

The carbohydrate may be selected from the group consisting of mono- carbohydrates, di-carbohydrates, tri-carbohydrates, oligo-carbohydrates and mixtures thereof. Suitable carbohydrates include carbohydrates selected from the group consisting of D-arabinose, L-arabinose, D-Cellobiose, 2-Deoxy-D-galactose, 2-Deoxy-D-ribose, D- Fructose, L-Fucose, D-Galactose, D-glucose, D-glycero-D-gulo-heptose, D-lactose, D- Lyxose, L-Lyxose, D-Maltose, D-Mannose, Melezitose, L-Melibiose, Palatinose, D- Raffinose, L-Rhamnose, D-Ribose, L-Sorbose, Stachyose, Sucrose, D-Trehalose, D- Xylose, L-Xylose and mixtures thereof.

Suitable carbohydrate oxidases include carbohydrate oxidases selected from the group consisting of aldose oxidase (IUPAC classification EC1. 1.3. 9), galactose oxidase (IUPAC classification EC1. 1.3. 9), cellobiose oxidase (IUPAC classification EC1. 1.3. 25), pyranose oxidase (IUPAC classification EC1. 1.3. 10), sorbose oxidase (IUPAC classification EC1. 1.3. 11) and/or hexose oxidase (IUPAC classification EC1. 1.3. 5), Glucose oxidase (IUPAC classification EC1. 1.3. 4) and mixtures thereof.

In some preferred embodiments, the cleaning compositions of the present

invention also include from about 0.01 to about 99.9, from about 0.01 to about 50, from about 0.1 to 20, or even from about 1 to about 15 weight percent a molecule comprising an ester moiety. Suitable molecules comprising an ester moiety may have the formula: R'O. [(R2)m (R3)n]p wherein Rl is a moiety selected from the group consisting of H or a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of the present invention, Rl may comprise from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon atoms, or even from 2 to 100 carbon atoms; each R is an alkoxylate moiety, in one aspect of the present invention, each R2 is independently an ethoxylate, propoxylate or butoxylate moiety; R3 is an ester-forming moiety having the formula : RCO-wherein R4 may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, in one aspect of the present invention, R4 may be substituted or unsubstituted alkyl, alkenyl, alkynyl, moiety comprising from 1 to 22 carbon atoms, an aryl, alkylaryl, alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22 carbon atoms or R4 may be a substituted or unsubstituted Cl-C22 alkyl moiety or R4 may be a substituted or unsubstituted Cl-C12 alkyl moiety; x is 1 when Rl is H; when Rl is not H, x is an integer that is equal to or less than the number of carbons in Rl p is an integer that is equal to or less than x m is an integer from 0 to 50, an integer from 0 to 18, or an integer from 0 to 12, and n is at least 1.

In one aspect of the present invention, the molecule comprising an ester moiety is an alkyl ethoxylate or propoxylate having the formula R'Ox [ (R) m (R3) n] p wherein:

Rl is an C2-C32 substituted or unsubstituted alkyl or heteroalkyl moiety; each R2 is independently an ethoxylate or propoxylate moiety; R3 is an ester-forming moiety having the formula: R4CO-wherein R4 may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, in one aspect of the present invention, R4 may be a substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety comprising from 1 to 22 carbon atoms, a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22 carbon atoms or R4 may be a substituted or unsubstituted Cl-C22 alkyl moiety or R4 may be a substituted or unsubstituted Cl-Cl2 alkyl moiety; x is an integer that is equal to or less than the number of carbons in'RI p is an integer that is equal to or less than x m is an integer from 1 to 12, and n is at least 1.

In one aspect of the present invention, the molecule comprising the ester moiety has the formula: R [ (R) m (R) n] p wherein Rl is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, said Ri moiety that comprises an amine moiety being selected from the group consisting of a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of Applicants' invention Rl may comprise from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon atoms, or even from 2 to 100 carbon atoms; each R2 is an alkoxylate moiety, in one aspect of the present invention each R2 is independently an ethoxylate, propoxylate or butoxylate moiety;

R3 is an ester-forming moiety having the formula: RCO-wherein R4 may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, in one aspect of the present invention, R4 may be a substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety comprising from 1 to 22 carbon atoms, a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22 carbon atoms or R4 may be a substituted or unsubstituted Cl-C22 alkyl moiety or R4 may be a substituted or unsubstituted Ci-Ci2 alkyl moiety; x is 1 when Rl is H; when Rl is not H, x is an integer that is equal to or less than the number of carbons in Rl p is an integer that is equal to or less than x m is an integer from 0 to 12 or even 1 to 12, and n is at least 1.

In any of the aforementioned aspects of the present invention, the molecule comprising an ester moiety may have a weight average molecular weight of less than 600,000 Daltons, less than 300,000 Daltons, less than 100,000 Daltons or even less than 60,000 Daltons.

Suitable molecules that comprise an ester moiety include polycarbohydrates that comprise an ester moiety.

The cleaning compositions provided herein will typically be formulated such that, during use in aqueous cleaning operations, the wash water will have a pH of from about 5.0 to about 11.5, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 and about 9.0. Granular laundry products are typically formulated to have a pH from about 9 to about 11. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids,

etc. , and are well known to those skilled in the art.

When the enzyme (s) of the present invention is/are employed in a granular composition or liquid, it may be desirable for the enzyme (s) to be in the form of an encapsulated particle to protect such enzyme from other components of the granular composition during storage. In addition, encapsulation is also a means of controlling the availability of the enzyme (s) during the cleaning process and may enhance performance of the enzyme (s). In this regard, the enzyme (s) may be encapsulated with any encapsulating material known in the art.

The encapsulating material typically encapsulates at least part of the enzyme (s).

Typically, the encapsulating material is water-soluble and/or water-dispersible. The encapsulating material may have a glass transition temperature (Tg) of 0°C or higher.

Glass transition temperature is described in more detail in WO 97/11151, especially from page 6, line 25 to page 7, line 2.

The encapsulating material may be selected from the group consisting of carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and combinations thereof. When the encapsulating material is a carbohydrate, it may be typically selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the encapsulating material is a starch. Suitable starches are described in EP 0 922 499; US 4,977, 252; US 5,354, 559 and US 5,935, 826.

The encapsulating material may be a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that can be used are those supplied by Expancel of Stockviksverken, Sweden under the trademark EXPANCEL@, and those supplied by PQ Corp. of Valley Forge, Pennsylvania U. S. A. under the

tradename PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES), LUXSILM), Q-CELt) and SPHERICELt@.

Processes of Making and Using the Cleaning Compositions of the Present Tnventinn The cleaning compositions of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U. S. 5,879, 584; U. S. 5,691, 297; U. S. 5,574, 005; U. S.

5,569, 645; U. S. 5,565, 422 Del Greco et al.; U. S. 5,516, 448; U. S. 5,489, 392; and U. S.

5,486, 303; all of which are incorporated herein by reference.

Adjunct Materials in Addition to the fnxymesnfthe Present Tnventtnn TTydrngen Peroxide, and/or Hydrogen Peroxide Source and Material Comprising an Ester Moiety While not essential for the purposes of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions and may be desirably incorporated in certain embodiments of the invention, for example to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dyes or the like. It is understood that such adjuncts are in addition to the enzymes of the present invention, hydrogen peroxide and/or hydrogen peroxide source and material comprising an ester moiety. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed

peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U. S. Patent Nos. 5,576, 282, 6,306, 812, and 6,326, 348, herein incorporated by reference.

The aforementioned adjunct ingredients may constitute the balance of the cleaning compositions of the present invention.

Snrfftctants-The cleaning compositions according to the present invention may comprise a surfactant or surfactant system wherein the surfactant can be selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof.

The surfactant is typically present at a level of from about 0. 1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject cleaning composition.

Rmlfjfrs-The cleaning compositions of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the subject cleaning composition will typically comprise at least about 1%, from about 3% to about 60% or even from about 5% to about 40% builder by weight of the subject cleaning composition.

Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders polycarboxylate compounds. ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3, 5-trihydroxy benzene-2, 4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid,

polymaleic acid, benzene 1, 3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.

Chelating Agents - The cleaning compositions herein may contain a chelating agent, Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof When a chelating agent is used, the cleaning composition may comprise from about 0. 1% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition. nepncition Aid-The cleaning compositions herein may contain a deposition aid.

Suitable deposition aids include, polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as Kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof.

Dye Transfer Inhibiting Admits-The cleaning compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N- vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.

When present in a subject cleaning composition, the dye transfer inhibiting agents maybe present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the cleaning composition.

Dispersants - The cleaning compositions of the present invention can also contain dispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

Enzymes - The cleaning compositions can comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits. Examples of

suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, B-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. A typical combination is cocktail of conventional applicable enzymes like protease, lipase, cutinase and/or cellulase in conjunction with amylase.

Enzyme Stabilizers - Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed herein can be stabilized by the presence of water- soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes.

Catalytic Metal Complexes - The cleaning compositions of the present invention may include catalytic metal complexes. One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof. Such catalysts are disclosed in U. S. 4,430, 243.

If desired, the compositions herein can be catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in U. S. 5,576, 282.

Cobalt bleach catalysts useful herein are known, and are described, for example, in U. S. 5,597, 936; and U. S. 5,595, 967. Such cobalt catalysts are readily prepared by known procedures, such as taught for example in U. S. 5,597, 936, and U. S. 5,595, 967.

Compositions herein may also suitably include a transition metal complex of a

macropolycyclic rigid ligand-abreviated as"MRL". As a practical matter, and not by way of limitation, the compositions and cleaning processes herein can be adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and will preferably provide from about 0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor.

Preferred transition-metals in the instant transition-metal bleach catalyst include manganese, iron and chromium. Preferred MRL's herein are a special type of ultra-rigid ligand that is cross-bridged such as 5, 12-diethyl-1, 5, 8, 12-tetraazabicyclo [6.6. 2] hexadecane.

Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00/332601, and U. S. 6,225, 464. menthe f T TRe The cleaning compositions disclosed herein of can be used to clean a situs inter alia a surface or fabric. Typically at least a portion of the situs is contacted with an embodiment of Applicants'cleaning composition, in neat form or diluted in a wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present invention, washing includes but is not limited to, scrubbing, and mechanical agitation.

The fabric may comprise most any fabric capable of being laundered in normal consumer use conditions. The disclosed cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5 °C to about 90 °C and, when the situs comprises a fabric, the water to fabric mass ratio is typically from about 1: 1 to about 30 : 1.

EXPERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be

construed as limiting the scope thereof.

In the experimental disclosure which follows, the following abbreviations apply: °C (degrees Centigrade); rpm (revolutions per minute); Ha0 (water); HC1 (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); gg and ug (micrograms); mg (milligrams); ng (nanograms); lil and ul (microliters); ml (milliliters); mm (millimeters); nm (nanometers); um and um (micrometer); M (molar); mM (millimolar); uM and uM (micromolar); U (units); V (volts); MW (molecular weight); sec (seconds); min (s) (minute/minutes); hr (s) (hour/hours) ; MgCl2 (magnesium chloride); NaCI (sodium chloride) ; OD2so (optical density at 280 nm); OD600 (optical density at 600 nm) ; PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]) ; SDS (sodium dodecyl sulfate); Tris (tris (hydroxymethyl) aminomethane); TAED (N, N, N'N'-tetraacetylethylenediamine) ; w/v (weight to volume) ; v/v (volume to volume); Per (perhydrolase); per (perhydrolase gene); Ms (M. smegmatis) ; MS (mass spectroscopy); BRAIN (BRAIN Biotechnology Research and Information Network, AG, Zwingenberg, Germany); TIGR (The Institute for Genomic Research, Rockville, MD); AATCC (American Association of Textile and Coloring Chemists) ; WFK (wfk Testgewebe GmbH, Bruggen-Bracht, Germany); Amersham (Amersham Life Science, Inc. Arlington Heights, IL); ICN (ICN Pharmaceuticals, Inc. , Costa Mesa, CA); Pierce (Pierce Biotechnology, Rockford, IL); Amicon (Amicon, Inc. , Beverly, MA); ATCC (American Type Culture Collection, Manassas, VA); Amersham (Amersham Biosciences, Inc., Piscataway, NJ); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, NJ); BioRad (BioRad, Richmond, CA); Clontech (CLONTECH Laboratories, Palo Alto, CA); Difco (Difco Laboratories, Detroit, MI); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, MD); Novagen (Novagen, Inc., Madison, WI); Qiagen (Qiagen, Inc., Valencia, CA); Invitrogen (Invitrogen Corp. , Carlsbad, CA); Genaissance (Genaissance Pharmaceuticals, Inc., New Haven, CT); DNA 2.0 (DNA 2.0, Menlo Park, CA); MIDI

(MIDI Labs, Newark, DE) InvivoGen (InvivoGen, San Diego, CA); Sigma (Sigma Chemical Co. , St. Louis, MO); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co. , Biotechnology Systems, Wilmington, DE); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Roche (Hoffmann La Roche, Inc. , Nutley, NJ); Agilent (Agilent Technologies, Palo Alto, CA); Minolta (Konica Minolta, Ramsey, NJ); and Zeiss (Carl Zeiss, Inc., Thornwood, NY).

In the following Examples, various media were used."TS"medium (per liter) was prepared using Tryptone (16 g) (Difco), Soytone (4 g) (Difco), Casein hydrolysate (20 g) (Sigma), K2HP04 (10 g), and d H20 (to 1 L). The medium was sterilized by autoclaving. Then, sterile glucose was added to 1.5% final concentration. Streptomyces Production Medium (per liter) was prepared using citric acid (H20) (2.4 g), Biospringer yeast extract (6 g), (NH4) 2SO4 (2.4 g), MgSO4-7 H20 (2.4 g), Mazu DF204 (5 ml), trace elements (5 ml). The pH was adjusted to 6.9 with NaOH. The medium was then autoclaved to sterilize. After sterilization, CaCb'2 H20 (2 mis of 100 mg/ml solution), KH2PO4 (200 ml of a 13% (w/v) solution at pH6.9), and 20 mls of a 50% glucose solution were added to the medium.

In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions. A reflectometer was used to measure the reflectance of the swatches. Unless otherwise indicated, protein concentrations were estimated by Coomassie Plus (Pierce), using BSA as the standard.

EXAMPLE 1 Enzyme Analysis In this Example, methods to assess enzyme purity and activity used in the subsequent Examples and throughout the present Specification are described.

Enzyme Activity Assay (pNB Assay) This activity was measured by hydrolysis ofp-nitrophenylbutyrate. The reaction mixture was prepared by adding 10 ul of 100 mM p-nitrophenylbutyrate in dimethylsulfoxide to 990 ml of 100 mM Tris-HCl buffer, pH 8.0 containing 0.1 % triton X-100. The background rate of hydrolysis was measured before the addition of enzyme at 410 nm. The reaction was initiated by the addition of 10 ul of enzyme to 990 ml of the reaction and the change of absorbance at 410 nm was measured at room temperate (-230C). The background corrected results are reported as 8A41o/min/ml or 8 0/min/mg protein.

Transesterification Transesterification was measured by GC separation of products in buffered aqueous reactions. Reactions to measure ethyl acetate transesterification with propanol contained in 1 ml of 50 mM KP04, pH 7.0 ; 200 mM ethyl acetate, 200 mM 1-propanol, and enzyme. Reactions to measure ethyl acetate transesterification with neopentyl glycol (NPG) contained in 1 ml of 50 mM KP04, pH 7.0 ; 303 mM ethyl acetate, 100 mM NPG, and enzyme. The reactions were incubated at the indicated temperatures and for the indicated times. Separations were preformed using a 30M FFAP column (Phenomenex).

The inlet split ratio was approximately 1: 25, the injector was 250°C, head pressure of 10 psi He, and detection was by FID at 250°C. The chromatography program was 40°C initial for 4 min, followed by a gradient of 15°C/min to 180°C. Components eluted in the following order and were not quantified; ethyl acetate, ethyl alcohol, propyl acetate, propyl alcohol, acetic acid, NPG diacetate, NPG monoacetate, and NPG.

Perhydrolase Used in Crystallography Studies This perhydrolase preparation was used for crystallography studies. In addition,

unlabelled protein was grown and purified in similar manner. A 500 ml preculture of E. coli BL21 (DE3)/pLysS/pMSATNcol-1 was grown in a baffled 2.8 L Fernbach flask on LB containing 100 ug/ml carbenicillin. After overnight culture at 37°C and 200 rpm on a rotary shaker, the cells were harvested by centrifugation and resuspended in M9 medium containing: glucose, 2 g/L; Na2HP04, 6 g/L ; KH2PO4, 3 g/L; NH4Cl, 1 g/L ; NaCl, 0.5 g/L; thiamine, 5 mg/L ; MgSO4, 2 mM; CaCl2, 100 uM; Citric acid. H20, 40 mg/L; MnSO4. H20, 30 mg/L ; NaCl, 10 mg/L; FeSO4g7H20, 1 mg/L; CoCl2#6H2O, 1 mg/L ; ZnS04. 7H20, 1 mg/L; CuS04-5H20, 100 ug/L; H3BO3g5H20, 100 ug/L ; and NaMoO4. 2H20, 100 ug/L; and supplemented with carbenicillin, 100 mg/L. The resuspended cells were used to inoculate six Fernbach flasks containing 500 ml each of M9 medium supplemented with carbenicillin (100 mg/L). The cultures were incubated at 20°C and 200 rpm on a rotary shaker until the OD600 reached about 0.7 at which time 100 mg/L of lysine, threonine, and phenylalanine and 50 mg/L of leucine, isoleucine, valine, and selenomethionine were added. After further incubation for 30 min, IPTG was added to a final concentration of 50 uM. The cultures were then incubated overnight (#15hr) and harvested by centrifugation. The cell pellet was washed 2 times with 50 mM KPO4 buffer, pH 6.8. The yield was 28.5 gm wet weight of cells to which was added 114 ml of 100 mM KPO4 buffer, pH 8.2 and 5 mg of DNase. This mixture was frozen at- 80°C and thawed 2 times.

The thawed cell suspension was lysed by disruption in a French pressure cell at 20K psi. The unbroken cells and cell membrane material were sedimented by centrifugation at 100K times g for 1 hour. The supernatant crude extract, 128 ml (CE) was then placed in a 600 ml beaker and stirred for 10 minutes in a 55°C water bath to precipitate unstable proteins. After 10 min the beaker was stirred in ice water for 1 min followed by centrifugation at 15K times g for 15 min. The supernatant from this procedure, HT, contained 118 ml. The HT extract was then made 20% saturating in (NH4) 2SO4 by the slow addition of 12.7 g of (NH4) 2SO4. This was loaded on to a 10 cm

X 11.6 cm Fast Flow Phenyl Sepharose (Pharmacia) column equilibrated in100 mM KP04 buffer, pH 6.8, containing 20% saturation (109 g/L) (NH4) 2SO4. After loading the extract the column was washed with 1700 ml of starting buffer and eluted with a two step gradient. The first step was a linear 1900 ml gradient from start buffer to the same buffer without (NH4) 2SO4, the second was a 500 ml elution with 100 mM KPO4, pH 6.8 containing 5% EtOH. Active fractions, 241 ml, were pooled, diluted 100 % with water and loaded onto a 1.6 mm X 16 mm Poros HQ strong anion exchange column equilibrated in 100 mM Tris-HCl, pH 7.6. After loading the extract, the column was washed with 5 column volumes of starting buffer. The protein was eluted with a 15 column volume gradient from start buffer to start buffer containing 175 mM KCI. The active fractions were pooled and concentrated using a Centriprep 30 (Millipore) to 740 RI.

Figure 6 provides a purification table showing the enzyme activity of the enzyme of the present invention through various steps in the purification process.

The present application must be used to determine the respective values of the parameters of the present invention.

Unless otherwise noted, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.

Enzyme components weights provided herein are based on total active protein.

All percentages and ratios were calculated by weight unless otherwise indicated. All percentages and ratios were calculated based on the total composition unless otherwise indicated.

EXAMPLE 2

Determination of Ratio Between Peracid and Acid Formation In this Example, methods for determining the ratio of perhydrolysis to hydrolysis are described. In particular, this Example provides methods for determining the ratio between peracid formation (i. e. , perhydrolysis) and acid formation (i. e., hydrolysis) resulting from enzyme activity on an ester substrate in the presence of peroxide in an aqueous system.

A. Determination of Perhydrocis tn FTadrniJrsis Ratin Preparation of Substrate The substrates were prepared as described herein. Ethyl acetate (EtOAc) and other water soluble esters were diluted in a desired buffer to a concentration of 10 mM of ester.

Tributyrin and other water insoluble substrates were prepared by making substrate swatches. Polyester swatches were cut from non-dyed polyester fabric (Polycotton, PCW 22) using a 5/8 inch punch and placed in a 24-well microtiter plate (Costar, Cell Culture Plate). The insoluble ester was diluted to 1.03 M in hexane. Then, 10 µL of the insoluble ester solution were then adsorbed onto the polyester swatch.

Determination of Hydrolysis (GC Assay) The hydrolytic assay described below was used to determine the amount of substrate hydrolysis. In this assay, the assay solution was comprised of 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, and 20 mM potassium chloride in a total volume of 0. 99ml and an amount of enzyme that would generate 20 nmoles of acetic acid per minute at 25°C.

For measuring water insoluble ester hydrolysis, the reaction mixture was added to the insoluble ester fabric swatch. The swatch was prepared as described above ("Preparation of Substrate"). All the other conditions for the assay were the same except

for exclusion of other ester substrates.

Hydrolytic activity was measured by monitoring the increase of acids generated by the enzyme from acyl donor substrates using gas chromatography coupled with flame ionization detection. The assay was conducted by first pipetting 50 gel of assay solution containing all the components except the enzyme into 200 mL of methanol (HPLC grade) to determine the amount of acid in the assay solution at time 0. Then, 10 gL of enzyme were added to the assay solution to a desired final concentration which produced approximately 20 nanomoles of acid per minute. A timer was started and 50 pL aliquots were taken from the assay solution and added to 200 uL of methanol at various times, typically 2,5, 10,15, 25,40, and 60 minutes, after addition of the enzyme.

These methanol-quenched samples were then injected into a gas chromatograph coupled with a flame ionization detector (Agilent 6890N) and analyzed for hydrolytic components, acetic, and butyric acids. Gas chromatography was conducted using a nitroterephthalic acid modified polyethylene glycol column (Zebron FFAP; with dimensions: 30 m long, 250 um diameter, 250 nm film thickness). A 3 ttL aliquot of sample was applied to the column by a splitless injection under constant a helium flow of 1.0 mL/minute. The inlet was maintained at a temperature of 250°C and was purged of any remaining sample components after 2 minutes. When analyzing acetic acid, the temperature of the column was maintained at 75°C for 1 minute after injection, increased 25°C/minute to 100°C, then increased 15°C/minute to 200°C.

When analyzing butyric acid, the temperature of the column was controlled as described above, except the temperature was additionally increased 25°C/minute to 225°C and held at 225°C for 1 minute. The flame ionization detector was maintained throughout the chromatography at 250°C and under constant hydrogen flow of 25 mL/minute, air flow of 200 mL/minute, and a combined column and makeup helium flow of 30 mL/minute. The amount of hydrolyzed acid in the sample was then determined by integrating the acid peak in the chromatogram for total ion counts and calculating the acid

from the ion count using a standard curve generated under the above conditions for acetic and butyric acids at varying concentrations in the assay solution (without enzyme).

Determination of Perhydrolysis (OPD Assay) The perhydrolytic activity assay described below was used to determine the amount of peracid formed in the reaction. In these assays, the solution comprised 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, 20 mM potassium chloride, and 10 mM O-phenylenediamine.

When using water insoluble ester as the acyl donor, an ester adsorbed fabric swatch was used as the substrate, prepared as described above ("Preparation of Substrate").

Perhydrolytic activity was measured by monitoring the absorbance increase at 458 nm of oxidized O-phenylenediamine (OPD) by peracid generated with the enzyme. The perhydrolytic activity assay solution was prepared in the same manner as the hydrolytic activity assay solution, except that OPD was added to the assay solution to a final concentration of 10mM. The OPD solution was prepared immediately before conducting the assay by dissolving 72mg OPD (Sigma-Aldrich, dihydrochloride) in 19.94 mL of the same buffer and the pH was adjusted by slowly adding 60 IlL of 13.5 M potassium hydroxide. The pH was measured and if needed, small quantities of potassium hydroxide were added to return the pH to the original pH of the buffer. Then, 495 gL of this OPD solution were added with the other assay components to a final assay volume of 0.990 mL. An assay quenching solution was also prepared by dissolving 36mg OPD in 20 mL 100 mM citric acid and 70% ethanol.

The assay was typically conducted at 25°C. The assay was started by pipetting 100 tiL of assay solution before the addition of the enzyme into 200 gel of quenching solution to determine the amount of perhydrolytic components and background absorbance in the assay solution at time 0. Then, 10 uL of enzyme were added to the

assay solution to a desired final concentration which produced approximately 10 nanomoles of peracid per minute. A timer was started and 100 liL aliquots were taken from the assay solution and added to 200 ItL of quenching solution at various times, typically 2,5, 10,15, 25,40, and 60 minutes, after adding the enzyme. The quenched assay solutions were incubated for 30 minutes to allow any remaining peracid to oxidize the OPD. Then, 100 pL of each quenched assay solution was transferred to a 96-well microtiter plate (Costar) and the absorbance of the solution was measured at 458 nm by a spectrophotometric plate reader (Molecular Devices, SpectraMAX 250). The amount of peracid in each quenched sample was calculated using a standard curve generated under the above conditions with peracetic acid at varying concentrations in the assay solution (without enzyme).

Perhydrolysis/Hydrolysis ratio: Perhydrolysis/Hydrolysis ratio= Perhydrolysis measured in the Perhydrolysis assay/ (Total acid detected in the hydrolysis assay-Perhydrolysis measured in the perhydrolysis assay) The results of these experiments are provided in Figures 7,10 and Figure 11.

Figure 7 provides a graph which shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 40 minutes. Figure 10 shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 4,10, and 30 minutes. Figure 11 shows the ratio of peracetic acid to acetic acid generated by various enzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10 minutes. The results obtained in these experiments indicated that M. smegmatis perhydrolase homologues exhibited a ratio above 1 in the OPD/GC assays described above, while other classes of enzymes exhibited ratios significantly below 1.

Table 2-lprovides data showing the perhydrolysis activity of various homologues described herein on triacetin, as compared to the wild-type M. smegmatis perhydrolase.

The results provided in Table 2-2 indicate that the perhydrolase has activity over a broad range of substrates. In addition to the results provided in these Tables, Figures 8 and 9 provide data showing that the perhydrolase of the present invention has broad pH and temperature range activities.

Table 2-1. Perhydrolysis Activity of Perhydrolase Homologues on Triacetin as Compared to M. smegmatis perhydrolase erhydrolysis Ratio homolog to xperiment Protein perhydrolase) pET26_Mlo 0. ET26b Mbo 0. 87 ET26_SmeII 2.1 ET26b Stm 0. 17 LO_SmeI 0.7 erhvdrolase 1. 0000 @lank 0.0660 ET26_S261_M2aA12 1.5 erhydrolase 1 @lank 0.3 et26 M40cD4 0.14 pet26 M44aA5 0. 16 Perhydrolase Blank _ 0. 01 Table 2-2. Peracid Production by 1 ppm Wild-Type Perhydrolase with 29 mM H202 and Various Esters nmol Peracetic Acid l min Ester lOmM of lOmM of lOmM of Ester Ester with Ester on Polycotton 0.5% Swatch Neodol Ethyl Acetate 5.00 Butyl Acetate 8.06 8.72 Hexyl Acetate 7.96 5.86 Octyl Acetate 8. 03 0. 48 Ethyl Propionate 0.90 1.43 Butyl Propionate 2.47 3.39 Hexyl Propionate 4.00 2.66 Isoamyl Acetate 7.83 17.69 Citronellyl Acetate 7.25 4.27 Citronellyl 2.85 3.21 Propionate Dodecyl Acetate 3.95 0.19 Neodol 23-3 2.25 8.77 Acetate Neodol 23-6.5 2.73 10.12 Acetate Neodol 23-9 2.97 10.20 Acetate Ethylene Glycol 13.30 Diacetate Propylene Glycol 13.17 Diacetate Triacetin 11.91 Tributyrin 0.66 2.70 Ethyl 0.49 Methoxyacetate Linalyl Acetate 0.30 Ethyl Butyrate 0.31 Ethyl Isobutyrate 0.10 Ethyl 2-0.11 methylbutyrate Ethyl Isovalerate 0. 37 Diethyl Maleate 0.75 Ethyl Glycolate 1.91

B. Typical Perhydrolase Peracid Generation Assay: Perhydrolase is active over a wide pH and temperature range and accepts a wide range of substrates for acyl transfer. Acceptors include water (hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classical acyl transfer). For perhydrolysis measurements enzyme was incubated in the buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide. Typical substrates used to measure perhydrolysis include ethylacetate, triacetin, tributyrin, ethoxylated neodol acetate esters, and others. In addition, the wild type enzyme was found able to hydrolyze nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. In some embodiments, peracid acid and acetic acid were measured by the ABTS or HPLC assays as described below. Nitrophenylester hydrolysis is also described below. r ARTS Acca (nnp milliliter, This assay provides a determination of peracetic acid produced by perhydrolase.

This protocol was adapted from Karst et al., Analyst, 122: 567-571 [1997] ). Briefly, a 100 J. L aliquot of solution to be analyzed was added to 1 mL 125 mM K+citrate pH 5,1 mM ABTS, 50 uM KI. Absorbance was measured at 420 nm for highest sensitivity.

However, multiple additional wavelengths were sometimes used over the broad absorption spectrum of ABTS. Calibration curves were constructed based on known peracid concentration series.

D. HPLC (Model - Agilent 1100) Determination of Perhydrolase Reaction Pro (nets For determination of the ratio of perhydrolysis to hydrolysis of the perhydrolase

reaction, perhydrolase reaction samples were quenched by acidification to a final concentration of 0.24% methanesulfonic acid, and the products were separated by reverse phase HPLC on a Dionex OA column (cat #062903 ; Dionex Corporation, Sunnyvale, CA). The mobile phase was 100 mM NaP04, pH 3.9 (buffer was prepared by titrating 100 mM Na2PO4 with methanesulfonic acid to pH 3.9) run under isocratic conditions at 30 C. Detection was at 210 nm. Concentrations of products were calculated by comparison of the integrated peak areas against calibration standards.

F, Nitrn henylester Hdrnlysis Kinete Assay Enzyme and substrate were incubated in 100 mM Tris/HCl pH 8.0 (or 50 mM B (OH) 3 pH 9.5 or another buffer). Absorbance at 402 nm was monitored. In some experiments, the assay was carried out in standard 1 mL cuvettes, while in other experiments, microtiter plate wells were used. The latter method was used for the screening of mutant libraries. Enzyme concentration was determined by comparison to standard curves obtained under the same reaction conditions.

Para-nitrophenylcaproate Hydrolysis Assay The pNC6 substrate solution was prepared by mixing 1mM pNC6 (100 mM stock solution), 1 ml DMSO, 19 mls 100mM Phosphate (pH8), and glycerol to a final concentration of 10%. To assay samples, 10 u. l of the cell lysate were added to 190 u. l of the substrate solution, and assayed at 405 nm for 15 minutes in a spectrophotometer. The results are presented as the average of two experiments.

G. Para-nitrophenyl Acetate (pNA) Hydrolysis Assay Aliquots of the lysed cell supernatant were diluted 1-100 in 100 mM phosphate buffer (pH 8). To assay the samples, 5 RI of the 1-100 diluted cell supernatant were

placed into each well of a microtiter plate. Then, 195 pi of reaction buffer/substrate mix (1 mM pNA, 100 mM phosphate, pH 8,10% glycerol) were added, and the absorbance rate at 405 nm was measured over 3 minutes (kinetics program, microtiter plate reader).

The results are presented as the average of two experiments.

EXAMPLE 3 Assays Including Detergent Compositions In this Example, assay systems used to screen for superior perhydrolase activity in detergents with particular substrates are provided. These assays include those that measure peracid degradation of perhydrolase, as well as the peracid synthesis activity of the enzyme.

Materials and Methods for Peracetic Acid Formation (PAF) and Peracetic Acid Degradation (PAD) Assays This section provides the materials and methods used to screen for a superior perhydrolases in Ariel with C9E20AC ester substrate Materials' Ariel Futur without bleach, perfume, or enzymes (P&G, Ariel"C") C9E20Ac (P&G) 30% Hydrogen Peroxide (Sigma) 32% Peroxyacetic acid ("peracid", PAA) (Sigma cat#) MW = 76.05 ; 4.208M Citric Acid, anhydrous MW=192. 12 Potassium Hydroxide MW=56.11 ABTS (Sigma cat# A1888) MW=548. 68 Potassium Iodide MW=166.0 Potassium Phosphate, mono and di-basic Stock solutions:

Ariel detergent stock: Ariel Futur without bleach, perfume, or enzymes ("Ariel C") was dissolved in water to 6.72 g/L. It was stirred at room temp for 30 minutes, then allowed to settle. Then, it was divided into convenient aliquots and stored at 4°C, until used.

When made and used fresh, the solution was filtered, instead of settled 100 mM C9E20Ac in Ariel detergent stock: First, 30 Ill C9E20Ac was added to 970, u1 Ariel detergent stock, using a positive displacement pipet. It was sonicated in a bath sonicator until a milky dispersion was formed (15-60 seconds). The dispersion was stable for about two hours. When used, 10 ttl of dispersion per ml of reaction mix were used.

42 mM Peroxyacetic acid stock: Right before use, the Sigma 32% PAA solution was diluted 1: 100 in water. Then 5.7 u. l of the 42 mM stock per ml of reaction mix was added.

2 M hydrogen peroxide: One ml of 30% Sigma hydrogen peroxide was added to 3.41 ml water. This solution was prepared fresh, right before use. It was used at 10 ul per ml of reaction mix.

125 mM Citrate buffer pH 5.0 : This was prepared to 24.0 grams per liter. It was made up in 800 ml, and titrated to pH 5.0 with 50% KOH. The volume was adjusted to 1 liter and stored at room temperature.

100 mM ABTS stock: This was prepared using 549 mg of ABTS in 10 ml of water. It was frozen at-80°C, in convenient aliquots in opaque Eppendorf tubes. The stock was stable indefinitely when kept frozen in the dark. ABTS will precipitate when thawed from-80°C but goes back into solution upon mixing. In use, 10 ul of ABTS stock was used per ml of ABTS reagent.

250 mM HI : This was prepared as 415 mg in 10 ml water. It was kept at 4°C. It was diluted to 25 mM working stock, and 2 ul of working stock was used per ml of ABTS reagent.

25 mM Potassium Phosphate buffer, pH 8.0 : Method: The night prior to performance of the assays, the plates containing lysed cells that contain perhydrolase were checked to be sure that they were frozen twice. On the day of

the assay, 30 to 45 minutes were allowed for the plates to thaw. The ABTS reagent was prepared and the Multidrop (Multidrop 384 instrument, ThermoElectron) to fill the detection plates with 200 ul per well. Store the filled plates covered at room temperature in the dark until needed. Dilutions of the standards were prepared so that when 20 , 1 of the diluted standard were added to the 180 gui of the reaction mix, the concentration in the well was 1 ppm. Four 4 two-fold serial dilutions were prepared to a set of six standards: 1,0. 5,0. 25,0. 125, and 0.0625 ppm final concentration in the wells.

To test, 20 p, l of the standards were added to the thawed 1: 10 dilution plate. The reaction mixtures were prepared and the Multidrop used to fill one reaction plate for each plate to be assayed (180111/well). Note that the reaction mixtures are different for the PAF and PAD assays.

Peracid Hydrolysis (Peracid Degradation, PAD) Assay: This assay measures the amount of peracetic acid remaining after a 100 minute incubation with enzyme in an Ariel detergent background. The amount of peracid remaining is detected by reacting an aliquot of the reaction mixture with the ABTS detection reagent.

In this assay, 20 ul enzyme samples from the thawed 1: 10 dilution plate were transferred, one column at a time with an 8 channel pipetter, into the corresponding column of the pre-filled PAD reaction plate. A timer was started as soon as transfer occurred from the first column ; subsequent columns were transferred at 15 second intervals (i. e. , the last column was finished 2 min. 45 sec. after starting the first one). The plate was mixed for 30 seconds on the thermomixer (750 rpm, to avoid splashing). The plate was then transferred to a humidified chamber at 25°C. The plate was incubated for a total of 100 minutes from the time the first column of enzyme was added. At 100 minutes incubation, the reaction plate was removed from the incubator. Then, 20 ul

aliquots of the reaction mixture were transferred to an ABTS reagent plate, in the same order and with the same 15 second time interval that the enzyme samples were originally added to the reaction plate. The ABTS plate was allowed to sit at room temperature for three minutes after the last column of reaction mixture was added. The plate was then read on the spectrophotometric plate reader at 420 and 740 nm.

Perhydrolysis (Peracid Formation, PAF) Assay Multidrop Optimized Protocol: Screening for a Superior Perhydrolysis in Ariel with C9E20AC Ester Substrate The same materials and stock solutions described above for PAD were used in these experiments, as indicated below.

Method: The methods were designed to assay 20 gel aliquots from a 1: 100 dilution plate.

The 20 gel 1: 100 dilution assay plates were produced during the process of obtaining the protein concentrations and were stored at-80°C. The plates were thawed for about 30 to 45 minutes before use. Dilutions of the S54V standards were prepared, so that when 2 of the diluted standard are added to the 20 RI of the 1: 100 diluted cell lysate, the concentration in the well was 0.1 ppm. Four two-fold serial dilutions were prepared to produced a set of six standards: 0.1, 0.05, 0.025, 0.0125, and 0.00625 ppm final concentration in the wells. Then, 2 ul of the standards were added to the thawed 20 ul 1: 100 dilution assay plates in the wells indicated.

Perhydrolysis (Peracid formation, PAF) Assay: This assay measures the amount of peroxyacetic acid that is produced in 10

minutes from the C9E20Ac substrate in an Ariel detergent background. The amount of peracid formed is detected after 10 minutes by reacting an aliquot of the reaction mixture with the ABTS detection reagent.

The Multidrop was used to deliver 180 ptl/well of the PAF reaction mix to the prepared 1: 100 dilution plate. The timer was started and the reaction plate was placed on the thermomixer, with the temperature set at 25°C. The plate was covered and the solutions mixed for 30 seconds at 750 rpm. The plate was then allowed to rest on the thermomixer without mixing, for a total of 10 minutes from the time the reaction mix was added.

At 10 minutes, the Multidrop was used to add 20, ul/well of the lOx ABTS reagent. The lOx reagent was a milky suspension. The thermomixer was used to briefly shake the plate. The ABTS reagent quickly went into solution. The plate was allowed to sit at room temperature for three minutes after the ABTS reagent was added. The plate was then read on the spectrophotometric plate reader at 420 nm.

EXAMPLE 4 Cloning of Mycobacterium smegmatis Perhydrolase In this Example, the cloning of M. smegmatis perhydrolase is described. An enzyme with acyltransferase activity was purified from Corynebacterium oxydans (now MycobacteriumparafortuitumATCC19686). Two peptide sequences were obtained from the purified protein. One peptide was determined by Edman degradation from cyanogen bromide cleavage of the purified enzyme using methods known in the art. The sequence of this peptide was determined to be KVPFFDAGSVISTDGVDGI (SEQ ID NO : 3). The second peptide was analyzed using N-terminal sequencing and was found to have the GTRRILSFGDSLTWGWIPV (SEQ ID NO : 4). A BLAST search against the

TIGR unfinished genome database identified a sequence of potential interest in Mycobacterium smegmatis, which is shown below: MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLS ARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALG MSVLVTQVLTSAGGVGTTYPAPKVLWSPPPLAPMPHPWFQLIFEGGEQKTTELA RVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL (SEQ ID NO : 2). The corresponding DNA sequence of the gene is : 5'- ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGTCCC CGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCTGGACC GGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAGGAGGGAC TGAGCGCGCGCACCACCAACATCGACGACCCCACCGATCCGCGGCTCAACGG CGCGAGCTACCTGCCGTCGTGCCTCGCGACGCACCTGCCGCTCGACCTGGTG ATCATCATGCTGGGCACCAACGACACCAAGGCCTACTTCCGGCGCACCCCGC TCGACATCGCGCTGGGCATGTCGGTGCTCGTCACGCAGGTGCTCACCAGCGC GGGCGGCGTCGGCACCACGTACCCGGCACCCAAGGTGCTGGTGGTCTCGCCG CCACCGCTGGCGCCCATGCCGCACCCCTGGTTCCAGTTGATCTTCGAGGGCG GCGAGCAGAAGACCACTGAGCTCGCCCGCGTGTACAGCGCGCTCGCGTCGTT CATGAAGGTGCCGTTCTTCGACGCGGGTTCGGTGATCAGCACCGACGGCGTC GACGGAATCCACTTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTCG CGGAACAGGTGCGGAGCCTGCTGTAA-3' (SEQ ID NO : 1) Primers were designed based on the gene sequence to amplify and clone the gene. The primers used for amplification were : MsRBSF : 5'- CTAACAGGAGGAATTAACCATGGCCAAGCGAATTCTGTGTTTCGGTGATTCC CTGACCT-3' (SEQ ID NO : 5)

MspetBamR : 5'- <BR> <BR> <BR> <BR> GCGCGCGGATCCGCGCGCTTACAGCAGGCTCCGCACCTGTTCCGCGAGGGCC ACCCCGA-3' (SEQ ID NO : 6) The amplification of the gene was done by PCR using Taq DNA polymerase (Roche) per the manufacturer's instructions, with approximately 500 ng of chromosomal DNA from Mycobacterium smegmatis as the template DNA and the addition of 1% DMSO to the PCR reaction mix. Thirty picomoles of each of the primers MsRBSF and MspetBamR were added to the mix. The amplification cycle was: 30 cycles of (95°C for 1 min, 55°C for 1 min, 72°C for 1 min).

The fragments obtained from the PCR reaction were separated on a 1.2% agarose gel and a single band of the expected size of 651 bp (coding sequence and stop codon) was identified. This band was cloned directly into the pCR2.1 TOPO cloning vector (Invitrogen) and transformed into E. coli Top 10 cells (Invitrogen) with selection on L agar (10 g/1 tryptone, 5 g/1 yeast extract, 5 g/l NaCl, 20 g/l agar) containing 100 micrograms/ml carbenicillin and X-gal (20 micrograms/ml, Sigma-Aldrich) for blue/white selection and incubated overnight at 37°C. Five white colonies were analyzed for the presence of the PCR fragment. Each colony was used to inoculate 5 mls of L broth (L agar without the addition of agar) containing 100 micrograms/ml carbenicillin and the cultures were grown overnight at 37°C with shaking at 200 rpm. Plasmid DNA was purified from the cultures using the Quikspin kit (Qiagen). The presence of the correct fragment was determined by restriction enzyme digest with EcoRl to release the fragment, and sequencing using primers supplied by the pCR2.1 manufacturer (Invitrogen). The correct plasmid was designated pMSATNcoI (See, Figure 12, for the map of this plasmid) ). The sequence of this plasmid is provided below

Construction of Perhydrolase T7 Expression Plasmid The primer pair used to create pMSATNcol was also used to create an NcoI site (CCATGG) in which the ATG is the start codon of the acyltransferase gene and a BamH1 (GGATCC) just after the TAA stop codon. The plasmid pMSATNcol was digested with NcoI/BamHl as recommended by the manufacturer (Roche) and the 658 bp fragment containing the perhydrolase gene was purified using standard procedures known in the art (e. g., Sambrook et al.). The fragment was ligated using standard procedures known in the art (e. g., Sambrook et al.) into the T7 promoter expression plasmid, pET16b (Novagen), also digested with NcoIlBamHl. The ligation reaction was transformed by standard procedures into E. coli Top 10 cells (Invitrogen) and selected on L agar containing 100 micrograms/ml carbenicillin overnight at 37°C. Ten colonies were picked from the several transformants and used to inoculate 5 ml of LB containing 100 micrograms/ml carbenicillin. Cultures were grown overnight at 37°C with shaking at 200 rpm. Plasmid DNA was purified from the cultures using the Qiagen Quikspin kit (Qiagen). The presence of the correct fragment was determined by restriction enzyme digest with NcoIlBamHl as directed by the manufacturer. The correct plasmid was designated pMSATNcol-1 (See, Figure 13, for the map of this plasmid). In this Figure, the following elements are indicated--LacI : gene encoding the LacI repressor protein, located at bpl455-2534, ori: plasmid origin of replication at bp 4471, bla: The P-lactamase gene located at bp 6089-5232 ; T7 promoter: located at bpl068-1052 ; T7 terminator: located at bp 259-213, per : the M. smegmatis perhydrolase gene located at 981-334. The sequence

of this plasmid is provided below:

This plasmid was transformed into the E. coli strain BL21 (#DE3) pLysS (Novagen), which contains the gene encoding the T7 RNA polymerase, with selection on

LA containing 100 micrograms/ml carbenicillin. Cells were grown overnight at 37°C.

One transformant was selected and the strain was designated MSATNcol.

Production of Perhydrolase in MSATNcol-1 Production of perhydrolase was done in cell culture. For example, 5 ml of LB with carbenicillin at a concentration of 100 micrograms/ml was inoculated with a single colony of MSATNcol and grown overnight at 37°C with shaking at 200 rpm. This culture was used to inoculate 100 ml of LB with carbenicillin at a concentration of 100 micrograms/ml (in a 250 ml baffled flask) to an OD600 of 0.1. The cultures were grown at 30°C with shaking at 200 rpm until they reached an OD600 of 0.4. The expression of the perhydrolase gene was then induced by the addition of 100 micromolar IPTG and the incubation continued overnight. Cultures were harvested by centrifugation (10 min at 7000 rpm, Sorvall SS34 rotor), the supernatant was removed and the pellets washed in 50 mM Kip04, pH 6.8. The cells were centrifuged again, the supernatants removed and the wet weight of the cells was determined. The cells were resuspended in 100 mM KP04 in a volume that was 4x the wet weight. The resuspended cells were frozen at-70°C. The cells were thawed and lysed in a French Pressure cell using standard procedures known in the art. The purification steps and assessment methods are provided in Example 1.

Figure 6 provides a purification table showing the enzyme activity of the perhydrolase of the present invention through various steps in the purification process.

M. smegmatis Perhydrolase is in an Operon In additional experiments, it was determined that the M. smegmatis perhydrolase is part of an operon. The gene (pAd) is the first gene in an operon that contains at least 2 genes, includingphd, that are separated by 10 bp (GGCTGGGGGC [SEQ ID NO : 7] ) not including the TAA stop codon of pAd. It is also possible that there are three genes in the operon, with the third being either 48 bp or 61 bp to the next ORF (open reading frame).

The latter two candidate genes have no significant homology to proteins in the database.

A putative promoter was identified for M. smegmatis pAd operon, TTGGGC (-35) SP (18) CCAGAT by sequence analysis and comparison with known M. smegmatis promoters (See e. g. , Salazar et al., Microbiol. , 149: 773-784 [2003] ). It is not intended that the present invention be limited to any particular promoter and/or construct design, as it is contemplated that other promoters and construct designs will find use in the present invention.

The second gene in the pAd operon encodes a protein (putative PBP-3) with the sequence: mhlrpaltwllwglfiswgcssspdpadrfsafaealgrkdaaaaaaqtsdpaaaeaait amlagingdaanvsvaaepee gddagatlkytwtwgegrdfgydttataaksgddwlitwsptvlhrdltpdlrfqyseds elqtpvldrtgqphntwqtvgvit verahpesaaplaallapfdpttttesvtaqlnsttddrvtvmklreddlgqvrdqlaqi pgvtvreqgelltadrqlsspaisgld elwhdritanagwsvylvdadgapaqqltstppkdtgpvrttldlrmqllaqqavaketr pavwaisgstggilaaaqnpaa dpqgaiafsglyppgstfktittaaaldaglatpdtpvacpgeltienrtipnddnfdlg tvplssafshscntsmaalsdelppn altdmakdfgigvdfmvpglttvtgrvpnadnaaqrvengigqgtvtvspfglavaeasl ahgstilptlvdgekttadtpsvp Ippnitdalrammrgtvtegtatalsdipdlggktgtaefgdnthshgwfagiagdiafa tlwggdssapavaisgdflrpala g (SEQ ID NO : 9) The corresponding DNA sequence of the gene encoding the putative PBP-3 : atgcacttacgtcccgctctgacgtggctcctggttgtcggtctgttcatatcggtcgtc ggatgttcgtcgtccccggatccggccg accggttctcggcgttcgccgaggcgctgggccgcaaggatgcggccgcggcggccgccc agaccagcgatccggcggcc gcggaggcggccatcaccgcgatgctggccgggatgggcgacgccgcgaacgtctcggtg gccgccgaacccgaggaagg cgacgacgcgggcgcgacgctgaagtacacgtggacctggggtgagggccgcgacttcgg ctacgacaccaccgcgacggc ggccaaatccggtgacgactggctgatcacctggtcccccaccgtgttgcaccgcgacct caccccggatctgcgcttccagtac agcgaggacagcgaattgcagaccccggtgctcgaccgcaccggccagccgttgatgaca tggcagaccgtcggtgtcatcac tgtcgaacgcgcacatccggagtcggccgcaccgctcgccgccctgctggcgcccttcga tccgaccaccaccaccgaatcgg tcaccgcacaactcaattcgacgaccgatgaccgcgtgacggtgatgaagctgcgcgagg acgatctgggtcaggtgcgcgat cagctcgcgcagatccccggcgtgaccgtgcgtgagcagggtgagctgctcaccgccgac cggcagctgtcctcgcccgccat cagcggcctggacgagctgtggcacgaccggatcaccgccaacgcgggctggtcggtgta cctggtcgacgccgacggtgca cccgcacaacagctcacgtccacgccgcccaaggacaccgggcccgtgcgcaccacgctg gacctgcgcatgcaactgctcg cgcagcaggccgtggccaaggagacccgcccggccgtggtggtcgcgatctccggatcga ccgggggcatcctggccgccg cacagaacccggccgccgatccgcaaggtgcgatcgcgttttcgggcctgtacccgccgg ggtcgacgttcaagaccatcacc acggcggcagccctcgacgcgggcctggccaccccggacacaccggtggcctgcccgggt gagctcaccatcgagaaccgc acgatccccaacgacgacaacttcgacctgggcaccgtgccgttgtcgtcggcgttctcg cactcctgcaacaccagcatggcc gccctgtccgacgagctgccgcccaacgcactgaccgacatggcaaaggacttcgggatc ggcgtcgacttcatggtgcccgg

A standard BLAST search against the protein database identified homology with several penicillin binding proteins, class 3 (PBP-3). By sequence alignment and comparison to literature (e. g., Goffin and Ghysen, Microbiol. Mol. Biol. Rev. , 66: 702-38 [2002] ) the PBP was found to contain the required bar codes (conserved protein sequences that define a class of proteins) to place it in the SxxK superfamily of acyl transferases, with a C-terminal domain acyl transferase and an N-terminal domain of unknown function, but with homology to the Pen (i. e. , penicillin resistant) protein fusions of class B-like II and m. This penicillin binding protein acyl transferase domain does not share significant homology with the perhydrolase of the present invention, although it does share homology with Co-A dependent acyl transferases known in the art.

The amino acid sequence is provided below.

The family-identifying bar codes provided in the above review were: (19) V (20)

G/A (140) PVxDRTG (142) TxDx3Q (22) TGGxLAx4PaxDP (13) SxxK (51) SCN (131) KTG (50) marked in bold letters in the above sequence. The letters represent the amino acid sequence defining the bar code; the numbers in brackets are the intervening number of amino acids between the particular bar codes ;"x"represents any amino acid, (i. e., the amino acids are not conserved within the bar code but the number of amino acids (e. g., x3 corresponding to 3 intervening amino acids) is conserved). Based on these results and other data, as described herein, it is clear that the perhydrolase of the present invention represents a unique enzyme class.

EXAMPLE 5 Expression of the Perhydrolase in P. citrea In this Example, methods used to express the perhydrolase in P. citrea are described. The plasmid pMSATNcoI was transformed into P. citrea by electroporation using the method essentially as known in the art (See e. g., Sambrook et al., supra) except that all cultures and recovery were done at 30°C. The transformants were plated on L agar + carbenicillin (200, ug/ml) and incubated overnight at 30'C. Three transformants were picked for analysis. Each colony was used to inoculate a 30 ml culture of LB + carbenicillin (200 gg/ml) and grown overnight at 30°C with shaking at 200 rpm. The cells were pelleted by centrifugation, washed one time in 50 mM phosphate buffer pH 7.2, and finally resuspended in 4x the wet cell weight of 100 mM phosphate buffer pH 8. 0. The cells were lysed by treatment with lysozyme (2 lofas0 mg/ml solution per one ml of P. citrea culture) at 37°C for one hour. The cell debris was pelleted at 13,000 rpm in a microfuge for 5 min. The resulting supernatant was used for further analysis in SDS-PAGE and Western blots, as well as assays for enzyme activity.

SDS-PAGE analysis was carried out as known in the art (See e. g., Sambrook et al., supra) on the supernatants. Detection of the perhydrolase protein by Western blot

was done using an anti-perhydrolase polyclonal anti-sera (prepared from purified perhydrolase protein by Covance). The blot was developed as per manufacturer's suggestions using the ECL plus kit (Amersham).

The enzymatic activity of the expressed perhydrolase was detected by the pNB (para-nitrophenylbutyrate) assay as described in Example 1, herein. The results are provided in the Table 5-1. Enzymatic Activity of Perhydrolase Expressed by P. citrea Concentration Clone OD405 Rate (mg/liter) P. citreal pMSATNcoI 3.1129 0.47948 7.1922 Control (P. citrea) 2.6187-9. 8312 0 The SDS-PAGE and Western blot results, as well as the assay results indicated that the perhydrolase is expressed by P. citrea and is active.

EXAMPLE 6 Expression of the Perhydrolase in Bacillus subtilis The perhydrolase was expressed intracellularly in B. subtilis. A variety of promoters find use in this embodiment, including but not limited to pSPAC, pAprE, pAmyE, pVeg, pHpaII. In some embodiments, the construct is present on a replicating plasmid (e. g., pBHl), while in other embodiments, it is integrated into the chromosome in one or more copies. Examples of sites for integration include, but are not limited to the aprE, the amyE, the veg or therm regions. Indeed, it is contemplated that other sites known to those skilled in the art will find use in the present invention.

A. Intracellular Expression of the Perhydrolase in Bacillus subtilis From

a Replicating Plasmid B. subtilis expresses a lipase/esterase encoded by the gene pnbA that hydrolyzes the pNB substrate used to detect activity of the perhydrolase. To identify B ; subtilis strains expressing the perhydrolase after transformation with replicating or integrating plasmids the pnbA gene (the entire coding sequence) was first deleted from the desired host using the loxP cassette deletion method described in WO 03/083125, herein incorporated by reference. It is also noted that other strains of Bacillus may contain one or more lipases/esterases capable of hydrolyzing the pNB or other substrate used as an indicator for perhydrolase activity. In some embodiments, for optimal expression and/or activity detection it is necessary to delete one or more of the lipases/esterases from the hosts. The Bacillus subtilis strain used in this Example has the genotype Bacillus subtilis comK pnbA (pnbdloxP-spec, aprE, nprE, degUHy32, oppA, spoIIE3501 and will be referred to as "B. subtilis pnbA" (See e.g., WO 03/083125, supra).

In these experiments, a consensus Bacillus ribosome binding site (RBS) was used.

It is not intended that the consensus RBS be the only sequence used for expression, as a non-consensus RBS also finds use in the present invention. The RBS of pMSATNcoI (See, Example 4) was changed to a Bacillus consensus RBS from the 16S rRNA (5'- ATAAGGAGGTGATC-3' [SEQ ID NO : 132] ) of B. subtilis and a HindIII site was added to the 5'end of the RBS by PCR using a primer (502rbsforward primer) containing the desired changes. The reaction was carried out using an MJ Research PCR machine with 30 cycles of (1 min at 95°C, 1 min at 55°C, and 1 min at 72°C). Template DNA (pMSATrbs) was added to a 50 µl reaction (10 ng) and 10 picomoles of each primer were used.

The PCR-generatedphd cassette was cloned into the PCR cloning vector, pCR- Script CM (Stratagene) and transformed into E. coli Top 10 cells (Invitrogen) to make pAH502R. The complete sequence of this plasmid is provided below.

Transformants were selected on L agar containing 100 llg/ml carbenicillin. The construct was confirmed by sequencing and biochemical assays (e. g., pNB activity assay) Primer set for pAH502R construction: 502rbsForward primer: 5'-ccaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcg-3' (SEQ ID NO : 134) 502Reverse Primer: 5'-ggggatccttttacagcaggctccgcacct-3' (SEQ ID NO : 135) The HindIII-RBS-phd-BamHI DNA fragment from pAH502R was cloned into the pSPAC containing vector, pMUTIN4 (See, Vagner et al., Microbiol. , 144,3097-3104 [1998]) creating the construct pAH503. The complete sequence of pAH503 is provided below:

The construction of pAH503 was confirmed by RFLP and pNB activity assays.

The pSPAC-RBS-phd DNA cassette was isolated as a BglII/SmaI digest and then subeloned into the replicating plasmid pBHl, digested with BamHllEcoRV (See e. g. , EP 0275509) to create pAH505 (See, Figure 14). The complete sequence of the plasmid is provided below.

The ligation mixture for pAH505 was transformed into Bacillus subtilis pnbA.

Correct transformants were verified by RFLP and sequencing of isolated plasmid DNA.

One transformant was selected for analysis (B. subtilis pnbAlpAH505).

Expression of the perhydrolase in Bacillus was assayed using the pNB Activity Assay described herein, after growth of the desired strain in shake flask. The data showed that the perhydrolase was expressed in B. subtilis pnbA.

B. Intracellular Expression of the Perhydrolase in B. subtilis pnbA by Integration into the Chromosome An additional construct useful to determine expression of the perhydrolase (act) gene integrated into the chromosome of B. subtilis pnbA involved use of the spo VG promoter, which was found to drive expression of the perhydrolase gene in a non- replicating (i. e. , integrating plasmid). In some embodiments, one site of integration is the aprE region of B. subtilis, although it is intended that integration occur at any suitable site. Indeed, it is not intended that the present invention be limited to this specific site nor this specific promoter, as various other suitable sites and promoters find use in the present invention.

The configuration of the promoter/gene at the aprE locus in the chromosome of Bacillus subtilis was as follows : pAprE-aprE first 7 codons-translation stop-pSpoVG-ATG-perhydrolase gene from second codon The clone was constructed as described below. The primers used were: Up5'F caggctgcgcaactgttgggaag (SEQ ID NO : 138) FuaprEAct34R agtagttcaccaccttttccctatataaaagcattagtgtatcaatttcagatccacaat tttttgcttctcactctttac (SEQ ID NO : 139) FuaprEAct4F Aattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcgaa ttctgtgtttcggtg (SEQ ID NO : 140) BsmI-DnAct504R gtgagaggcaitcggatccttttacagcaggctccg (SEQ ID N0 : 141) PCR fusion is a technique well known in the art, in which two or more fragments of DNA are generated either by restriction digest or by PCR amplification. The fragments have overlapping segments, usually at least 18 bases long. In the instance that two fragments are used, the 3'end of fragment #1 has an overlapping sequence with the 5'end of fragment #2. The two fragments are used as template in a PCR reaction in which the primer set used hybridizes to the 5'end of fragment #1 (forward primer) and the 3'end of fragment #2 (reverse primer). During the amplification, the two regions of overlap hybridize forming a single template from which the two primers can amplify a full length fragment, a"fusion"of fragments #1 and #2. Multiple fragments of any length can be used in such a reaction, limited only by the ability of the chosen polymerase to

amplify long DNA pieces.

In the current example, the above construct was made by PCR fusion of two PCR products the above construct was made by PCR fusion of two PCR products. The first was a construct with the spoVG promoter added upstream of the phd gene. The second was the aprE promoter and first 7 codons of aprE, followed by a stop codon. Regions of 20 bp overlap were added on the 5'and 3'ends of the products respectively, to allow the PCR fusion reaction. The primer set FuaprEAct4F/BsmI-DnAct504R was used to amplify the perhydrolase gene from pAH505 as described above, which added the spo VG promoter sequence (contained within the primer) to the 5'end of the gene and changed the start codon from ATG to GTG. To create the second product (pAprE plus the first 7 codons of aprE) for the fusion, the primer set Up5'F/FuaprEAct34R was used to amplify a fragment from pBSFNASally. Figure 15 provides a map of this plasmid. The complete sequence of pBSFNASally is provided below.

The two PCR products were subjected to fusion PCR as known in the art to create the 1.5 kb fusion. The resulting fusion product was then cloned into PCR2. 1TOPO to produce pCP609 (See, Figure 16) and sequence below).

The plasmid PCP609 was digested with BamHl/XmaI to release the fragment containing the pAprE-aprE-stop-pSpoVG-phd construct and ligated into pBSFNASally digested withXmaI/BclI to give the plasmid pCP649. Figure 17 provides a map of pCP649. The complete sequence of pCP649 is provided below.

All constructs were confirmed by sequence analysis. PCR reactions were done using Hercules polymerase (Roche) as per the manufacturer's directions. pCP649 was transformed into B. subtilis comKpnbA and integrants selected on L agar containing chloramphenicol (5u. g/ml). The activity of the expressed perhydrolase was determined by the pNB activity assay as described herein. The results indicated that the perhydrolase was expressed and active EXAMPLE 7 Expression of the Perhydrolase in Streptomyces.

In this Example, experiments conducted to assess the expression of the perhydrolase in Streptomyces are described. To test expression of the perhydrolase in Streptomyces, a replicating plasmid was constructed with the phd gene being expressed from either the glucose isomerase (GIT) or the A4 promoter (See e. g., US/PCT/, filed November 18,2004, herein incorporated by reference).

However, it is not intended that the present invention be limited to these specific promoters, as any suitable promoter will find use with the present invention. Also, although the strain used for perhydrolase expression in this Example was Streptomyces lividans TK-23, it is contemplated that any Streptomyces will find use in the present invention.

The Streptomyces strains were transformed and manipulated using methods known in the art (See e. g. , Kieser et al., Practical Streptomyces Genetics, John Innes [2000]).

Construction of pSECGT-MSAT and pSECA4-MSAT Using standard methods known in the art, therm coding sequence (See, Example 4) was cloned into pSECGT to place the gene under control of the GI promoter.

Similarly, the gene was cloned in the same plasmid with the A4 promoter using methods known in the art (See e. g., US/PCT/, filed November 18,2004, herein incorporated by reference). Transformants were first selected in E. coli, verified by sequence analysis, and then transformed into S. lividans TK-23 using methods known in the art (See e. g., Kieser et al., [2000], supra). The correct clones expressed from the GI promoter and the A4 promoter were designated"pSECGT-MSAT"and"pSECA4-phd." The sequence of pSECGT-MSAT is provided below, while Figure 18 provides a map of the plasmid. ctagagtcgaccacgcaggccgccaggtagtcgacgttgatctcgcagccgagcccggcc ggaccggcggcgctgagcgcg aggccgacggcgggacggccggcaccggtacgcggtggcgggtcgagttcggtgagcagc ccaccggcgatcaggtcgtcg

acggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgct ggagtggaacctctcgcgctg gcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcg gcagatgctcgggctcgacgg cggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcgggga gctgcgggccggggtcgccg tgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccggg ccgccgaaggcaaggacggc aacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcg gccgacacagtggtggtgctc acggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgc gaggaagcaactgcacgccg acggegagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgc ccggcacatcgcccggctc gcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgt acggcgggggtcggtgacgtacg cggcgacggcggccggggtcgaagcegcgggagtaatcctgggattactcgcccggggtc ggccccgccggcacttcgtgca ggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggcca tgagcaccaccacccccaggga cgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcgg ccggagccgggactactgccg ccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgc cgtggcgtcggcagtcgctcg ccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcg ggccgcaggtcagagcacggttcc gcccgctccggccctgccggacecccggctgcagctcgcccggccgccggtccccctgcc gtccggcccgtcccagaggca gcgtcggcggctcctgcctcccccgcccggcgccgaccgggacecgcaaaccccttgatc cgctgtcgggggtgatcactacg gtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggacca ggcgttagcggcagtgctggc cgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgc cgtggcggtcggcatggcgccc gccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtc cgccggttccgccggcatcttgc ccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcg acggccgccacctcatcgcgg cagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcac cgcgacggcgggggtacgge gctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctc cccgctgccctgtcggcgage acaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgc ccccgtcgtgtgcgcgggccg gtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggc cgcgcggcccgctcgcgg gccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcga tcct (SEQ ID NO : 145) Figure 19 provides a map of pSEGT-phdA4, while the sequence is provided below : ctagagatcgaacttcatgttcgagttcttgttcacgtagaagccggagatgtgagaggt gatctggaactgctcaccctcgttggt ggtgacctggaggtaaagcaagtgacccttctggcggaggtggtaaggaacggggttcca cggggagagagagatggccttg acggtcttgggaaggggagcttcngcgcgggggaggatggtcttgagagagggggagcta gtaatgtcgtacttggacaggga gtgctccttctccgacgcatcagccacctcagcggagatggcatcgtgcagagacagacc cccggaggtaaccatggccaagc gaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcac ccaccgagcggttcgcccccgacgt gcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggaggg actgagcgcgcgcaccaccaa catcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgc gacgcacctgccgctcgacctgg tgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgaca tcgcgctgggcatgtcggtgctcgt cacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgct ggtggtctcgccgccaccgetg gcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccact gagctcgcccgcgtgtacagcgc gctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacgg cgtcgacggaatccacttcaccgag gccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacaa tggggatccgcgagcggatcgg

Two colonies of S. lividans TK-23 pSECA4-phd were inoculated in 10 ml of TS medium + 50 ppm thiostrepton and incubated at 37°C with shaking at 200 rpm for 2 days.

Three mIs of broth were used to inoculate 50 ml of Streptomyces Production medium 1 and the culture was incubated for 4 days at 37°C with shaking at 200 rpm.

A sample was taken to assay perhydrolase activity measurement as follows: 10 Ills of 20 mg/ml lysozyme were added to 200 p1 of sample. After 1 hour of incubation at 37°C, samples were centrifuged and activity was measured using the pNB activity assay described herein. SDS-PAGE and Western blots were also prepared using both clones (pSECA4-phd and pSECGT-MSAT), as known in the art. Briefly, after SDS-PAGE, the proteins were transferred to PVDF membrane and Western blot analysis was conducted.

The perhydrolase was detected using an anti-perhydrolase polyclonal anti-sera (1: 500 dilution) prepared against purified perhydrolase protein by Covance. The blot was developed using the ECL kit from Amersham. The results indicated that Streptomyces lividans strains were capable of expressing active perhydrolase.

EXAMPLE 8 Site-Scanning Mutagenesis of the M. smegmatis Perhydrolase Gene In this Example, experiments involving site-scanning mutagenesis of the M. smegmatis perhydrolase gene are described. In these experiments, the QuikChange@ site- directed mutagenesis (QC; Stratagene) kit or the QuikChange Multi Site-Directed mutagenesis (QCMS; Stratagene) kit was used to create site-saturation libraries at each codon in the entire M. smegmatis perhydrolase gene contained in the pMSAT-NcoI plasmid. Each perhydrolase codon was mutagenized by replacement with the NNG/C (NNS; 32 combinations) degenerate codon, which encodes for all 20 amino acids and one stop codon. In the case of the QC method, complementary overlapping primers were designed for each codon of interest with 18 bases flanking the NNS codon (See, Tables 8- 1 and 8-2). A comparison of cartridge purified versus purified primers (desalted only) revealed a better representation of amino acids in the libraries made with purified primers (15-19 amino acids versus 11-16 with unpurified primers). Thus, a majority of the libraries were created with the QC method and purified primers. A small number of the libraries were made using the QCMS method and a single 5'phosphorylated forward primer containing 18 bases flanking both sides of the NNS codon (See, Table 8-1), however this method resulted in a greater wild type background and fewer amino acid substitutions per site compared to the QC methods. Libraries"nsa301"and"nsa302" were made using the QCMS method, but a trinucleotide mix made up of a single codon for each of the 20 amino acids (i. e. , rather than 32 possibilities encoded by NNS for the 20 amino acids) was incorporated within the primers at the sites of interest.

Table 8-1. Site-Saturation Forward Primers Residue Primer Primer Sequence M1 nsa202F taaca a aattaaccnns ccaa cgaattct t (SEOID NO : 147) sa203F caggaggaattaaccatgnnsaagcgaattct ttc (SEO ID NO : 148) Table 8-2 Site-Saturation Reverse Primer Sequences Residue Primer Primer Sequence ACACAGAATTCGCTTGGCSNNGGTTAATTCCTCCTGTTA mi nsa202R SE ID NO : 363) GAAACACAGAATTCGCTTSNNCATGGTTAATTCCTCCTG A2 nsa203R SEO ID NO : 364) ACCGAAACACAGAATTCGSNNGGCCATGGTTAATTCCTC K3 nsa204R (SEO ID NO : 365) ATCACCGAAACACAGAATSNNCTTGGCCATGGTTAATTC M nsa205R (SEO ID NO : 366) GGAATCACCGAAACACAGSNNTCGCTTGGCCATGGTTAA I5 nsa206R (SEO ID NO : 367) CAGGGAATCACCGAAACASNNAATTCGCTTGGCCATGGT L6 nsa207R SEO ID NO : 368) GTCAGGGAATCACCGAASNNCAGAATTCGCTTGGCCAT C7 nsa208R SEO ID NO : 369) CCAGGTCAGGGAATCACCSNNACACAGAATTCGCTTGGC sa209R (SEO ID N0 : 370)

QC Method to Create Site-Saturation Libraries The QC reaction consisted of 40. 25 uL of sterile distilled H2O, 5 I1L of PfuTurbo lOx buffer from the kit, luL dNTPs from the kit, 1. 25 gel of forward primer (100ng/µL), 1.25 µL reverse primer (100ng/µL), 0. 25 gL of pMSAT-NcoI miniprep DNA as template (-50ng), and 1 uL of PfuTurbo from the kit, for a total of 50 tir. The cycling conditions were 95°C for Imin, once, followed by 19-20 cycles of 95°C for 30 to 45 sec, 55°C for lmin, and 68°C for 5 to 8 min. To analyze the reaction, 5uL of the reaction was run on a 0.8% E-gel (Invitrogen) upon completion. Next, DpnI digestion was carried out twice sequentially, with 1 IL and 0. 5 ptL of enzyme at 37°C for 2 to 8 hours. A negative control was carried out under similar conditions, but without any primers. Then, 1 gel of the DpnI-digested reaction product was transformed into 50 µL of one-shot TOP10 electrocompetent cells (Invitrogen) using a BioRad electroporator. Then, 300 uL of SOC provided with the TOP 10 cells (Invitrogen) were added to the electroporated cells and incubated with shaking for 1 hour before plating on LA plates containing l Oppm kanamycin. The plates were incubated at 37°C overnight. After this incubation, 96

colonies from each of the libraries (i.e., each site) were inoculated in 200pL of LB containing 10-50ppm of kanamycin in 96-well microtiter plates. The plates were frozen at-80°C after addition of glycerol to 20% final concentration, and they were used for high throughput sequencing at Genaissance with the M13F and M13R primers.

QCMS Method to Create Site-Saturation Libraries The QCMS reaction consisted of 19.25 µl of sterile distilled HiO, 2.5 I1L of 10x buffer from the kit, 1 pL dNTPs from the kit, l llL of 5'phosphorylated forward primer (lOOng/pL), 0.25 µL of pMSAT-NcoI miniprep DNA as template (#50ng), and luL of the enzyme blend from the kit for a total of 25 I1L. The cycling conditions were 95°C for Imin once, followed by 30 cycles of 95°C for Imin, 55*C for lmin, and 68°C for 8 min.

To analyze the reaction product, 5pL of the reaction were run on a 0.8% E-gel (Invitrogen) upon completion. Next, DpnI digestion was carried out twice sequentially, with 0.5 uL of enzyme at 37°C for 2 to 8 hours. The controls, transformation, and sequencing was performed as for the QC method described above.

Details of Screening Plate Preparation Using a sterilized stamping tool with 96 pins, the frozen clones from each sequenced library plate were stamped on to a large LA plate containing l Oppm kanamycin. The plate was then incubated overnight at 37°C. Individual mutant clones each representing each one of the 19 substitutions (or as many that were obtained) were inoculated into a Costar 96-well plate containing 195pL of LB made with 2 fold greater yeast extract and l Oppm kanamycin. Each mutant clone for a given site was inoculated in quadruplicate. The plate was grown at 37°C and 225 rpm shaking for 18 hrs in a humidified chamber. In a separate 96-well plate, 26, L of BugBuster (Novagen) with DNase were added to each well. Next, 125, L of the library clone cultures were added to the BugBuster-containing plate in corresponding wells and the plate was frozen at-80°C.

The plate was thawed, frozen and thawed again before use of the lysates in the peracid formation and peracid hydrolysis assays described herein.

Combinatorial Libraries and Mutants From the screening of the single site-saturation libraries, the important sites and substitutions were identified and combined in different combinatorial libraries. For example, libraries described in Table 8-3 were created using the following sites and substitutions: L12C, Q, G T25S, G, P L53H, Q, G, S S54V, L, A, P, T, R A55G, T R67T, Q, N, G, E, L, F K97R V125S, G, R, A, P F154Y F196G

TABLE 8-3. Libraries Library Description Parent Method Template NSAA1 L12G S54 (NNS) L12G QC NSAA2 S54V L12 (NNS) S54V QC NSAA3 L12 (NNS) S54 (NNS) WT QCMS NSAB1 S54V T25 (NNS) S54V QC NSAB2 S54V R67 (NNS) S54V QC NSAB3 S54V V125 (NNS) S54V QC NSAB4 L12I S54V T25 (NNS) L12I S54V QC NSAB5 L12I S54V R67 (NNS) L12I S54V QC NSAB6 L12I S54V V125 (NNS) L12I S54V QC NSAC1 S54 (NNS) R67 (NNS) WT QCMS V125 (NNS) NSAC2 43 primer library, 10 sites S54V QCMS (lOOng total primers) NSAC3 same as nsaC2 but 300ng S54V QCMS total primers NSAC4 32 primer library, 8 sites S54V QCMS (lQOng total primers) NSAC5 same as nsaC4 but 300ng S54V QCMS total primers NSAC6 8 primers, 7 substitutions, S54V QCMS 5 sites (100ng total primers) NSAC7 same as nsaC6 but 300ng S54V QCMS total primers *NNS indicates site-saturation library **All parent templates were derived from the pMSAT-NcoI plasmid and contained mutations at the indicated codons with in the M. smegmatis perhydrolase gene The QC or QCMS methods were used to create the combinations. The QC reaction was carried out as described above, with the exception being the template plasmid, which consisted of 0.25µL of miniprep DNA of the L12G mutant, S54V mutant, or the L12I S54V double mutant plasmid derived from pMSAT-NcoI. The QCMS

reaction was also carried out as described above, with the exception of template and primers. In this case, 0. 25gel of the pMSAT-Ncol template were used for NSAC1 and NSAA3 or S54V template for NSAC2-C7 libraries. The NSAA3 and the NSAC1 libraries were made using 100 ng of each of the primers shown in the Table 8-4. The NSAC2, NSAC4, and NSAC6 libraries were made with a total of 100ng of all primers (all primers being equimolar), and NSAC3, NSAC5, NSAC7 libraries were made with a total of 300ng of all primers (all primers being approximately equimolar) Table 8-4. Libraries Primer 'r i rin ; _e_quence NSAC1_ i5 N S-FP-72 SACI 54NNS-FP atc a a ct c c caccaccaacatc SE m N0 : 579 SAC1 67NNS-FP acgaccccaccgatcc ctcaacggc c a cta S I1380) C 12 NNS-FP ctcacca c c c cnns cac ac ccc a SE N : S 1 NSAC2-C5 L12C ct c attccTGCacct c cccc SE NOS 2 NSAC2-C7 L12 ct c attccCAGacct ct cccc SE m NO 83 NSAC2-C5 L12I ct c attccATCacct ct cccc (SEO ID N0 : 584) NSAC2-C3 L 2M ct c attccATGacct ct cccc SE ID NO : 585) NSAC2-C3 L12T ct c attccACGacct ct cccc (SEO ID NO : 586) NSAC2-C5 T25SgtcgaagacsggscacccAGCsagcggttcgcccccgac fSEOID N0 : 587) NSAC2-C5 r25G gtogaagacggggcacccGGCzaRcrgttcRccocczgac (SEOIDNO : 588) NSAC2-C3 T25PgtcsaagacgssscacccCCGsagcggttcscccccgac f SEO ID N0 : 589) NSAC2-C'L53H gaggtRategaggagRgaCACagogogcgeaccaccaac fSEOIDNO : 590) SAC2-C3 L53 a atc a a aCAGa c c c caccaccaac SE m N0 : 591) NSAC2-C3 L53GgagstgatcgassagEeaGGCascecgcgcaccaccaac (SEO ID N0 : 592) NSAC2-C'L53S gaggtgatczaggagggaAGCagcgcgogeaccaccaac (SEO ID NO : 593) NSAC2-C'L53HS54V gaggtgatogagRagggaCACGTGgcgcRcaccaccaac (SEO ID NO : 594) NSAC2-C3 L530S54V gagggaCAGGTGgcgcgcaccaccaac (SEQ ID NO : 595 NSAC2-C3 L53GS54V ga atcgaggagggaGGCGTGgcgcgcaccaccaac SEOm N0 : 596) NSAC2-C3 L53SS54V gaggtgatcgagga aAGCGTGgcgcgcaccaccaac (SEO ID N0 : 597 NSAC2-C7 S54V gtgatzgagzagrgactgGTGgcWgogoaccaccaacatc (SEO ID NO : 598 NSAC2-C5 S54L gtgatzzaggagggactgCTGgcgxcaccaccaacatc fSEO ID NO : 599) NSAC2-C5 A55G atcgaggagggactgagcGGCcgcaccaccaacatc ac (SEO ID N0 : 600)

Screening of Combinatorial Libraries and Mutants For each of the NSAB 1-B6 libraries, a 96-well plate full of clones was first sequenced. Once the sequencing results were analyzed, the mutants obtained for each library were inoculated in quadruplicate, similar to the site-saturation libraries described above. For the NSAC1-C7 libraries, 96 colonies per/plate/library were initially inoculated, and each plate was screened without sequencing. Upon screening, some libraries looked better than others. Several plates for each of the NSAC1, C2, C4, C6 libraries were screened. The"winners"from these single isolate screening plates were

then streaked out for singles or directly screened in quadruplicate just like the site- saturation libraries (i. e. , as described above). Only the"winners"identified were sequenced.

EXAMPLE 9 Improved Properties of Multiply Mutated Perhydrolase Variants In this Example, experiments conducted to assess the properties of multiply- mutated perhydrolase variants are described. In these experiments, combinatorial mutants obtained from combinatorial libraries were tested in their performance in perhydrolysis, peracid hydrolysis and perhydrolysis to hydrolysis ratio. These parameters were measured in the HPLC or ABTS assays described in Example 2, above. Combinatorial variants tested were: L12I S54V, L12M S54T, L12T S54V, L12Q T25S S54V, L53H S54V, S54P V125R, <BR> <BR> <BR> <BR> S54V V125G,<BR> <BR> <BR> <BR> <BR> S54V F196G, S54V K97R V125G, and A55G R67T K97R V125G, As is indicated in Table 9-1 below, all of these variants were better than wild type enzyme in at least one of the properties of interest.

Table 9-1 Results for Multiple Variants Multiple Variant ! Fold-Improvement in Property Perhydrolysis Peracid Hydrolysis Ratio L12I S54V 2 2. 5 L12M S54T 1. 6 3 L12T S54V 1. 5 2. 5 L120 T25S S54V 4 to 5 L53H S54V 2 4 to 5 S54P V125R 4 S54V V125G 2 4 S54V F196G 2 S54V K97R V125G 2 A55G R67T K97R 1.6 4 to 5 V125G

EXAMPLE 10 PAF and PAD Assays of Perhydrolase Variants In this Example, assay results for PAF and PAD testing of perhydrolase variants are provided. The tests were conducted as described in Example 1, above. In addition, Tables are provided in which the protein expression of the variant was greater than wild- type under the same culture conditions (described herein). These results are indicated as the"protein performance index. "Thus, a number greater than"1"in the protein performance index indicates that more protein was made for the particular variant than the wild-type. In the following Tables, "WT"indicates the wild-type amino acid residue; "Pos"indicates the position in the amino acid sequence;"Mut."and"Var"indicate the amino acid residue substituted at that particular position ;"prot."indicates"protein ; and "Perf. Ind"indicates the performance index. Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results PAF PAF Position..,. Vanant Perf. Positton.,.., Variant Pert. slutatlon Mutatio Ind. Ind. 3 K003Y Y 1. 058244 17 V017R R 1. 09735 3 K003I I 1. 053242 17 V017A A 1. 012116 3 K003L L 1. 038686 18 P018Y Y 1. 332844 3 K003T T 1. 009071 18 P018N N 1. 331062 3 K003H H 1. 00528 18 P018C C 1. 261104 4 R0040 0 1. 025332 18 P018E E 1. 217708 5 IOOST T 1. 12089 18 P018V V 1. 185736 5 IOOSS S 1. 023576 18 P018R R 1. 16328 6 L006V V 1. 072388 18 P0180 0 1. 124133 6 L006I 1 1. 066182 18 P018H H 1. 120443 6 L006T T 1. 062078 18 P018G G 1. 068272 7 C007K K 2. 687956 19 V019G G 1. 317001 7 C007Y 2. 08507 19 V019S S 1. 235759 7 C007I 1 1. 758096 19 V019R R 1. 025471 7 C007H H 1. 731475 19 V019L L 1. 002833 7 C007A A 1. 423943 21 D021K K 1. 062138 7 C007G G 1. 393781 21 D021W W 1. 040173 7 C007M M 1. 126028 22 G022A A 1. 554264 10 D010L L 3. 97014 22 G022T T 1. 032118 10 DOlOW W 3. 179778 22 G022S S 1. 022133 10 DOIOK K 2. 133852 25 T025G G 1. 857878 10 DOILY Y 1. 508981 25 T025S S 1. 59954 10 D010T T 1. 473387 25 T025A A 1. 327579 10 DOlOI 1 1. 281927 25 T025I 1 1. 019417 12 L0120 0 2. 651732 26 E026M M 2. 002044 12 L012C C 2. 289224 26 E026A A 1. 927 ;) 99 12 L012A A 1. 100171 26 E026R R 1. 484814 15 G015A A 1. 543799 26 E026K K 1. 464368 15 G015S S 1. 05273 26 E026T T 1. 441939 17 V017G G 1. 173641 26 E026C C 1. 403045 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results WT/Pos7 WT/Pos/ Position Variant Perf. Position Variant Perf. Ind. Ind. ind. Ind. 26 E026V V 1. 392881 30 P030E E 1. 006761 26 E026N N 1. 366419 31 D031 W 1. 834044 26 E026H H 1. 329562 31 D031L L 1. 81056 26 E026L L 1. 295378 31 D031T T 1. 450556 26 E026G G 1. 283477 31 D031G G 1. 441703 26 E026S S 127140 31 D031F F 143826E 26 E026W W 1. 251752 31 D031N N 1. 339422 27 R027K K 1. 215697 31 D031V V 1. 280091 28 F028M M 1. 331874 31 D031A A 1. 240923 28 F028A A 1. 269493 31 D031R R 1. 222181 28 F028W W 1. 156698 31 DO 1 S 1. 152736 28 F028L L 1. 08849 31 D031E E 1. 132795 28 F028S S 1. 046063 31 D0310 O 1o06979} 29 A029W W 1. 912244 32 V032K K 1. 08606 29 A029V V 1. 799733 32 V032R R 1. 045435 29 A029R R 1. 757225 33 R033S S 1. 000491 29 A029Y Y 1. 697554 36 G0361 1 29 A029G G 1. 595061 36 G036K K 1. 265563 29 A029S S 1. 486877 36 G036L L 1. 237473 29 A029T T 1. 424584 38 L038L L 6. 528092 29 A029E E 1. 115768 38 L038V V 5. 735873 29 A029C C 1. 07522 38 L038C C 4. 182031 30 P030K K 1. 207673 38 L038K K 4. 135067 30 P030R R 1164892 38 L038A A 3. 844719 30 P030V V 1. 063047 38 L038S S 2. 467764 30 P030T T 1. 05383 40 0040K K 2. 613726 30 P030A A 1. 045476 40 00401 1 2. 576806 30 P030S S 1. 031747 40 0040W W 2. 394926 30 P0300 1. 013468 40 0040L L 2. 144687 30 P030H H 1. 012332 40 0040T T 2sO0648 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results PAF PAF Position.,,,. Variant Perf. Position-,,,. Variant Perf. utatio d, utatio Ind. Ind. 40 0040R R 1. 885154 43 G043R R 1. 215829 40 0040Y Y 1. 825366 43 G043S S 1. 178103 40 0040G G 1. 785768 43 G043H H 1. 169457 40 0040S S 1. 565973 43 G043P P 1. 080176 40 0040N N 1. 528677 44 A044F F 2. 84399 40 0040D D 1. 16151 44 A044V V 2. 133682 40 0040E E 1. 075259 44 A044C C 1. 796096 41 0041K K 1. 381385 44 A044L L 1. 607918 41 0041R R 1. 190317 44 A044W W 1. 395243 41 0041W W 1. 141041 44 A044M M 1. 199028 41 0041H H 1. 123719 45 D045K K 1. 342858 41 0041S S 1. 107641 45 D045T T 1. 268367 41 0041Y Y 1. 091652 45 D045R R 1. 158768 41 0041V V 1. 070265 45 D045W W 1. 145157 41 0041A A 1. 032945 45 D045S S 1. 133098 41 0041L L 1. 000416 45 D045G G 1. 12761 42 L042K K 2. 463086 45 D045H H 1. 127539 42 L042W W 2. 056507 45 D045F F 1. 11152 42 L042H H 1. 917245 45 D045L L 1. 054441 42 L042R R 1. 378137 45 D045V V 1. 050576 42 L042G G 1. 172748 45 D0450 0 1. 04498 42 L042T T 1. 079826 45 D045A A 1. 037993 42 L042F F 1. 072948 46 F046E E 1. 247552 43 G043A A 1. 49082 46 F046D D 1. 174794 43 G043C C 1. 47701 46 F046G G 1. 016913 43 G043K K 1. 424919 46 F046K K 1. 003326 43 G043M M 1. 371202 47 E047R R 2. 448525 43 G043Y Y 1. 262703 47 E047T T 1. 960505 43 G043E E 1. 250311 47 E047P P 1. 361173 43 G043L L 1. 216516 47 E047S S 1. 278809 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results WT/Pos/P) A-F Vff/Pos/PAF Position.,,.. Variant Perf. Position.,... Variant Perf. Ind. Ind. Ind. Ind. 47 E047H H 1. 266229 54 S054I I 4. 775938 47 E047G G 1. 197541 54 S054V V 4. 722033 47 E047K K 1. 19183 54 S054A A 3. 455902 47 E047F F 1. 092281 54 S054R R 3. 375793 47 E047I I 1. 030029 54 S054L L 2. 015828 49 1049G G 1. 342918 54 S054T T 1. 459971 49 I049H H 1. 265204 54 S054K K 1. 438715 49 I049S S 1. 238211 54 S054G G 1. 429605 49 I049K K 1. 230871 54 S054C C 1. 259773 49 I049V V 1. 203314 54 S0540 0 1. 03365 49 I049L L 1. 136805 55 A055G G 1. 694814 49 I049Y Y 1. 068104 55 A055T T 1. 692885 49 I049R R 1. 052285 57 T057S S 1. 633613 49 I049E E 1. 015762 57 T057R R 1. 605072 49 I049M M 1. 00526 57 T057V V 1. 281788 50 E050L L 1. 191901 57 T057I I 1. 189062 50 E050M M 1. 178039 59 N059W W 1. 035044 50 E050A A 1. 124087 59 N059R R 1. 002315 51 E051V V 1. 471315 60 I060H H 1. 02415 51 E051 A A 1. 279983 60 I060R R 1. 003947 51 E051G G 1. 217963 61 D061H H 1. 439407 51 E051 T T 1. 182792 61 D061 S S 1. 259714 51 E051L L 1. 112889 61 D061R R 1. 105425 51 E051I 1 1. 072835 61 D061I I 1. 076937 53 L053H H 5. 05321 61 D061F F 1. 00566 53 L0530 1. 480206 62 D062E E 1. 019293 53 L053G G 1. 317357 63 P063G G 1. 709657 53 L053S S 1. 161011 63 P063T T 1. 499483 53 L053T T 1. 019146 63 P063M M 1. 460336 54 S054P P 5o198689 63 P063S S 1a416192 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results WT/Pos/ WT/Pos/PAF Position Variant Perf. Position Variant Perf. Ind. Ind. 99 63 P063K K 1. 404615 67 R0670 0 1. 164899 63 P063A A 1. 347541 67 R067W W 1. 066028 63 P063Y Y 1. 346046 67 R067E E 1. 044676 63 P063W W 1. 34587 67 R067P P 1. 012761 63 P063V V 1. 313631 68 L068E E 1. 435218 63 P063R R 1. 310696 68 L068W W 1. 209193 63 P063F F 1. 246299 68 L068I 1 1. 125898 63 P063L L 1. 146416 68 L068G G 1. 092454 63 P0630 0 1. 093179 68 L068V V 1. 088042 64 T064G G 1. 234467 68 L068H H 1. 051612 64 T064S S 1. 114348 68 L068T T 1. 032331 65 D065A A 1. 312312 69 N069V V 1. 989028 65 D065S S 1. 166849 69 N069K K 1. 71908 65 D065H H 1. 096335 69 N069R R 1. 493163 66 P066R R 1. 846257 69 N0691 1 1. 469946 66 P066V V 1. 828926 69 N069H H 1. 357968 66 P066H H 1. 589631 69 N069T T 1. 351305 66 P066I I 1. 588219 69 N069L L 1. 299547 66 P066G G 1. 499901 69 N069S S 1. 205171 66 P0660 0 1. 463705 69 N069G G 1. 19653 66 P066T T 1. 410091 69 N0690 1. 074622 66 P066S S 1. 390845 69 N069W W 1. 049602 66 P066Y Y 1. 330684 69 N069C C 1. 048373 66 P066L L 1. 137635 71 A071S S 1. 751794 66 P066N N 1. 122261 71 A071T T 1. 700442 67 R067N N 1. 580401 71 A071H H 1. 697558 67 R067G G 1. 390129 71 A071G G 1. 58881 67 R067T T 1. 284643 71 A071I 1 1. 507841 67 R067F F 1. 25763 71 A071E E 1. 445699 67 R067L L 1. 203316 71 A071K K 1. 441146 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results WT/Pos/PAF //Pos/! P Position Variant Perf. Position Variant Perf. Mutation,. Mutation,. Ind. Ind. 71 A071R R 1. 401499 79 A079I I 1. 592463 71 A071N N 1. 232241 79 A079M M 1. 49963 71 A071L L 1. 231991 79 A079N N 1. 475806 71 A071F F 1. 12753E 79 A0790 O 1. 47248i 71 A071C c. 1. 00977 79 A079R R 1. 465943 72 S072L L 1. 257945 79 A079W 1. 270538 72 S072H H 1. 208899 79 A079T T 1. 169146 72 S072G G 1. 198197 79 A079E E 1. 123457 72 S072T T 1. 10065 80 T080C C 1. 310752 72 S072V V 1. 080089 80 T080V V 1. 230659 72 S072Y Y 1. 066178 80 T080G G 1. 16031 73 Y073R R 1. 2555 80 T080A A 1. 000722 73 Y0730 1. 23429 82 L082P P 1. 456374 73 Y073S S 1. 165683 82 L082G G 1. 379439 73 Y073K K 1. 070678 82 L082R R 1. 339485 76 S076P P 1. 229172 82 L082H H 1. 332844 77 C077T T 1. 120603 82 L082K K 1. 1909 77 C077V V 1. 052586 82 L082T T 1. 17992 77 C077G G 1. 013806 82 L082I I 1. 171013 78 L078G G 4. 975852 82 L082S S 1. 153417 78 L078H H 4. 824004 82 L082V V 1. 019854 78 L078E E 3. 007159 83 P083K K 1. 369406 78 L078N N 2. 683604 83 P083G G 1. 313431 78 L078T T 1. 867711 83 P083H H 1. 265876 78 L0780 0 1. 726942 83 P083R R 1. 194464 78 L078V V 1. 534239 83 P083S S 1. 171208 78 L078I 1 1. 434206 84 L084K K 1. 099089 78 L078Y Y 1. 387889 84 L084H H 1. 008187 79 A079H H 1. 927914 85 D085 0 3. 093245 79 A079L L 1. 796126 85 D085R R 2. 379647 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results WT/Pos/PAF WT/Pos/PAF WT/Po< :/ WT/Pos/ Position.,,.."Variant Perf. Position.,... Variant Perf. Ind. Ind. Ind. Ind. 85 D085S S 2. 284009 104 P104C C 1. 951282 85 D085H H 1. 548556 104 P104E E 1. 837373 85 D085N N 1. 539497 104 P104F F 1. 785718 85 D085G G 1. 413812 104 P104N N 1. 624722 85 D085T T 1. 329395 104 P104R R 1. 618032 85 D085E E 1. 117228 104 P1040 0 1. 343174 85 D085F F 1. 008028 104 P104M M 1. 093185 86 L086A A 1. 376284 105 L105P P 1. 713219 86 L086C C 1. 156625 105 L105C C 1, 557999 86 L086G G 1. 145834 105 L105F F 1. 29575 95 D095E E 2. 044825 105 L105W W 1. 283998 96 T096S S 1. 044425 105 L105G G 1. 078743 97 K097R R 2. 798748 106 D106K K 1. 278457 97 K0970 1. 136975 106 D106L L 1. 198148 100 F100W W 1. 082799 106 D106G G 1. 178297 100 F100E E 1. 0116 106 D106H H 1. 090134 101 RlOlK K 1. 244945 106 D106E E 1. 084931 103 T103W W 1. 261503 106 D 106T T 1. 061622 103 T103Y Y 1. 193299 106 D106I 1 1. 036191 103 T103G G 1. 113343 106 D106F F 1. 021513 103 T103K K 1. 093573 106 D106C C 1. 005553 103 T103I 1 1. 076338 107 I107E E 2. 551108 103 T103L L 1. 050734 107 I107S S 2. 044692 104 P104H H 2. 837034 107 I107N N 1. 810584 104 P 104T T 2. 696977 107 I107G G 1. 764761 104 P104G G 2. 672719 107 I107V V 1. 001703 104 P104V V 2. 585315 108 A108L L 1. 407382 104 P104S S 2. 481687 108 A108T T 1. 050964 104 P104I 1 2. 431309 109 L109N N 1. 523277 104 P 104W W 2. 05178 z 109 L109W W 1. 296964 --| Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results WT/Pos/PAF WT/POS/PAF Position Mut°S Variant Perf. Position MTios Variant Perf. Ind. i Ind. Ind. Ind. 109 L109 1. 182653 117 117T T 2. 233854 109 L109Y Y 1. 155328 117 0117Y Y 2. 227983 109 L109I 1 1. 053129 117 0117W W 2. 155359 109 L109D D 1. 003394 117 0117V V 2. 154646 111 Ml l lK K 1. 977248 117 117G G 2. 080223 ill iiii I'-.. L949343 117 0117A A 2. 048752 111 M111L L 1. 546317 117 0117S S 1. 949232 111 Ml l 1T T 1. 48980E 117 0117F F 1. 573776 111 Mil IF F 1. 467344 117 0117R R 1. 564466 111 Mil in V 1. 466478 117 0117M M 1. 541944 111 M111Y Y 1. 42589 117 0117E E 1. 145341 111 Mills S 1. 031939 118 V118Y Y 1. 25067 112 S112L L 1. 027928 118 V118K K 1. 125917 112 S112H H 1. 001485 118 VI 18G G 1. 083422 113 V113L L 1. 503622 120 T120S S 1. 089798 113 V113H H 1. 339003 121 S121L L 1. 348931 113 V113K K 1. 192607 121 S121W W 1. 333741 113 V113R R 1. 133751 121 S121R R 1. 25879 113 V113Y Y 1. 113256 121 S121K K 1. 241105 113 V113F F 1. 045057 121 S121G G 1. 204547 113 V 113 1. 032496 121 S121C C 1. 177769 115 V115W W 1. 234 121 S121N N 1. 143954 115 V115T T 1. 145757 121 S121T T 1. 132507 115 V115L L 1. 117398 121 S121A A 1. 120633 115 V115G G 1. 089596 121 S121V V 1. 120454 115 V115I I 1. 050387 122 A122H H 1. 137861 115 V115Y Y 1. 032052 122 A122I 1 1. 133601 116 T116G G 1. 095496 122 A122T T 1. 083131 116 T116A A 1. 006702 122 A122K K 1. 082552 117 0117H H 2. 327857 122 A122V V 1. 041449 Table 10-1. PAF Assay Results Table 10-1. PAF Assay Results PAF PAF WT/Pn</WT/Pn</ Position.,. 'Variant Perf. Position.,..."Variant Perf. Ind. Ind. inu. inu. 122 A122S S 1. 031411 148 P148L L 1. 638438 124 G124L L 1. 91642 148 P148A A 1. 637334 124 G124I 1 1. 853337 148 P148R R 1. 509086 124 G124T T 1. 63716 148 P148T T 1. 501359 124 G124H H 1. 588068 148 P148Y Y 1. 459512 124 G124V V 1. 441979 148 P148S S 1. 45564 124 G124F F 1. 320782 148 P148E E 1. 417449 124 G124S S 1. 269245 148 P148F F 1. 367568 124 G124Y Y 1. 234423 148 P14 1. 334517 124 G124R R 1. 144212 148 P148D D 1. 030185 124 G1240 0 1. 123498 150 F150L L 1. 290835 125 V125G G 2. 948291 150 F150E E 1. 228159 125 V125S S 1. 942881 153 I153K K 1. 618543 125 V125A A 1. 689696 153 I153H H 1. 464262 125 V125P P 1. 50166 153 I153T T 1. 271928 125 V125R R 1. 301534 153 I153L L 1. 270149 125 V125D D 1. 238852 153 I153F F 1. 227821 125 V125Y Y 1. 080394 153 I153A A 1. 194659 125 V125I 1 1. 010779 154 F154Y Y 1. 323693 126 G126T T 1. 577938 196 F196H H 1. 774774 126 G126P P 1. 171092 196 F196L L 1. 768072 126 G126L L 1. 169527 196 F196C C 1. 738263 127 T127H H 1. 57251 196 F196M M 1. 647608 127 T127V V 1. 073821 196 F196G G 1. 590716 127 T127I 1 1. 063668 196 F196S S 1. 577837 127 T127S S 1. 046984 196 F196Y Y 1. 414589 128 T128L L 1. 064623 196 F196V V 1. 395387 128 T128K K 1. 062947 196 F196I 1 1. 320955 148 P148V V 2. 426937 196 F196W W 1. 014435 148 P148K K 1. 786508

The following Table provides variants with PAF results that were better than those observed for wild-type M. smegmatis perhydrolase. In this Table, the middle column indicates the amino acid residue in the wild-type perhydrolase (WT), followed by the position number and the variant amino acid in that position (Var). Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var-WT Pos Var WT-- 2A002W 1. 75 8F008G 1. 09 2A002D 1. 30 8F008H 1. 02 2A002F 1. 24 10DO10L 3. 97 2A002I 1. 18 10DOlOW 3. 18 2A002G 1. 15 lODOlOK 2. 13 2A002S 1. 01 10DO10Y 1. 51 3K003Y 1. 06 lODOlOT 1. 47 3K003I 1. 05 10D010I 1. 28 3K003L 1. 04 12L012Q 2. 65 3K003T 1. 01 12L012C 2. 29 3K003H 1. 01 12L012A 1. 10 4R004Q 1. 03 15GO15A 1. 54 5I005T 1. 12 15GO15S 1. 05 5I005S 1. 02 17V017G 1. 17 6L006V 1. 07 17V017R 1. 10 6L006I 1. 07 17V017A 1. 01 6L006T 1. 06 18P018Y 1. 33 7C007K 2. 69 18P018N 1. 33 7C007Y 2. 09 18P018C 1. 26 7C007I 1. 76 18P018E 1. 22 7C007H 1. 73 18P018V 1. 19 7C007A 1. 42 18P018R 1. 16 7C007G 1. 39 18P018Q 1. 12 7C007M 1. 13 18P018H 1. 12 8F008R 1. 43 18PEG 1. 07 8F008V 1. 18 19V019G 1. 32 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 19V019S 1. 24 26E026K 1. 46 19V019R 1. 03 26E026T 1. 44 19VO19L 1. 00 26E026C 1. 40 20E020W 2. 94 26E026V 1. 39 20E020G 2. 36 26E026N 1. 37 20E020T 2. 22 26E026H 1. 33 20E020L 2. 20 26E026L 1. 30 20E020H 2. 17 26E026G 1. 28 20E020V 2. 11 26E026S 1. 27 20E020S 2. 01 26E026W 1. 25 20E020C 1. 57 27R027K 1. 22 20E020N 1. 40 28F028M 1. 33 20E020A 1. 29 28F028A 1. 27 20E020Q 1. 27 28F028W 1. 16 21D021K 1. 58 28F028L 1. 09 21D021W 1. 55 28F028S 1. 05 21D021L 1. 46 29A029W 1. 91 21D021A 1. 46 29A029V 1. 80 21D021G 1. 37 29A029R 1. 76 21D021Y 1. 30 29A029Y 1. 70 21D021F 1. 30 29A029G 1. 60 21D021S 1. 24 29A029S 1. 49 22G022A 1. 55 29A029T 1. 42 22G022T 1. 03 29A029E 1. 12 22G022S 1. 02 29A029C 1. 08 25T025G 1. 86 30P030K 1. 21 25T025S 1. 60 30P030R 1. 16 25T025A 1. 33 30P030V 1. 06 25T025I 1. 02 30P030T 1. 05 26E026M 2. 00 30P030A 1. 05 26E026A 1. 93 30P030S 1. 03 26E026R 1. 48 30P030Q 1. 01 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 30P030H 1. 01 39A039W 1. 23 30P030E 1. 01 39A039V 1. 21 31D031W 1. 83 39A039G 1. 17 31D031L 1. 81 39A039R 1. 17 31D031T 1. 45 39A039E 1. 09 31D031G 1. 44 40Q040K 2. 61 31 D03 IF 1. 44 40Q040I 2. 58 31D031N 1. 34 40Q040W 2. 39 31D031V 1. 28. 40Q040L 2. 14 31DNA 1. 24 40Q040T 2. 01 31D031R 1. 22 40Q040R 1. 89 31D031S 1. 15 40Q040Y 1. 83 31DO31E 1. 13 40Q040G 1. 79 31D031Q 1. 07 40Q040S 1. 57 32V032K 1. 09 40Q040N 1. 53 32V032R 1. 05 40Q040D 1. 16 33R033S 1. 00 40Q040E 1. 08 36G036I 1. 32 41Q041K 1. 38 36G036K 1. 27 41Q041R 1. 19 36G036L 1. 24 41Q041W 1. 14 37V037S 1. 40 41Q041H 1. 12 37V037I 1. 26 41Q041S 1. 11 37V037A 1. 25 41Q041Y 1. 09 37V037H 1. 21 41Q041V 1. 07 37V037L 1. 16 41Q041A 1. 03 37V037C 1. 09 41Q041L 1. 00 37V037T 1. 05 42L042K 2. 46 39A039L 1. 43 42L042W 2. 06 39A039K 1. 36 42L042H 1. 92 39A039Y 1. 36 42L042R 1. 38 39A039I 1. 26 42L042G 1. 17 39A039T 1. 26 42L042T 1. 08 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/PosJ relative to Pos Var WT Pos Var WT 42 L042F 1. 07 46 F046G 1. 02 43 G043A 1. 49 46 F046K 1. 00 43 G043C 1. 48 47 E047R 2. 45 43 G043K 1. 42 47 E047T 1. 96 43 G043M 1. 37 47 E047P 1. 36 43 G043Y 1. 26 47 E047S 1. 28 43 G043E 1. 25 47 E047H 1. 27 43 G043L 1. 22 47 E047G 1. 20 43G043R 1. 22 47elk 1. 19 43G043S 1. 18 47E047F 1. 09 43G043H 1. 17 47E047I 1. 03 43 G043P 1. 08 49I049G 1. 34 44A044F 2. 84 49I049H 1. 27 44A044V 2. 13 49I049S 1. 24 44A044C 1. 80 49I049K 1. 23 44A044L 1. 61 49I049V 1. 20 44A044W 1. 40 49I049L 1. 14 44A044M 1. 20 49I049Y 1. 07 45 D045K 1. 34 49I049R 1. 05 45 D045T 1. 27 49I049E 1. 02 45D045R 1. 16 49I049M 1. 01 45 D045W 1. 15 50E050L 1. 19 45 D045S 1. 13 50E050M 1. 18 45 D045G 1. 13 50E050A 1. 12 45 D045H 1. 13 51 E051V 1. 47 45 D045F 1. 11 51 E051A 1. 28 45D045L 1. 05 51 E051 G 1. 22 45D045V 1. 05 51 E051T 1. 18 45 D045Q 1. 04 51 E051L 1. 11 45 D045A 1. 04 51 E051I 1. 07 46 F046E 1. 25 53 L053H 5. 05 46F046D 1. 17 53L053Q 1. 48 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 53L053G 1. 32 62D062E 1. 02 53L053S 1. 16 63P063G 1. 71 53L053T 1. 02 63P063T 1. 50 54S054P 5. 20 63P063M 1. 46 54S054I 4. 78 63P063S 1. 42 54S054V 4. 72 63P063K 1. 40 54S054A 3. 46 63P063A 1. 35 54S054R 3. 38 63P063Y 1. 35 54S054L 2. 02 63P063W 1. 35 54S054T 1. 46 63P063V 1. 31 54S054K 1. 44 63P063R 1. 31 54S054G 1. 43 63P063F 1. 25 54S054C 1. 26 63P063L 1. 15 54S054Q 1. 03 63P063Q 1. 09 55A055G 1. 69 64T064G 1. 23 55A055T 1. 69 64T064S 1. 11 57T057S 1. 63 65D065A 1. 31 57T057R 1. 61 65D065S 1. 17 57T057V 1. 28 65D065H 1. 10 57T057I 1. 19 66P066R 1. 85 59N059W 1. 13 66P066V 1. 83 59N059R 1. 09 66P066H 1. 59 59N059T 1. 07 66P066I 1. 59 59N059S 1. 06 66P066G 1. 50 59N059Q 1. 02 66P066Q 1. 46 60I060H 1. 02 66P066T 1. 41 60I060R 1. 00 66P066S 1. 39 61D061H 1. 44 66P066Y 1. 33 61D061S 1. 26 66P066L 1. 14 61D061R 1. 11 66P066N 1. 12 61D061I 1. 08 67R067N 1. 58 61D061F 1. 01 67R067G 1. 39 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 67R067T 1. 28 71A071K 1. 44 67R067F 1. 26 71A071R 1. 40 67R067L 1. 20 71A071N 1. 23 67R067Q 1. 16 71A071L 1. 23 67R067W 1. 07 71A071F 1. 13 67R067E 1. 04 71A071C 1. 01 67R067P 1. 01 72S072L 1. 26 68L068E 1. 44 72S072H 1. 21 68L068W 1. 21 72S072G 1. 20 68L068I 1. 13 72S072T 1. 10 68L068G 1. 09 72S072V 1. 08 68L068V 1. 09 72S072Y 1. 07 68L068H 1. 05 73Y073R 1. 26 68L068T 1. 03 73Y073Q 1. 23 69N069V 1. 99 73Y073S 1. 17 69N069K 1. 72 73Y073K 1. 07 69N069R 1. 49 74L074S 2. 72 69N069I 1. 47 74L074G 1. 95 69N069H 1. 36 74L074W 1. 38 69N069T 1. 35 75P075R 1. 60 69N069L 1. 30 75P075S 1. 39 69N069S 1. 21 75P075T 1. 28 69N069G 1. 20 75P075Q 1. 21 69N069Q 1. 07 75P075G 1. 16 69N069W 1. 05 75P075H 1. 05 69N069C 1. 05 75P075W 1. 04 71A071S 1. 75 76S076P 1. 23 71A071T 1. 70 77C077T 1. 12 71A071H 1. 70 77C077V 1. 05 71A071G 1. 59 77C077G 1. 01 71A071I 1. 51 78L078G 4. 98 71A071E 1. 45 78L078H 4. 82 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Posl relative to WT/Pos./relative to Pos Var WT Pos Var WT 78L078E 3. 01 82L082G 1. 38 78 L078N 2. 68 82 L082R 1. 34 78L078T 1. 87 82L082H 1. 33 78L078Q 1. 73 82L082K 1. 19 78L078V 1. 53 82L082T 1. 18 78L078I 1. 43 82L082I 1. 17 78L078Y 1. 39 82L082S 1. 15 79A079H 1. 93 82L082V 1. 02 79A079L 1. 80 83P083K 1. 37 79A079I 1. 59 83P083G 1. 31 79A079M 1. 50 83P083H 1. 27 79A079N 1. 48 83P083R 1. 19 79A079Q 1. 47 83 P083S 1. 17 79A079R 1. 47 84L084K 1. 10 79A079W 1. 27 84L084H 1. 01 79A079T 1. 17 85D085Q 3. 09 79A079E 1. 12 85 D085R 2. 38 80T080C 1. 31 85D085S 2. 28 80T080V 1. 23 85 D085H 1. 55 80T080G 1. 16 85D085N 1. 54 80T080A 1. 00 85D085G 1. 41 81 H081K 1. 52 85D085T 1. 33 81 H081L 1. 23 85D085E 1. 12 81 H081N 1. 17 85 D085F 1. 01 81 H081G 1. 17 86L086A 1. 38 81 H081A 1. 15 86L086C 1. 16 81 H081 C 1. 13 86L086G 1. 15 81 H081W 1. 13 88I088H 1. 20 81 H081V 1. 10 88I088T 1. 03 81 H081F 1. 10 88I088G 1. 01 81 H081S 1. 04 90M090T 1. 27 82L082P 1. 46 90M090I 1. 13 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 90M090V 1. 08 103T103K 1. 09 90M090S 1. 06 103T103I 1. 08 90M090L 1. 02 103T103L 1. 05 91 L091G 1. 21 104P104H 2. 84 91L091T 1. 06 104P104T 2. 70 92G092V 1. 49 104P104G 2. 67 92G092S 1. 26 104P104V 2. 59 93 T093Y 5. 26 104P104S 2. 48 93 T093F 3. 52 104P104I 2. 43 93T093A 1. 38 104P104W 2. 05 93 T093C 1. 08 104P104C 1. 95 95 D095E 2. 04 104P104E 1. 84 96T096S 1. 04 104P104F 1. 79 97K097R 2. 80 104P104N 1. 62 97K097Q 1. 14 104P104R 1. 62 98 A098L 2. 22 104P104Q 1. 34 98A098H 2. 09 104P104M 1. 09 98 A098I 2. 05 105 L1OSP 1. 71 98A098Y 2. 02 105L105C 1. 56 98A098S 1. 73 105L105F 1. 30 98A098T 1. 72 105L105W 1. 28 98A098G 1. 57 105L105G 1. 08 98A098C 1. 30 106D106K 1. 28 98A098N 1. 24 106D106L 1. 20 98A098D 1. 11 106D106G 1. 18 98 A098P 1. 10 106 D106H 1. 09 10OF10OW 1. 08 106D106E 1. 08 100 F 1 OOE 1. 01 106D106T 1. 06 1 O 1 Rl O l I 1. 24 106D106I 1. 04 103T103W 1. 26 106D106F 1. 02 103T103Y 1. 19 106D106C 1. 01 103T103G 1. 11 107I107E 2. 55 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 107I107S 2. 04 115 Vl 15G 1. 09 107I107N 1. 81 115VllSI 1. 05 107I107G 1. 76 115 V 11 SY 1. 03 107I107V 1. 00 116T116G 1. 10 108A108L 1. 41 116T116A 1. 01 108 A108T 1. 05 117 Q117H 2. 33 109L109N 1. 52 117Q117T 2. 23 109L109W 1. 30 117Q117Y 2. 23 109L109Q 1. 18 117Q117W 2. 16 109L109Y 1. 16 117Q117V 2. 15 109 L1091 1. 05 117Q117G 2. 08 109L109D 1. 00 117Q117A 2. 05 11IM111K 1. 98 117Q117S 1. 95 111 M111I 1. 95 117Q117F 1. 57 111 M111L 1. 55 117Q117R 1. 56 111MI11T 1. 49 117Q117M 1. 54 IIIMI11F 1. 47 117Q117E 1. 15 lllMlllV 1. 47 118V118Y 1. 25 lllMlllY 1. 43 118V118K 1. 13 111 M111S 1. 03 118V118G 1. 08 112 S112L 1. 03 120T120S 1. 09 112S112H 1. 00 121 S121L 1. 35 113 V113L 1. 50 121 S121W 1. 33 113V113H 1. 34 121 S121R 1. 26 113 V113K 1. 19 121 S121K 1. 24 113V113R 1. 13 121 S121G 1. 20 113V113Y 1. 11 121 S121C 1. 18 113V113F 1. 05 121 S121N 1. 14 113V113Q 1. 03 121 S121T 1. 13 115V115W 1. 23 121 S121A 1. 12 115V115T 1. 15 121 S121V 1. 12 115V115L 1. 12 122A122H 1. 14 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 122A122I 1. 13 127T127H 1. 57 122A122T 1. 08 127T127V 1. 07 122A122K 1. 08 127T127I 1. 06 122A122V 1. 04 127T127S 1. 05 122A122S 1. 03 128T128L 1. 06 123G123D 1. 73 128T128K 1. 06 123G123V 1. 40 130P130T 1. 19 123G123P 1. 32 130P130H 1. 17 123G123E 1. 13 130P130K 1. 16 123G123T 1. 06 130P130G 1. 16 123G123H 1. 00 130P130S 1. 16 124G124L 1. 92 130P130V 1. 15 124G124I 1. 85 130P130W 1. 15 124G124T 1. 64 130P130I 1. 12 124G124H 1. 59 130P130L 1. 12 124G124V 1. 44 130P130R 1. 11 124G124F 1. 32 130P130F 1. 08 124G124S 1. 27 130P130E 1. 00 124G124Y 1. 23 131A131L 1. 83 124G124R 1. 14 131A131R 1. 76 124G124Q 1. 12 131A131H 1. 72 125V125G 2. 95 131A131G 1. 66 125V125S 1. 94 131A131W 1. 61 125V125A 1. 69 131A131V 1. 59 125V125P 1. 50 131A131P 1. 52 125V125R 1. 30 131A131Y 1. 50 125V125D 1. 24 131A131S 1. 48 125V125Y 1. 08 131A131E 1. 36 125V125I 1. 01 131A131D 1. 31 126G126T. 1. 58 131A131Q 1. 29 126G126P 1. 17 132P132Y 1. 57 126G126L 1. 17 132P132S 1. 13 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 133K133Y 1. 12 142L142K 1. 60 133K133L 1. 05 142L142F 1. 05 133K133H 1. 02 143A143K 3. 16 134V134G 1. 71 143A143H 2. 90 134V134T 1. 25 143A143L 2. 51 134V134N 1. 18 143A143V 2. 45 134V134S 1. 16 143A143W 2. 27 134VAL 1. 13 143A143T 2. 18 134V134I 1. 12 143A143R 2. 15 136V136T 1. 13 143A143S 1. 77 137V137M 1. 22 143A143Q 1. 74 137V137L 1. 09 143A143F 1. 56 137V137T 1 08 143A143P 1. 53 137V137A 1. 07 143A143G 1. 48 137V137G 1. 02 143A143D 1. 45 138S138I 1. 15 143A143E 1. 43 138S138G 1. 05 143A143C 1. 39 140P140A 1. 90 143A143N 1. 30 140P140T 1. 74 144ply 2. 34 140P140S 1. 31 144P144K 2. 09 141P141L 2. 32 144P144H 1. 94 141P141I 2. 29 144P144F 1. 82 141P141H 2. 07 144P144R 1. 76 141P141V 1. 96 144P144S 1. 69 141P141T 1. 84 144P144T 1. 46 141P141S 1. 70 144PEG 1. 45 141P141R 1. 65 144PAD 1. 45 141P141G 1. 64 144P144N 1. 44 141P141Q 1. 39 144P144L 1. 43 141P141N 1. 32 144P144Q 1. 37 141P141A 1. 10 144P144M 1. 24 142L142W 2. 41 144P144A 1. 09 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/PosJ relative to Pos Var WT Pos Var WT 145M145L 1. 72 151 Q151K 1. 07 145M145F 1. 49 151 Q151H 1. 06 145M145R 1. 15 151 Q151S 1. 05 145M145W 1. 15 151 Q151C 1. 05 145M145C 1. 02 151 Q151Y 1. 01 145M145T 1. 01 152L152V 1. 22 147H147A 1. 28 152L152K 1. 21 147H147S 1. 26 152L152R 1. 20 147H147T 1. 20 152L152W 1. 18 147H147P 1. 12 152L152T 1. 12 147H147E 1. 11 152L152S 1. 12 148P148V 2. 43 152L152Y 1. 09 148P148K 1. 79 152L152H 1. 09 148P148L 1. 64 152L152G 1. 08 148P148A 1. 64 152L152E 1. 08 148P148R 1. 51 152L152Q 1. 07 148P148T 1. 50 152L152D 1. 07 148P148Y 1. 46 152L152I 1. 04 148P148S 1. 46 152L152C 1. 00 148P148E 1. 42 153 I153K 1. 62 148P148F 1. 37 153I153H 1. 46 148P148Q 1. 33 153I153T 1. 27 148P148D 1. 03 153I153L 1. 27 150F150L 1. 29 153 I153F 1. 23 150F150E 1. 23 153 I153A 1. 19 151 Q151D 1. 47 154F154Y 1. 32 151 Q151R 1. 36 155E155T 1. 49 151 Q151P 1. 35 155E155R 1. 47 151 Q151A 1. 29 155E155L 1. 31 151 Q151T 1. 24 155E155Y 1. 27 151 Q151M 1. 24 155E155K 1. 23 151 Q151E 1. 14 155E155G 1. 17 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 155E155S 1. 08 158E158T 1. 45 155E155D 1. 08 158E158P 1. 41 155E155F 1. 07 158E158N 1. 41 156G156P 1. 44 158E158M 1. 39 156G156T 1. 15 158E158I 1. 38 156G156K 1. 10 158E158D 1. 35 156G156M 1. 09 159Q159R 1. 15 156G156C 1. 07 159Q159C 1. 13 156G156N 1. 07 159Q159S 1. 10 156G156R 1. 05 159Q159D 1. 09 156G156H 1. 04 159Q159A 1. 08 156G156S 1. 02 159Q159M 1. 07 157G157T 1. 74 159Q159P 1. 06 157G157R 1. 51 159Q159L 1. 02 157G157S 1. 30 161T161R 3. 61 157G157K 1. 28 161T161Y 2. 40 157G157F 1. 27 161T161H 1. 82 157G157V 1. 23 161T161W 1. 41 157G157H 1. 14 161T161I 1. 40 157G157I 1. 11 161T161V 1. 27 158E158H 2. 40 161T161L 1. 25 158E158K 2. 08 161T161Q 1. 04 158E158F 2. 06 162T162K 1. 22 158E158R 1. 99 162T162R 1. 17 158E158Y 1. 77 162T162W 1. 15 158E158W 1. 77 162T162Y 1. 03 158E158L 1. 59 162T162H 1. 02 158E158S 1. 57 163E163L 1. 50 158E158V 1. 52 163E163Y 1. 41 158E158Q 1. 49 163E163H 1. 32 158E158C 1. 46 163E163G 1. 25 158E158A 1. 45 163E163W 1. 21 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 163E163V 1. 13 167V167H 1. 03 163E163R 1. 12 168Y168G 1. 89 163E163S 1. 12 168Y168T 1. 51 163E163A 1. 11 168Y168V 1. 19 163E163C 1. 11 169S169Y 1. 26 163E163F 1. 07 169S169R 1. 24 165A165R 1. 70 169S169K 1. 21 165A165K 1. 35 169S169I 1. 16 165A165F 1. 23 169S169T 1. 15 165A165Q 1. 21 169S169L 1. 08 165A165V 1. 21 169S169C 1. 03 165A165Y 1. 20 169S169Q 1. 02 165A165T 1. 18 170A170K 1. 71 165A165I 1. 17 170A170G 1. 59 165A165P 1. 14 170A170I 1. 59 165A165L 1. 08 170A170S 1. 47 165A165G 1. 05 170A170F 1. 44 165A165N 1. 01 170A170T 1. 40 165A165S 1. 00 170A170E 1. 28 166R166Y 1. 29 170A170D 1. 27 166R166L 1. 27 170A170N 1. 21 166R166I 1. 26 170A170V 1. 20 166R166W 1. 25 170A170C 1. 15 166R166H 1. 20 170A170Q 1. 15 166R166T 1. 19 170A170L 1. 05 166R166V 1. 17 170A170W 1. 04 166R166K 1. 17 170A170M 1. 03 166R166S 1. 16 171L171K 2. 05 166R166G 1. 15 171L171H 1. 67 167V167T 1. 13 171L171T 1. 54 167V167I 1. 08 171L171I 1. 53 167V167Y 1. 07 171L171S 1. 43 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 171 L171F 1. 30 175M175W 1. 25 171 L171G 1. 26 176K176W 1. 19 171 L171Y 1. 20 176K176T 1. 04 171 L171V 1. 02 176K176Y 1. 04 172A172I 1. 70 176K176V 1. 04 172A172S 1. 59 176K176G 1. 01 172A172W 1. 43 178P178L 1. 82 172A172G 1. 41 178P178Y 1. 38 172A172V 1. 40 178P178K 1. 34 172A172T 1. 25 178P178W 1. 14 172A172L 1. 20 178P178G 1. 09 172A172C 1. 20 179F179L 1. 15 173 S173Y 1. 19 179F179Y 1. 05 173 S173K 1. 17 180F180L 1. 30 173 S173W 1. 16 180F180I 1. 20 173 S173L 1. 15 180F180V 1. 14 173 S173R 1. 09 180F180Y 1. 12 173S173H 1. 07 180F180W 1. 11 173 S173T 1. 06 180F180K 1. 08 174F174G 1. 60 180F180T 1. 01 174F174P 1. 54 181 D181A 1. 35 174F174Q 1. 42 181 D181K 1. 33 174F174C 1. 32 181D181Y 1. 29 174F174S 1. 16 181 D181W 1. 26 174F174L 1. 05 181 D181L 1. 25 175M175T 2. 21 181 D181R 1. 23 175M175G 2. 04 181D181S 1. 21 175M175V 1. 93 181 D181Q 1. 14 175M175L 1. 61 181D181E 1. 10 175M175Q 1. 56 181D181G 1. 09 175M175R 1. 55 181D181C 1. 09 175M175N 1. 39 181 D181P 1. 03 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 181D181T 1. 02 187S187R 1. 04 182A182T 1. 14 187S187G 1. 03 184 S184Y 1. 06 187S187F 1. 02 184S184F 1. 05 188T188Y 1. 48 184 S 184T 1. 04 188 T188V 1. 22 184S184H 1. 02 188T188S 1. 16 185V185K 1. 37 188T188I 1. 13 185V185Y 1. 37 188T188H 1. 11 185V185W 1. 36 188T188R 1. 01 185V185H 1. 30 189D189L 1. 30 185V185L 1. 23 189I) 189H 1. 25 185V185R 1. 15 189D189W 1. 09 185V185G 1. 12 190G19OW 1. 88 185V185T 1. 11 190G190K 1. 01 185V185S 1. 09 191 V191Y 1. 32 185V185I 1. 07 191 V191H 1. 30 185V185F 1. 02 191 V191W 1. 20 186I186G 1. 86 191 V 191 S 1. 20 186I186T 1. 51 191 V19lK 1. 17 186I186A 1. 46 191V191I 1. 14 186I186S 1. 39 191V191F 1. 13 186I186V 1. 28 191 V191R 1. 05 186I186L 1. 17 191 V191L 1. 04 186I186F 1. 01 196F196H 1. 77 187 S 187K 1. 45 196 F196L 1. 77 187 S187Y 1. 43 196F196C 1. 74 187su871 1. 38 196F196M 1. 65 187S187L 1. 37 196F196G 1. 59 187 S187W 1. 30 196F196S 1. 58 187S187H 1. 29 196fly 1. 41 187 S187V 1. 23 196F196V 1. 40 187S187T 1. 12 196F196I 1. 32 Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/Pos./relative to WT/Pos./relative to Pos Var WT Pos Var WT 196F196W 1. 01 201N201G 1. 08 197T197L 1. 21 202R202W 1. 97 198E198R 1. 82 202R202F 1. 89 198E198I 1. 80 202R202E 1. 69 198E198V 1. 60 202R202H 1. 64 198E198W 1. 59 202R202T 1. 55 198E198L 1. 57 202R202S 1. 49 198E198P 1. 52 202R202A 1. 48 198E198Y 1. 48 202R202C 1. 44 198E198C 1. 38 202R202M 1. 43 198E198F 1. 37 202R202L 1. 43 198E198Q 1. 28 202R202G 1. 39 198E198T 1. 25 202R202I 1. 33 198E198N 1. 24 203D203L 2. 42 198E198M 1. 18 203D203R 2. 23 198E198S 1. 06 203D203I 1. 99 199A199C 1. 77 203D203W 1. 99 199A199K 1. 72 203D203F 1. 92 199A199E 1. 56 203D203H 1. 84 199A199L 1. 38 203D203C 1. 78 199A199T 1. 33 203D203S 1. 66 199A199R 1. 33 203D203V 1. 66 199A199V 1. 32 203D203G 1. 63 199A199D 1. 31 203D203Q 1. 60 199A199H 1. 27 203D203A 1. 53 199A199Y 1. 24 203D203E 1. 34 199A199F 1. 23 203D203N 1. 05 199A199S 1. 20 199A199G 1. 14 199A199M 1. 07 201N201Y 1. 29 201N201F 1. 16

The following Table, provides variants with a PAF PI greater than 1.5. Ta 1- >. Wild-Type Wild-Type Wild-Type Wild-Type Residue/Pos. Variant Amino Acid (t Residue/Pos. Variant Amino Acid (s) L8 E N TT H I Y A79 H I DIP K. L. W. Y H81E. L12 5 H N R G15 A T93 F. Y E20 C S T V W D95 E 1 W K97 9 GH L TY G22 _ A 98 G ! H, I, L. S. T, Y T2 S C, E, F, G, H, I, N, R, S, 26 A. M P104 T. V. W A29 G R V W Y L105 p D31 L. W I107 E, GN. S G, I, K, L, N, R, S, T, W, L109 N Q40 M111 I L L42 H, K W V113 L A44 C F L V A, F, G, H, M, R, S, T, E47 R T 117 V W Y L53 H G123 D H I L T S54 A I L P R V G124 I L A55 G T V 125 A G P S T57 R S G126 P63 G T127 H P66 H I R V A131 G. H L P R V W Y R67 N P132 y N69 K*V V134 G A71 GHIST P140 AT L74 S P141 G. H. 1. L. R. S. T. V P75 R L142 Table 10-3. PAF PI > 1. 5 Wild-Type Residue/Pos. Variant Amino Acid F, H, K, L, P, Q, R, S, T, A143 V W P144 F. H K. R. S. Y M145 L P148 A. K L. R. T. V 1153K G157 R. T E158 F. H*K. L. R^S. V. WY T161 H R Y A165 T Y168 G. T A170 G. I. K L171 H. I. K. T A1 A172 I. S F174 G P M175 G. L. 0. R. T. V P178 L F196 C, G, H, L, M, S G190 W E198 I, L, P, R, V, W A199 C, E, K R202 E, F, H, T, W A, C, F, G, H, I, L, Q, R, D203 S, V, W V206 E, F, G, H, K, R, S, A209 K E210 H, K, S, T, V, W Q211 K V212 W Table 10-4 provides variants with PAF PI values greater than 2.0. Table 10-4. Variants with PAF PI > 2. 0 Wild-Type Residue/Pos. Amino Acid Variant (s) C7 Y D10 K. L W L12 O E20 G. H. L S. T V W E26 M 040 I. K. L. T. W L42 K W A44 Fs V E47 R L53 H S54 A. I. L. P. R. V L74 S L78 E. G. H. N D85 R. S T93 F. Y D95 E K97 R A98 H I. L Y P104 G. H. I. S. T. V. W I107 E S 117 A G H T V W Y V125 G P141 H I L L142 W A143 H K L R T V W P144 K Y P148 V E158 F H K T161 R Y L171 K M175 G, T D203 L, R V206 E, F, K E210 T The following Table provides PAD assay results for various variants. Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results Position WT/Pos/Variant PAD Perf. Position WT/Pos/Variant PAD Perf. Position utatio Ind. position utation Vanan. Mutation Ind. Mutation Ind. 1 MOOlA A <0. 01 5 I005P P <0. 01 1 MOOlE E <0. 01 5 I005R R <0. 01 1 MOTIF F <0. 01 5 I005W W <0. 01 1 MOOIG G <0. 01 5 I005F F 0. 15045 1 M001K K <0. 01 5 I005S S 0. 367738 1 MOOlN N_ <0. 01 _ 5 I005H H 0. 626022 1 M001P P 0. 01 5 1005T T 0. 7212 1 MOOR R <0. 01 5 1005V V 0. 917243 1 M001 S S <0. 01 6 L006S S <0. 01 1 M001T T <0. 01 6 L006K K <0. 01 1 M001W W 0. 01 6 L006G G <0. 01 1 M001 V V 0. 944944 6 L006H H <0. 01 3 K003V V 0. 835476 6 L006R R <0. 01 4 R004L L <0. 01 6 L006W W <0. 01 4 R004V V 0. 079216 6 L006E E <0. 01 4 R004I 1 0. 153122 6 L0060 0 <0. 01 4 R004W W 0. 484006 6 L006V V 0. 352616 4 R004G G 0. 78952 6 L006T T 0. 354148 4 R004S S 0. 907174 6 L006I 1 0. 819654 4 R004E E 0. 970668 7 C007S S <0. 01 4 R004Y Y 0. 983327 7 C007R R <0. 01 4 R004H H 0. 986096 7 C007L L <0. 01 4 R0040 0 0. 98766 7 C007P P <0. 01 4 R004T T 0. 999841 7 C007T T <0. 01 5 I005G G <0. 01 7 C007W W <0. 01 5 I005N N <0. 01 7 C007Y Y 0. 544454 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. W'T/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 7 C007M M 0. 678238 12 L012V V <0. 01 7 C007G G 0. 686018 12 L012S S <0. 01 10 DOlOW W <0. 01 12 L012G G <0. 01 10 DOlOK K <0. 01 12 L012R R <0. 01 10 DOlOY Y <0. 01 12 L012D D <0. 01 10 D010T T <0. 01 12 L012P P <0. 01 10 DOlOI I <0. 01 <0. 01627385 10 DOlOV V <0. 01 12 L012W W 75856614 10 DOlOS S <0. 01 12 L012T T 0. 064264 10 DO 1 OG G <0. 01 12 L012A A 0. 074567 10 DOOR R <0. 01 12 L012K K 0. 134919 10 D010A A <0. 01 12 L012H H 0. 164894 10 D010M M <0. 01 12 L012F F 0. 171369 10 DOlON N <0. 01 12 L0120 0 0. 219754 10 DOLLOP P <0. 01 12 L012C C 0. 221492 10 D010E E 0. 147899 12 L012N N 0. 655242 11 S011T T <0. 01 13 T013F F <0. 01 11 SO1 1V V <0. 01 13 T013R R <0. 01 11 S011D D <0. 01 13 T013W W <0. 01 11 SOllE E <0. 01 13 T0130 0 0. 508867 11 SO11F F <0. 01 13 T013V V 0. 625148 11 S011G G <0. 01 13 T013S S 0. 682494 11 SOUL L <0. 01 13 T013G G 0. 768701 11 S0110 0 0. 01 14 W014I I <0. 01 11 SO1 1R R <0. 01 14 W014S S <0. 01 11 SO11H H 0. 332012 14 W014G G <0. 01 11 SOUK K 0. 399168 14 W014K K <0. 01 11 S011A A 0. 528328 14 W014V V <0. 01 11 SO11I I 0. 562735 14 W014L L <0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Position utation Variant d, Position utatio Variant Ind. Mutation Ind. Mutation Ind. 14 W014T T <0. 01 16 W016M M 0. 370155 14 W014R R <0. 01 16 W016A A 0. 553088 14 W014N N <0. 01 16 W016D D 0. 569713 14 W014P P <0. 01 16 W016E E 0. 647375 14 W014E E 0. 150043 16 W016V V 0. 875327 14 W014F F 0. 218073 17 V017A A 0. 675391 14 W014A A 0. 271277 17 V017E E 0. 749717 14 W014Y Y 0. 64896 17 V017G G 0. 838345 14 W014W W 0. 989643 17 V017K K 0. 844479 15 G015C C <0. 01 17 V017F F 0. 847091 15 G015N N <0. 01 17 V017T T 0. 861827 15 G015D D <0. 01 17 V017Y Y 0. 876678 15 G015E E <0. 01 17 V017R R 0. 936013 15 G015H H <0. 01 17 V017P P 0. 956795 15 G015K K <0. 01 17 V017I 1 0. 993337 15 G015L L <0. 01 17 V017L L 0. 996217 15 G015P P <0. 01 18 P018A A <0. 01 15 G015R R <0. 01 18 P018M M <0. 01 15 G015Y y <0. 01 18 P018S S 0. 066689 15 G015A A 0. 614319 19 V019P P <0. 01 15 G015S S 0. 631317 19 V019M M 0. 117174 16 W016S S <0. 01 19 V019R R 0. 343385 16 W016G G <0. 01 19 VOl90 0 0. 395965 16 W016H H <0. 01 19 V019A A 0. 554598 16 W016N N <0. 01 19 V019G G 0. 55596 16 W016R R <0. 01 19 V019S S 0. 573928 16 W016T T <0. 01 19 V019E E 0. 620236 16 W016P P 0. 150383 19 V019Y Y 0. 696626 16 W0160 0 0. 312038 19 V019D D 0. 785756 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Position utatio Vaant Ind. Position utation Vanant Mutation Ind. Mutation Ind. 19 V019L L 0. 910961 24 P024I I 0. 853247 19 V019K K 0. 965611 24 P024R R 0. 907892 21 D021V V <0. 01 24 P024H H 0. 969695 21 D021P P 0. 534939 25 T025P P <0. 01 21 D021S S 0. 689672 25 T025H H <0. 01 21 D021E E 0. 864655 25 T025L L <0. 01 21 D021F F 0. 876655 25 T025R R <0. 01 21 D021W W 0. 894205 25 T025M M <0. 01 21 D021L L 0. 971454 25 T025E E <0. 01 22 G022K K <0. 01 25 T025D D <0. 01 22 G022W W 0. 231005 25 T025K K 0. 133406 22 G022R R 0. 563069 25 T025W W 0. 144315 22 G022V V 0. 850851 25 T025I 1 0. 350917 22 G022S S 0. 981692 25 T025G G 0. 426214 23 A023R R 0. 283095 25 T025C C 0. 509792 23 A023S S 0. 335177 25 T025V V 0. 514769 23 A023G G 0. 350575 25 T025S S 0. 576256 23 A023F F 0. 438047 25 T025A A 0. 863346 23 A023V V 0. 598414 26 E026S S 0. 280953 23 A0230 0 0. 732052 26 E026T T 0. 39705 23 A023P P 0. 733451 26 E026W W 0. 471182 23 A023W W 0. 801206 26 E026N N 0. 47572 23 A023M M 0. 946802 26 E026R R 0. 813632 23 A023Y Y 0. 962455 26 E026G G 0. 869755 24 P024S S 0. 614708 26 E026C C 0. 939981 24 P0240 0 0. 652848 26 E026V V 0. 966156 24 P024T T 0. 663925 26 E026P P 0. 993535 24 P024A A 0. 681992 27 R027W W <0. 01 24 P024G G 0. 755229 27 R027T T <0. O1497896 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. 1 77895526 27 R027P P 0. 483512 27 R027P P 0. 483512 27 R027C C 0. 58498 27 R027C C 0. 58498 27 R027S S 0. 686775 27 R027S S 0. 686775 27 R027G G 0. 836174 27 R027G G 0. 836174 27 R027E E 0. 925988 27 R027E E 0. 925988 27 R027V V 0. 943209 27 R027V V 0. 943209 28 F028G G <0. 01 28 F028G G <0. 01 28 F028H H <0. 01 28 F028H H <0. 01 28 F028I I <0. 01 28 F028I I <0. 01 28 F028R R <0. 01 28 F028R R <0. 01 28 F028P P 0. 385272 28 F028P P 0. 385272 28 F028V V 0. 531941 28 F028V V 0. 531941 28 F028S S 0. 696363 28 F028S S 0. 696363 29 A029V V 0. 43718 29 A029V V 0. 43718 29 A029T T 0. 467508 29 A029T T 0. 467508 29 A029S S 0. 546873 29 A029S S 0. 546873 29 A029Y Y 0. 593264 29 A029Y Y 0. 593264 29 A029P P 0. 622623 29 A029P P 0. 622623 29 A029R R 0. 728312 29 A029R R 0. 728312 29 A029W W 0. 738583 29 A029W W 0. 738583 29 A029M M 0. 768108 29 A029M M 0. 768108 29 A029G G 0. 802278 29 A029G G 0. 802278 29 A029E E 0. 844095 29 A029E E 0. 844095 29 A029D D 0. 996225 29 A029D D 0. 996225 30 P030M M 0. 78893 30 P030M M 0. 78893 30 P0300 0 0. 905135 30 P0300 o o. 905135 30 P030A A 0. 918048 30 P030A A 0. 918048 31 D031E E 0. 882779 31 D031E E 0. 882779 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Posl PAD Perf. Position utation Variant Ind. posihon utation Variant Mutation Ind. Mutahon tnd. 32 V032P P <0. 01 36 G036V V 0. 375828 32 V032R R 0. 715259 36 G036M M 0. 536338 33 R033D D <0. 01 36 G036N N 0. 557724 33 R033E E <0. 01 36 G036W W 0. 682701 33 R033H H <0. 01 36 G0360 0. 712029 33 R033P P <0. 01 36 G036R R 0. 897684 33 R033W W <0. 01 38 L038K K <0. 01 33 R033V V 0. 935183 38 L038G G <0. 01 34 W034R R <0. 01 38 L038E E <0. 01 34 W034E E <0. 01 38 L038P P <0. 01 34 W034K K <0. 01 38 L0380 0 <0. 01 34 W0340 0 0. 041311 38 L038R R <0. 01 34 W034S S 0. 079486 38 L038W W <0. 01 34 W034T T 0. 153641 40 0040P P 0. 01 34 W034V V 0. 72591 41 0041V V 0. 01 34 W034G G 0. 880049 41 0041S S 0. 222419 34 W034I 1 0. 93831 41 0041P P 0. 662368 35 T0350 0 <0. 01 41 041Y Y 0. 701492 35 T035N N <0. 01 41 0041W W 0. 878483 35 T035R R <0. 01 42 L042W W <0. 01 35 T035K K <0. 01 42 L042H H <0. 01 35 T035L L <0. 01 42 L042T T <0. 01 35 T035P P <0. 01 42 L042D D <0. 01 35 T035W W <0. 01 42 L0420 0 0. 280991 35 T035Y Y <0. 01 42 L042S S 0. 450557 35 T035V V 0. 344374 42 L042R R 0. 64188 36 G036P P <0. 01 42 L042I 1 0. 658658 36 G036S S 0. 25722 42 L042V V 0. 725221 36 G036T T 0. 326076 42 L042M M 0. 73687 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 42 L042G G 0. 759964 45 D045P P 0. 531241 43 G043S S 0. 233902 45 D0450 0 0. 568687 43 G043P P 0. 310899 45 D045W W 0. 582004 43 G043V V 0. 332639 45 D045H H 0. 779564 43 G0430 0 0. 475759 45 D045L L 0. 781626 43 G043R R 0. 585481 45 D045M M 0. 78286 43 G043C C 0. 725373 45 D045G G 0. 839279 43 G043I 1 0. 766408 45 D045A A 0. 841569 43 G043K K 0. 856798 45 D045C C 0. 844725 43 G043M M 0. 877674 45 D045K K 0. 867296 43 G043Y Y 0. 944457 46 F046H H <0. 01 43 G043H H 0. 957156 46 F046T T 0. 429962 44 A044S S <0. 01 46 F046W W 0. 633171 44 A044Y Y <0. 01 46 F046S S 0. 656356 44 A044T T <0. 01 46 F046V V 0. 786355 44 A044R R <0. 01 46 F046I 1 0. 882982 44 A044D D <0. 01 46 F046G G 0. 944614 44 A044H H <0. 01 47 E047P P 0. 357072 44 A044P P <0. 01 47 E047R R 0. 620501 44 A044E E 0. 028463 47 E047N N 0. 627512 44 A044V V 0. 504951 47 E047S S 0. 628088 44 A044F F 0. 803847 47 E047M M 0. 703134 44 A044W W 0. 847767 47 E047A A 0. 757492 44 A044M M 0. 975188 47 E047F F 0. 763159 44 A044L L 0. 99381 47 E047C C 0. 772744 45 D045S S 0. 382964 47 E047T T 0. 837562 45 D045T T 0. 438291 47 E047D D 0. 975388 45 D045R R 0. 492492 47 E047H H 0. 99217 45 D045V V 0. 500129 48 V048R R <0. 01 NCQ Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 48 V048W W <0. 01 52 G052T T <0. 01 48 V048S S 0. 423613 52 G052M M <0. 01 48 V048G G 0. 873544 52 G052F F <0. 01 48 V048N N 0. 980906 52 G052I I 0. 069022 48 V048E E 0. 987222 52 G052P P 0. 242545 49 I049P P 0. 161279 52 G052L L 0. 244397 49 I049R R 0. 29139 52 G0520 0 0. 283827 49 I049W W 0. 676641 52 G052R R 0. 349923 49 I049H H 0. 740799 52 G052E E 0. 549067 49 I049S S 0. 789362 52 G052A A 0. 793929 49 I049E E 0. 876247 53 L053R R <0. 01 49 I049V V 0. 972022 53 L053W W <0. 01 50 E050R R <0. 01 53 L053P P <0. 01 50 E050W W 0. 14091 <0. 01328259 50 E050V V 0. 425221 53 L053D D 968325 50 E050I 1 0. 575369 53 L053E E 0. 191623 50 E050S S 0. 645021 53 L053K K 0. 237686 50 E0500 0. 906441 53 L053S S 0. 260431 50 E050L L 0. 967983 53 L053G G 0. 32712 51 E051R R <0. 01 53 L053V V 0. 652864 51 E051P P <0. 01 53 L053I 1 0. 659806 51 E051I 1 0. 044391 53 L0530 0 0. 717093 51 E051W W 0. 165053 53 L053T T 0. 842042 51 E05 1V V 0. 367755 54 S054F F <0. 01 51 E0510 0 0. 761883 54 S054W W <0. 01 51 E051L L 0. 927544 54 S054H H <0. 01 52 G052H H <0. 01 54 S054K K 0. 083519 52 G052S S <0. 01 54 S054I 1 0. 116295 52 G052V V <0. 01 54 S054Y Y 0. 124722 Table 10-5. PAD Assay Results Table 10-S. PAD Assay Results W°T/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 54 S054G G 0. 170484 56 R056W W <0. 01 54 S054L L 0. 258821 56 R056Y Y <0. 01 54 S054V V 0. 285755 56 R056S S 0. 123501 54 S054E E 0. 296919 56 R056L L 0. 237933 54 S054T T 0. 329279 56 R056N N 0. 267811 54 S054R R 0. 354857 56 R056A A 0. 68802 54 S054M M 0. 482666 57 T057R R <0. 01 54 S0540 0 0. 531633 57 T057P P <0. 01 54 S054D D 0. 647787 57 T057W W <0. 01 54 S054C C 0. 87772 57 T057N N 0. 245605 55 A055V V <0. 01 57 T057C C 0. 398001 55 A055I I <0. 01 57 T057Y Y 0. 551709 55 A055P P <0. 01 57 T057H H 0. 605386 55 A055W W <0. 01 57 T057A A 0. 651879 55 A055Y Y 0. 176777 57 T057L L 0. 762087 55 A055R R 0. 245648 57 T057V V 0. 86913 55 A055T T. 0. 415054 57 T057I 1 0. 870692 55 A055G G 0. 731513 58 T058E E <0. 01 55 A055L L 0. 866592 58 T058G G <0. 01 55 A055S S 0. 866756 58 T058K K <0. 01 55 A055H H 0. 921909 58 T058P P <0. 01 56 R056C C <0. 01 58 T058R R <0. 01 56 R056G G <0. 01 58 T058W W <0. 01 56 R056T T <0. 01 58 T058Y Y <0. 01 56 R056E E <0. 01 58 T058M M 0. 026886 56 R056H H <0. 01 58 T058A A 0. 361258 56 R056K K <0. 01 58 T058V V 0. 955494 56 R056P P <0. 01 58 T058S S 0. 964758 56 R0560 0 <0. 01 59 N059R R <0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Posl PAD Perf. WT/Posl PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 59 N059M M <0. 01 62 D062T T <0. 01 59 N059P P <0. 01 62 D062I I <0. 01 59 NOS90 0. 165409 62 D062V V <0. 01 59 N059T T 0. 501362 62 D062H H <0. 01 59 N059S S 0. 651989 62 D062W W <0. 01 59 N059K K 0. 731191 62 D062S S <0. 01 59 N059E E 0. 879272 62 D062L L <0. 01 59 N059V V 0. 887341 62 D062G G <0. 01 59 N059G G 0. 890006 62 D062R R <0. 01 59 N059F F 0. 911279 62 D062M M <0. 01 _ 59 N059A A 0. 929578 62 D062P P <0. 01 59 N059Y Y 0. 99189 62 D0620 0 <0. 01 59 N059C C 0. 99959 62 D062A A 0. 113753 60 I060P P 0. 318965 62 D062C C 0. 490736 60 I060D D 0. 660273 62 D062E E 0. 602369 60 I060C C 0. 668516 63 P063A A 0. 598416 60 I060M M 0. 682237 63 P063R R 0. 801911 60 I060A A 0. 788799 63 P063S S 0. 898408 60 I060R R 0. 809655 63 P063M M 0. 908904 60 I060L L 0. 913226 63 P063F F 0. 925844 60 I060E E 0. 923286 63 P063Y Y 0. 948378 60 I060K K 0. 959958 64 T064R R 0. 106209 60 I060S S 0. 999829 64 T064D D 0. 640095 61 D061F F 0. 698154 64 T064W W 0. 691185 61 D061A A 0. 708121 64 T0640 0. 865168 61 D061C C 0. 848446 64 T064C C 0. 876862 61 D061Y Y 0. 948278 64 T064P P 0. 936023 61 D061 V V 0. 968066 64 T064H H 0. 960718 61 D061N N 0. 999276 64 T064N N 0. 983933 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutatiom Ind. Mutation Ind. 64 T064S S 0. 987972 67 R067E E 0. 113415 65 D065V V 0. 199467 67 R067V V 0. 1203 65 D065R R 0. 215599 67 R0670 0 0. 126838 65 D065H H 0. 398178 67 R067L L 0. 156654 65 D065Y Y 0. 42301 67 R067A A 0. 215271 65 D065P P 0. 423122 67 R067T T 0. 315404 65 D065S S 0. 468174 67 R067N N 0. 333066 65 D065W W 0. 50219 67 R067G G 0, 40823 65 D065T T 0. 5039 67 R067K K 0. 986487 65 D065G G 0. 51655 68 L068G G <0. 01 65 D065I 1 0. 617391 68 L068A A <0. 01 65 D065A A 0. 723321 68 L068M M 0. 02834 66 P066N N 0. 381273 68 L068C C 0. 05996 66 P0660 0 0. 422614 68 L068S S 0. 071622 66 P066G G 0. 444859 68 L068N N 0. 100981 66 P066R R 0. 508806 68 L068E E 0. 131505 66 P066C C 0. 523524 68 L068H H 0. 222734 66 P066A A 0. 563865 68 L0680 0 0. 254448 66 P066F F 0. 672865 68 L068F F 0. 254797 66 P066Y Y 0. 699931 68 L068T T 0. 324904 66 P066D D 0. 718749 68 L068P P 0. 35297 66 P066I 1 0. 844376 68 L068D D 0. 443469 66 P066V V 0. 89302 68 L068Y Y 0. 447862 66 P066H H 0. 947771 68 L068R R 0. 465293 66 P066L L 0. 987271 68 L068V V 0. 507389 <0. 01497362 68 L068W W 0. 561612 67 R067F F 60903786 68 L068I I 0. 727312 <0. 01713297 69 N069Y Y 0. 173925 67 R067W W 32205367 69 N069W W 0. 55063 67 R067P P 0. 036575 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results W°T/Pos/PAD Perf. W'T/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 69 N069P P 0. 591783 71 A071C C 0. 993683 69 N069R R 0. 828172 72 S072Y Y 0. 069096 69 N069G G 0. 976332 72 S072W W 0. 339835 70 G070M M <0. 01 72 SO ? 2P P 0. 555612 70 G070T T <0. 01 72 S0720 0 0. 655328 70 G070P P <0. 01 72 S072L L 0. 703483 70 G070V V <0. 01 72 S072R R 0. 742354 70 G070C C <0. 01 72 S072D D 0. 800127 70 G070R R <0. 01 72 S072V V 0. 82827 70 G070Y Y <0. 01 72 S072E E 0. 930527 70 G070K K <0. 01 72 S072T T 0. 973836 70 G070N N <0. 01 73 Y073P P <0. 01 70 G0700 0 <0. 01 73 Y073R R 0. 262561 70 G070F F <0. 01 73 Y073L L 0. 497588 70 G070I 1 0. 270463 73 Y073G G 0. 509699 70 G070E E 0. 33356 73 Y073H H 0. 515737 70 G070S S 0. 638917 73 Y073I 1 0. 641914 71 A071P P <0. 01 73 Y073S S 0. 676285 71 A071N N 0. 613838 73 Y073V V 0. 73535 71 A071D D 0. 646588 73 Y073N N 0. 758401 71 A071G G 0. 675895 73 Y073D D 0. 803442 71 A071S S 0. 693249 73 Y0730 0 0. 866092 71 A071R R 0. 771492 73 Y073K K 0. 944166 71 A071H H 0. 781953 76 S076W W <0. 01 71 A071I 1 0. 786894 76 S076Y Y 0. 177113 71 A071T T 0. 79386 76 S076F F 0. 461095 71 A071E E 0. 809505 76 S0760 0. 900789 71 A071L L 0. 838126 77 C077Y Y <0. 01 71 A071F F 0. 985677 77 C077R R 0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutatton Ind. Mutatton Ind. 77 C077W W <0. 01 79 A079W W 0. 530746 77 C077F F <0. 01 79 A079T T 0. 598368 77 C077N N <0. 01 79 A079I I 0. 673986 77 C077P P <0. 01 79 A079S S 0. 779628 77 C077G G 0. 181068 79 A079G G 0. 915372 77 C077L L 0. 734708 79 A079P P 0. 94147 77 C077S S 0. 764136 79 A079L L 0. 958677 77 C077V V 0. 802259 80 T080W W <0. 01 77 C077A A 0. 912937 80 T080L L <0. 01 78 L078E E <0. 01 80 T080K K <0. 01 78 L078N N <0. 01 80 T080R R <0. 01 78 L078A A <0. 01 80 T080E E <0. 01 78 L078P P <0. 01 80 T080P P <0. 01 78 L078R R <0. 01 80 T080H H 0. 049717 78 L078S S <0. 01 80 T080Y Y 0. 107973 78 L078M M 0. 477538 80 T080I 1 0. 146188 78 L0780 0 0. 519566 80 T080N N 0. 529867 78 L078C C 0. 779536 82 L082R R <0. 01 78 L078Y Y 0. 809511 82 L082S S <0. 01 78 L078V V 0. 827484 82 L082W W <0. 01 79 A079H H <0. 01 82 L082V V 0. 187819 79 A079F F <0. 01 82 L082G G 0. 310823 79 A079V V <0. 01 82 L082T T 0. 377413 79 A079C C 0. 026887 82 L082H H 0. 468806 79 A079 0 0. 268704 82 L082I 1 0. 508005 79 A079E E 0. 272158 82 L082K K 0. 508537 79 A079N N 0. 281684 82 L082P P 0. 516154 79 A079M M 0. 284387 82 L082A A 0. 976228 79 A079R R 0. 321618 83 P083T T <0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Position utation Variant Ind. Position utation Variant 83 P083V 83 P083V V 0. 186837 85 D085T T 0. 708548 83 P083L L 0. 211018 85 D085N N 0. 781957 83 P083H H 0. 611439 85 D0850 0 0. 988545 83 P083W W 0. 621496 86 L086H H <0. 01 83 P083G G 0. 677444 86 L086S S <0. 01 83 P083S S 0. 789585 86 L086R R <0. 01 83 P0830 0 0. 818267 86 L086E E <0. 01 83 P083D D 0. 831344 86 L086F F <0. 01 83 P083F F 0. 99445 86 L0860 0 <0. 01 84 L084W W <0. 01 86 L086W W 0. 077717 84 L084V V 0. 416576 86 L086V V 0. 120133 84 L084P P 0. 43025 86 L086T T 0. 284184 84 L084T T 0. 438956 86 L086G G 0. 696393 84 L084A A 0. 453182 86 L086Y Y 0. 815121 84 L0840 0 0. 516002 86 L086P P 0. 987233 84 L084S S 0. 550862 87 V087S S <0. 01 84 L084R R 0. 565943 87 V087G G <0. 01 84 L084N N 0. 665228 87 V087Y Y <0. 01 84 L084K K 0. 79008 87 V087R R <0. 01 84 L084D D 0. 85276 87 V087K K <0. 01 84 L084I 1 0. 870124 87 V087D D <0. 01 84 L084H H 0. 993217 87 V087F F 0. 103908 85 D085I 1 0. 100248 87 V087T T 0. 147618 85 D085L L 0. 241561 87 V087A A 0. 16806 85 D085V V 0. 25268 87 V087M M 0. 751854 85 D085W W 0. 341677 89 I089H H <0. 01 85 D085P P 0. 543807 89 I089S S <0. 01 85 D085Y Y 0. 554364 89 I089G G <0. 01 85 D085S S 0. 675803 89 I089W W <0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Position utation ariant d. Position utation Variant Mutation Ind. Mutation md. 89 10890 0 <0. 01 95 D095S S <0. 01 89 I089D D <0. 01 95 D095T T <0. 01 89 I089E E <0. 01 95 D095V V <0. 01 89 I089R R <0. 01 95 D095W W <0. 01 89 I089F F 0. 745747 95 D095Y Y <0. 01 89 I089V V 0. 820031 95 D095E E 0. 754335 89 I089T T 0. 900425 96 T096I I <0. 01 94 N094L L <0. 01 96 T096W W <0. 01 94 N094T T <0. 01 96 T096Y Y <0. 01 94 N094V V <0. 01 96 T096R R 0. 136108 94 N094H H <0. 01 96 T096V V 0. 58611 94 N094R R <0. 01 96 T096S S 0. 786547 94 N094W W <0. 01 96 T096P P 0. 885134 94 N094M M 0. 031458 97 K0970 0 <0. 01 94 N094C C 0. 072751 97 K097G G <0. 01 94 N094Y Y 0. 123924 97 K097I I <0. 01 94 N094G G 0. 532837 97 K097W W <0. 01 94 N094A A 0. 74316 97 K097L L <0. 01 94 N094P P 0. 789771 97 K097V V <0. 01 94 N094S S 0. 877698 97 K097Y Y <0. 01 95 D095A A <0. 01 97 K097S S <0. 01 95 D095C C <0. 01 97 K097T T <0. 01 95 D095G G <0. 01 97 K097D D <0. 01 95 D095H H <0. 01 97 K097M M 0. 216645 95 D095K K <0. 01 97 K097A A 0. 227977 95 D095L L <0. 01 97 K097P P 0. 26585 95 D095N N <0. 01 97 K097R R 0. 587184 95 D0950 0 <0. 01 99 Y099R R 0. 291941 95 D095R R <0. 01 99 Y099V V 0. 311502 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 99 Y099S S 0. 367181 102 R102D D 0. 684234 99 Y099W W 0. 566038 102 R102P P 0. 894709 99 Y099H H 0. 591623 102 R102S S 0. 960127 99 Y099I 1 0. 60574 103 T103W W <0. 01 99 Y099G G 0. 700083 103 T103Y Y <0. 01 99 Y099P P 0. 813989 103 T103G G <0. 01 99 Y099A A 0. 822549 103 T103K K <0. 01 99 Y099L L 0. 856204 103 T103I I <0. 01 100 F100W W <0. 01 103 T103L L <0. 01 100 F100K K <0. 01 103 T103H H <0. 01 100 F100D D <0. 01 103 T103A A <0. 01 100 F1OOE E 0. 152427 103 T103V V <0. 01 100 F100S S 0. 852784 103 T103S S <0. 01 101 R101W W <0. 01 103 T103C. C <0. 01 101 R101K K 0. 068708 103 T103R R <0. 01 101 R1010 0 0. 107171 103 T103N N <0. 01 101 R101V V 0. 442582 103 T103F F <0. 01 101 R101D D 0. 800722 103 T103P P <0. 01 101 R101Y y 0. 803109 104 P104R R <0. 01 101 R101P P 0. 855496 104 P104A A <0. 01 101 R101N N 0. 918012 104 P104L L <0. 01 101 R101C C 0. 946306 104 P104W W 0. 232802 101 R101I 1 0. 955711 104 P104T T 0. 333526 101 R101F F 0. 965422 104 P104S S 0. 529113 102 R102W W <0. 01 104 P1040 0 0. 847699 102 R102F F 0. 226881 104 P104F F 0. 863543 102 R102G G 0. 270733 104 P104G G 0. 984538 102 R102C C 0. 363718 105 L105V V <0. 01 102 R102V V 0. 60605 105 L105A A <0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Position utation ariant Ind. position utation Variant Mutatton Ind. Mutation tnd. 105 L105M M <0. 01 108 A108N N <0. 01 105 L105E E 0. 528458 108 A108P P <0. 01 105 L105S S 0. 609931 108 A108R R <0. 01 105 L105Y Y 0. 620029 108 A108E E 0. 60726 105 L105T T 0. 638962 108 A1080 0 0. 734472 105 L1 05P P-0. 902642 108 A108T T 0. 865471 106 D106R R 0. 559786 108 A108V V 0. 950481 106 D1060 0 0. 617485 109 L109W W <0. 01 106 D106P P 0. 632087 109 L109D D 0. 106206 106 D106N N 0. 642667 109 L109I I 0. 144257 106 D106M M 0. 855673 109 L109E E 0. 194168 106 D106I I 0. 915931 109 L109R R 0. 210346 106 D106L L 0. 99561 109 L109H H 0. 220153 107 I107E E <0. 01 109 L109 0. 222755 107 I107G G <0. 01 109 L109F F 0. 317718 107 I107F F <0. 01 109 L109A A 0. 323528 107 I1070 O <0. 01 109 L109S S 0. 378623 107 I107R R <0. 01 109 L109P P 0. 434661 107 I107H H <0. 01 109 L109G G 0. 51022 107 I107W W <0. 01 109 L109V V 0. 539733 107 I107P P 0. 318743 109 L109M M 0. 628881 107 I107Y Y 0. 524182 109 L109N N 0. 658369 107 I107A A 0. 795478 109 L109T T 0. 79132 107 I107N N 0. 929935 109 L109Y Y 0. 825105 107 I107V V 0. 96863 110 G110T T <0. 01 108 A108D D <0. 01 110 G110L L <0. 01 108 A108F F <0. 01 110 G110W W <0. 01 108 A108H H <0. 01 110 G110Y Y <0. 01 108 A108I I <0. 01 110 G110P P 0. 224284 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. vtutatton Ind. 110 G110I I 0. 232219 112 S112F F 0. 878895 110 G110S S 0. 30218 112 S112A A 0. 943049 110 G1100 0 0. 343918 113 V113S S 0. 572415 110 Gil OR R 0. 476072 113 V113G G 0. 579385 110 G110H H 0. 73456 113 V113K K 0. 716865 110 Gil ON N 0. 770851 113 V113H H 0. 763416 110 G110M M 0. 816422 113 V113W W 0. 803685 III M111R R <0. 01 13 V113L L 0. 854963 III Milles S 0. 139078 113 V113T T 0. 861744 111 M111H H 0. 192733 113 V1 13D D 0. 871104 111 M111G G 0. 315165 113 V113E E 0. 936465 111 M111P P 0. 566892 113 V113C C 0. 937598 Ill M111E E 0. 668985 113 V113F F 0. 959822 11 M111L L 0. 67115 113 V113Y Y 0. 981976 III M111K K 0. 706165 114 L114H H <0. 01 111 IT T 0. 763332 114 L114E E <0. 01 111 M11 1F F 0. 776934 114 L114F F <0. 01 111 M111D 1D D 0. 78777 114 L114K K <0. 01 111 Mil IV V 0. 92522 114 L114R R <0. 01 112 S112Y Y <0. 01 114 L114W W <0. 01 112 S112R R <0. 01 114 Ll 14Y Y <0. 01 112 S112P P <0. 01 114 L1140 Q 0. 115737 112 S112H H 0. 380254 114 L114P P 0. 275464 112 S112V V 0. 479716 114 L114S S 0. 545726 112 S112M M 0. 564157 114 L1 14V V 0. 595416 112 S112W W 0. 582165 114 L114N N 0. 77333 112 S112K K 0. 678369 115 V1 15H H <0. 01 112 S112T T 0. 721644 115 V1 15K K <0. 01 112 S I 12N_I. N 0. 850159 115 VI 151 1 0. 994833 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Imd. Mutation Ind. 116 T116Y Y 0. 466112 119 L119G G <0. 01 116 T116V V 0. 571817 119 L119S S <0. 01 116 T116R R 0. 619823 119 L119F F <0. 01 116 T116L L 0. 681201 119 L119R R <0. 01 116 T116W W 0. 748358 119 Ll 19P p <0. 01 116 Til 61 I 0. 760474 119 L119T T 0. 102922 116 T1160 0 0. 768867 119 L119N N 0. 113151 116 T116P P 0. 836786 119 L119V V 0. 150373 116 T116G G 0. 901886 119 L119W W 0. 203313 116 Tl 16E E 0. 906124 119 Ll 19C c 0. 244106 116 T116A A 0 952003 119 L119D D 0. 280381 116 T116S S 0. 963005 119 19E E 0. 322167 117 0117W W 0. 707035 119 L119I 1 0. 427476 117 117V V 0. 761971 119 L119H H 0. 462912 117 0117G G 0. 794858 119 L119Y Y 0. 556343 117 0117S S 0. 86512 120 T120P P <0. 01 118 V118K K <0. 01 120 T120H H 0. 498304 118 V118W W <0. 01 120 T120R R 0. 599376 118 V118E E <0. 01 120 T120A A 0. 663543 118 V118R R 0. 069623 120 T1200 0 0. 781096 118 V118P P 0. 222399 120 T120C C 0. 924433 118 V1 18D D 0. 40168 121 S121P P 0. 384623 118 V1 18I I 0. 545694 121 S121R R 0. 701237 118 V118G G 0. 559239 121 S121W W 0. 772781 118 V118S S 0. 815888 121 S121K K 0. 77795 118 V1 18A A 0. 852723 121 S121G G 0. 992545 118 V118T T 0. 91759 122 A122G G <0. 01 118 V118M M 0. 933469 122 A122D D 0. 059137 118 V118F F 0. 998467 122 A122F F 0. 148369 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Posl PAD Perf. Mutation Ind. utation Ind. Mutation Ind. Mutation md. 122 A122H H 0. 169443 124 G124F F 0. 950801 122 A122R R 0. 396041 125 V125W W 0. 24527 122 A122S S 0. 431258 125 V125E E 0. 385171 122 A122K K 0. 450105 125 V125R R 0. 466062 122 A122E E 0. 467766 125 V125C C 0. 541228 122 A122T T 0. 520454 125 V125D D 0. 541318 122 A122P P 0. 548155 125 V125P P 0. 622352 122 A122I 1 0. 647406 125 V125F F 0. 627367 122 A122N N 0. 704284 125 V125S S 0. 790998 122 A1220 0 0. 741587 125 V125Y Y 0, 813593 122 A122W W 0. 862265 125 V125A A 0. 925641 122 A122V V 0. 886387 125 V125I 1 0. 941326 <0. 01042634 122 A122M M 0. 938855 <0. 01042634 124 G124I I <0. 01 126 G126I 1 7441542 124 G124H H <0. 01 126 G126V V 0. 175001 124 G124M M <0. 01 126 G126Y Y 0. 234673 124 G124W W <0. 01 126 G126L L 0. 540613 124 G124P P <0. 01 126 G126A A 0. 552538 124 G124A A 0. 031196. 126 G126E E 0. 599533 124 G1240 0 0. 208313 126 G126P P 0. 673809 124 G124T T 0. 315233 126 G126T T 0. 737666 124 G124V V 0. 329769 126 G126R R 0. 761417 124 G124R R 0. 409769 126 G126N N 0. 846727 124 G124L L 0. 536625 126 G126S S 0. 902662 124 G124S S 0. 555215 126 G126C C 0. 980807 124 G124Y Y 0. 559199 127 T127L L <0. 01 124 G124N N 0. 599171 127 T127E E <0. 01 124 G124D D 0. 63784 127 T1270 0 0. 151533 124 G124C C 0. 672179 127 T127I 1 0. 203586 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Positlo. Vanant Positio. Variant nIutatlon Ind. Hutatlon Ind. 127 T127H H 0. 60105 149 W149R R <0. 01 127 T127D D 0. 61747 149 W149E E <0. 01 127 T127M M 0. 639504 149 W149P P <0. 01 127 T127C C 0. 653314 149 W149C C 0. 1164 127 T127V V 0. 683337 149 W149I I 0. 235936 127 T127G G 0. 710564 149 W149A A 0. 311848 127 T127P P 0. 773291 149 W149S S 0. 329233 127 T127S S 0. 828003 149 W149 0 0. 402387 128 T128D D 0. 662836 149 W149T T 0. 440303 129 Y129W W <0. 01 149 W149G G 0. 44856 129 Y129G G <0. 01 149 W149M M 0. 494615 129 Y129K K <0. 01 149 W149F F 0. 495779 129 Y129V V 0. 01 149 W149L L 0. 637667 129 Y129T T 0. 138769 149 W149Y Y 0. 747652 129 Y129A A 0. 173554 150 F150P P 0. 31768 129 Y129R R 0. 178362 150 F150N N 0. 362798 129 Y129M M 0. 211662 150 F150G G 0. 458431 129 Y129D D 0. 228506 150 F150V V 0. 511676 129 Y129L L 0. 270643 150 F150A A 0. 539571 129 Y129N N 0. 530034 150 F150T T 0. 580879 129 Y129P P 0. 588917 150 F150W W 0. 622886 129 Y129C C 0. 610384 150 F150M M 0. 625886 129 Y129S S 0. 692051 150 F150E E 0. 727755 129 Y129F F 0. 713199 150 F150C C 0. 778063 146 P146W W 0. 680806 150 F150I 1 0. 78431 146 P146T T 0. 756105 150 F150K K 0. 848249 146 P146V V 0. 768041 153 Il 53N N 0. 890296 146 P146S S 0. 956673 154 F154T T <0. 01 148 P1480 0. 975963 154 F154D D <0. 01 Table 10-5. PAD Assay Results Table 10-5. PAD Assay Results WT/Pos/PAD Perf. WT/Pos/PAD Perf. Mutation Ind. Mutation Ind. Mutation Ind. Mutation Ind. 154 F154E E <0. 01 196 F 196M M 0. 709899 154 F154G G <0. 01 196 F196Y Y 0. 970105 154 F154L L <0. 01 154 F154P P <0. 01 154 F154V V <0. 01 154 F154S S 0. 287767 154 F1540 0 0. 973299 194 I194S S <0. 01 194 I194A A <0. 01 194 I194C C <0. 01 194 I194P P <0. 01 194 I194F F <0. 01 194 I194W W <0. 01 194 I194R R <0. 01 194 I194Y Y <0. 01 194 I194G G 0. 044503 194 I194L L 0. 577811 194 I194V V 0. 780569 196 F196H H <0. 01 196 F196G G <0. 01 196 F196S S <0. 01 196 F1960 0 <0. 01 196 F196A A <0. 01 196 F196K K <0. 01 196 F196N N <0. 01 196 F196R R <0. 01 196 F196W W 0. 38122 196 F196P P 0. 385754 196 F196V V 0. 675769

The following Table provides variants that are better than wild-type at degrading peracids (i.e., the performance index for the variant is better than the wild-type). Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 1 MOOlI 1. 19 5I005M 1. 09 1 MOO1 L 2. 11 SIOOSE 1. 59 2A002D 1. 05 51005L 1. 63 2A002R 1. 17 5 I005A 1. 88 2A002W 1. 17 5I005C 2. 47 2A002P 1. 17 5I005D 3. 11 2A002Q 1. 29 6L006C 1. 22 2A002E 1. 38 6L006M 1. 44 3 K003T 1. 03 6 L006A 1. 99 3K003S 1. 17 7C007A 1. 03 3K003Q 1. 19 7C007H 1. 37 3 K003R 1. 29 7 C007I 1. 48 3 K003Y 1. 39 7 C007E 1. 63 3 K003M 1. 44 7 C007K 2. 95 3K003P 1. 45 8F008M 1. 11 3 K003C 1. 52 8 F008L 1. 31 3 K003L 1. 84 8F008A 1. 33 3 K003H 1. 89 8 F008C 4. 01 3 K003A 2. 14 lODOlOL 2. 04 3K003I 2. 44 13T013I 1. 05 3K003E-3. 51 13T013E 1. 09 3K003G 3. 74 13T013L 1. 47 4R004D 1. 18 13T013M 1. 47 4R004C 1. 34 13T013C 1. 55 4R004P 1. 44 13T013A 1. 88 4R004A 1. 64 13T013N 2. 61 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos. Nar. PAD PI Pos. WT/Pos./Var. PAD PI 13T013P 2. 73 21D021K 1. 80 16W016K 1. 03 21D021Y 2. 01 16W016I 1. 06 22G022I 1. 03 16W016Y 1. 09 22G022T 1. 16 16W016L 1. 16 22G022E 1. 19 17V017S 1. 04 22G022L 1. 35 18P018N 1. 42 22G022P 1. 36 18 PO18Q 3. 26 22 G022Q 1. 44 18P018R 3. 97 22G022A 1. 66 18P018C 4. 16 23A023H 1. 04 18P018Y 4. 17 23A023L 1. 30 18P018V 4. 85 24P024C 1. 04 18P018E 4. 87 24P024K 1. 36 18P018G 4. 96 24P024L 1. 51 18P018H 6. 05 26E026M 1. 10 18P018L 7. 40 26E026H 1. 19 20E020D 1. 14 26E026D 1. 39 20E020S 1. 18 26E026A 1. 45 20E020H 1. 20 26E026K 1. 47 20E020T 1. 25 26E026L 1. 71 20E020V 1. 27 27R027I 1. 41 20E020A 1. 28 27R027K 1. 55 20E020W 1. 30 27R027L 2. 60 20E020N 1. 34 27R027A 2. 78 20E020P 1. 43 28F028E 1. 04 20E020Q 1. 56 28F028W 1. 17 20E020C 1. 76 28F028C 1. 21 21D021S 1. 11 28F028Y 1. 36 21D021E 1. 39 28F028M 1. 37 21D021F 1. 41 28F028A 1. 48 21D021W 1. 44 28F028L 2. 02 21D021L 1. 57 28F028D 2. 07 21D021A 1. 75 29A029C 1. 15 21 D021G 1. 76 30P030H 1. 08 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 30P030G 1. 09 33 R033N 1. 30 30P030R 1. 14 33R033A 1. 32 30P030L 1. 17 33 R033C 1. 73 30P030E 1. 24 33 R033G 2. 63 30P030Y 1. 31 33R033K 2. 72 30P030I 1. 38 33R033L 2. 90 30P030K 1. 39 34W034P 1. 21 30P030S 1. 49 34W034M 1. 22 30P030T 1. 64 34W034C 1. 49 30P030V 1. 74 34W034A 2. 29 31D031V 1. 08 35T035M 2. 72 31 D031T 1. 11 35T035A 3. 85 31 D031Q 1. 13 35T035C 4. 72 31D031W 1. 14 35T035I 5. 38 31 D031G 1. 16 35T035E 5. 73 31 D031A 1. 18 36G036C 1. 06 31 D031 S 1. 23 36G036A 1. 07 31 D031F 1. 39 36G036H 1. 10 31D031R'1. 49 36G036K 1. 71 31 D031N 1. 55 36G036I 1. 81 31 D031L 1. 61 36G036L 2. 49 32V032S 1. 09 36G036D 2. 50 32V032N 1. 61 37V037I 1. 04 32V032W 1. 71 37V037L 1. 16 32V032Q 1. 74 37V037S 1. 49 32V032G 2. 65 37V037N 1. 52 32V032M 3. 41 37V037C 1. 63 32V032I 3. 51 37V037A 2. 00 32V032A 3. 64 37V037P 2. 10 32V032E 3. 92 38L038V 1. 12 32V032D 4. 19 39A039W 1. 02 32V032L 4. 72 39A039Y 1. 13 32V032K 4. 73 40Q040N 1. 00 33R033S 1. 01 40Q040I 1. 10 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PADPI 40Q040E 1. 28 47E047K 1. 06 40Q040R 1. 48 47E047G 1. 10 40Q040L 1. 49 47E047I 1. 15 40Q040D 1. 59 48V048Q 1. 39 40Q040S 1. 65 48V048F 1. 42 40Q040T 1. 81 48V048A 1. 63 40Q040Y 2. 02 48V048M 1. 79 40Q040G 2. 17 48V048C 2. 25 40Q040W 2. 59 48V048L 2. 29 40Q040K 3. 64 48V048P 3. 08 41Q041G 1. 09 49I049Y 1. 02 41Q041H 1. 14 49I049M 1. 02 41Q041R 1. 27 49I049L 1. 03 41Q041K 1. 61 49I049G 1. 12 41Q041L 1. 92 49I049K 1. 26 41Q041A 2. 58 49I049A 1. 87 42 L042F 1. 02 50E050P 1. 02 42L042P 1. 34 50E050M 1. 04 42L042K 1. 41 50E050G 1. 11 42 L042C 1. 43 50EO50D 1. 22 43G043A 1. 07 50E050A 1. 23 43G043L 1. 82 51 E051T 1. 17 43G043E 1. 88 51E051M 1. 20 44A044C 1. 92 51E051D 1. 28 45D045F 1. 04 51E051G 1. 34 46F046C 1. 16 51E051K 2. 00 46F046A 1. 25 51 E051A 2. 72 46F046E 1. 31 52G052W 2. 47 46 F046D 1. 39 53 L053H 1. 70 46F046M 1. 42 54S054N 1. 29 46F046K 1. 46 54S054P 1. 30 46F046P 1. 50 54S054A 1. 41 46 F046L 1. 54 55 AOSSN 1. 05 47E047L 1. 02 55A055K 1. 08 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos. lVar. PAD PI 55A055C 1. 26 63P063Q 1. 05 57T057S 1. 01 63P063W 1. 11 57T057G 1. 05 63P063G 1. 22 58T058L 1. 12 63P063L 1. 23 58 T058H 1. 49 63 P063T 1. 32 59N059Q 1. 86 64T064G 1. 08 59NO59T 5, 63 64TO64M 1. 09 59N059S 7. 32 64T064A 1. 20 59N059K 8. 21 64T064L 1. 22 59N059E 9. 88 66P066S 1. 02 59N059V 9. 97 66P066T 1. 10 59NOS 10. 00 69N069D 1. 11 59N059F 10. 23 69N069A 1. 13 59N059A 10. 44 69N069Q 1. 14 59N059Y 11. 14 69N069C 1. 20 59N059C 11. 23 69N069L 1. 20 59N059D 11. 72 69N069S 1. 42 59N059W 12. 80 69N069T 1. 43 59N059L 14. 74 69N069H 1. 52 60I060G 1. 04 69N069K 1. 59 60I060V 1. 06 69N069V 1. 73 60I060H 1. 07 69 N0691 1. 75 60I060Y 1. 19 70G070L 1. 01 61D061P 1. 13 70G070A 1. 41 61D061Q 1. 16 70G070H 1. 90 61 D061L 1. 20 71 A071K 1. 01 61D061G 1. 25 71A071M 1. 11 61D061S 1. 35 72S072F 1. 15 61D061R 1. 59 72S072G 1. 76 61 D061I 1. 66 72S072M 2. 13 61D061H 1. 67 72S072C 2. 18 61D061K 1. 72 72S072H 2. 48 63P063K 1. 02 72S072N 2. 85 63P063V 1. 04 72S072A 3. 52 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 73Y073M 1. 13 80T080C 1. 15 73Y073C 1. 20 80T080S 1. 40 73 Y073A 1. 40 80T080G 1. 50 74L074F 1. 13 81H081N 1. 00 74L074M 1. 21 81H081L 1. 03 74L074A 2. 90 81H081W 1. 09 75P075E 1. 19 81H081C 1. 09 75P075L 1. 19 81H081A 1. 45 75P075W 1. 31 81H081M 1. 54 75P075Y 1. 32 82L082M 1. 06 75P075V 1. 39 83P083C 1. 01 75 P075C 1. 42 83 P083R 1. 09 75P075D 2. 09 83P083N 1. 10 76S076C 1. 06 83P083K 1. 16 76S076T 1. 11 83P083E 1. 26 76S076A 1. 11 83P083M 1. 88 76 S076H 1. 11 83 P083A 2. 36 76S076P 1. 20 84L084F 1. 01 76S076V 1. 35 84L084G 1. 01 76 S076K 1. 53 85 D085R 1. 03 76S076M 1. 61 85D085A 1. 09 76S076D 1. 94 85D085H 1. 24 76S076E 2. 09 85D085E 1. 25 76S076G 2. 15 85D085C 1. 50 76S076L 4. 70 85D085G 1. 60 77 C077T 1. 03 85 D085F 1. 98 77C077D 1. 05 86L086C 2. 44 78L078T 1. 10 86L086A 3. 32 78L078I 1. 11 87V087P 1. 64 78L078G 1. 38 87V087C 2. 22 78L078H 1. 57 87V087L 4. 30 80T080V 1. 01 88I088M 1. 09 80T080Q 1. 07 88I088P 3. 51 80T080A 1. 11 89IL 1. 22 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 89I089A 1. 83 104P104V 1. 02 89I089P 1. 91 104P104H 1. 03 90M090C 1. 09 104P104N 1. 44 90M090E 1. 15 104P104C 1. 83 90M090A 1. 41 104P104E 1. 97 90M090D 2. 88 104P104I 2. 05 91 L09lI 1. 05 104P104M 2. 24 91 L091C 1. 27 105 L105Q 1. 04 91 L091A 1. 45 105L105H 1. 23 91 L091D 1. 47 105L105R 1. 25 92G092C 2. 05 105L105G 1. 40 93 T093A 1. 05 105 L1OSW 1. 71 96T096F 1. 24 105L105F 1. 73 96T096G 1. 28 105L105C 1. 92 96T096L 1. 93 106D106S 1. 02 96T096M 2. 53 106D106W 1. 07 96T096C 3. 76 106D106E 1. 09 96T096A 4. 20 106D106C 1. 10 98A098Y 1. 15 106D106A 1. 13 98A098P 1. 26 106D106H 1. 18 98A098N 1. 40 106D106K 1, 24 98A098C 1. 42 106D106T 1. 38 98A098L 1. 47 106D106F 1. 45 98A098D 2. 19 106D106G 1. 45 100 F 1 OOC 1. 28 106D106V 1. 68 100 F 1 OOT 1. 42 107I107L 1. 04 10OF10ON 1. 45 107I107S 1. 33 100 F 1 OOA 2. 02 107I107C 1. 41 100F100M 2. 19 107I107T 1. 53 101 R101L 1. 12 108A108S 1. 00 102R102Q 1. 19 108A108G 1. 13 102R102Y 1. 29 108A108L 2. 56 102R102L 1. 64 108A108K 2. 97 102R102A 1. 79 11OG110A 1. 01 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 110 Gl l OD 1. 40 115VllSY 2. 07 110G110C 1. 43 115V115D 2. 21 11OG110E 1. 76 115V115P 2. 21 1 lOGl lOF 2. 29 115 V 115W 2. 48 111 M111C 1. 01 116T116N 1. 05 111M111A 1. 02 116T116C 1. 05 111 M111I 1. 03 116T116H 1. 08 111 Ml l lY 1. 06 116T116M 1. 39 111M111W 1. 23 117Q117F 1. 02 lllMlllN 1. 31 117Q117R 1. 05 112S112L 1. 00 117Q117T 1. 10 112S112E 1. 16 117Q117H 1. 12 113V113M. 1. 06 117Q117Y 1. 13 113V113Q 1. 11 117Q117P 1. 13 113V113R 1. 11 117Q117E 1. 21 113V113P 1. 14 117Q117A 1. 73 113V113N 1. 22 117Q117M 1. 89 113V113A 1. 31 118V118L 1. 05 114L114T 1. 05 118V118C 1. 14 114L114A 1. 07 118V118Y 1. 34 114L114G 1. 14 118V118Q 1. 50 114L114C 1. 14 119L119A 1. 02 114L114I 1. 17 120T120V 1. 07 114L114M 1. 28 120T120S 1. 07 115V115C 1. 08 120T120K 1. 09 115 V 115S 1. 14 120T120M 1. 22 115V115Q 1. 15 120T120L 1. 26 115USA 1. 19 120T120N 1. 42 115V115T 1. 28 120T120E 1. 53 115VAL 1. 30 120T120I 1. 56 115V115M 1. 32 120T120Y 1. 61 115VI15R 1. 63 121S121E 1. 04 115 V 11 SF 1. 69 121S121N 1. 06 115V115G 1. 76 121 S121Q 1. 09 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. NW/Pos. Nar. PAD PI 121S121T 1. 26 132P132Y 4. 78 121S121L 1. 49 132P132G 4. 98 121S121A 1. 55 132P132S 5. 05 121S121V 1. 59 132P132C 5. 68 121S121C 1. 64 132P132A 6. 08 122A122L 1. 02 132PI32Q 6. 15 123G123K 1. 12 133K133Y 1. 44 123G123A 1. 19 133K133L 1. 92 123 G123Y 1. 24 134V134C 1. 37 123 G123M 1. 38 134V134G 1. 42 123G123L 1. 38 134V134S 1. 44 123G123W 1. 39 134VAL 1. 45 125V125G 1. 09 134V134A 1. 64 126G126M 1. 17 134V134P 1. 71 126G126D 1. 22 134V134M 1. 89 127T127A 1. 10 134V134N 2. 80 128T128M 1. 06 135L135D 2. 90 128T128H 1. 08 136V136T 1. 13 128T128V 1. 15 136V136L 1. 13 128T128P 1. 16 136V136C 1. 23 128T128W 1. 23 136V136A 1. 60 128T128S 1. 27 137V137M 1. 13 128T128A 1. 31 137VAL 1. 27 128T128Q 1. 34 137V137C 1. 42 128T128N 1. 36 137V137A 1. 46 128T128K 1. 57 138S138G 1. 11 128T128R 1. 70 138S138C 1. 18 128T128F 1. 71 138sa 1. 28 128T128L 1. 72 138S138N 1. 31 128T128Y 1. 81 138S138P 1. 39 131A131R 1. 04 140P140C 1. 07 132P132N 1. 05 140P140A 1. 83 132P132L 2. 24 140P140H 2. 25 132P132E 3. 02 140P140F 2. 89 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 140P140G 3. 11 147H147D 1. 18 141P141A 1. 08 147H147P 1. 21 143A143C 1. 07 147H147N 1. 25 143A143E 1. 13 147H147L 1. 29 143A143D 1. 22 147H147M 1. 44 143A143L 1. 28 148P148V 1. 04 143A143H 1. 36 148P148A 1. 06 143A143K 1. 37 148P148T 1. 09 144P144M 1. 01 148P148E 1. 19 144P144F 1. 08 148P148G 1. 20 144P144Q 1. 08 148P148S 1. 21 144P144K 1. 09 148P148R 1. 25 144P144R 1. 14 148P148K 1. 30 144P144L 1. 15 148PAD 1. 34 144P144D 1. 38 148P148Y 1. 37 144P144N 1. 49 148P148L 1. 39 144P144H 1. 60 148P148F 1. 50 144ply 1. 65 149W149H 1. 01 146P146N 1. 00 150F150Y 1. 07 146P146G 1. 04 150 F 1 SOH 1. 18 146P146R 1. 06 150F150L 1. 30 146P146M 1. 23 151Q151P 1. 91 146P146A 1. 36 151Q151E 2. 07 146P146Y 1. 44 151Q151K 2. 19 146P146F 1. 53 151Q151H 2. 19 146P146H 1. 57 151Q151S 2. 25 146P146C 1. 69 151Q151R 2. 32 146P146L 2. 00 151Q151T 2. 37 147H147Q 1. 03 151Q151C 2. 55 147H147W 1. 05 151Q151Y 2. 75 147H147K 1. 06 151Q151D 2. 81 147H147E 1. 10 151Q151A 2. 93 147H147Y 1. 12 151 Q151M 6. 36 147H147C 1. 17 152L152M 1. 10 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 152L152C 1. 14 156G156H 1. 40 152L152E 1. 23 156G156Y 1. 40 152L152A 1. 29 156G156T 1. 53 152L152Y 1. 37 156G156M 1. 62 152L152W 1. 55 156G156D 1. 62 153I153V 1. 15 157G157I 1. 33 153I153A 1. 49 157G157F 1. 42 153I153L 1. 50 157G157K 1. 47 153I153T 1. 62 157G157H 1. 57 153I153S 1. 66 158E158H 1. 01 153I153F 1. 75 158E158P 1. 19 153I153P 1. 87 158E158Q 1. 24 153I153H 2. 00 158E158S 1. 27 153I153K 2. 44 158E158A 1. 28 154F154Y 4. 96 158E158R 1. 29 155E155S 1. 12 158E158W 1. 31 155E155G 1. 12 158E158C 1. 37 155E155T 1. 19 158E158N 1. 58 155E155D 1. 24 158E158M 1. 73 155E155K 1. 33 158 E158F 1. 77 155E155N 1. 79 158E158K 1. 88 155E155L 2. 07 158E158L 1. 96 155E155A 2. 59 158E158Y 2. 48 155E155P 2. 60 159Q159H 1. 48 155E155Y 2. 65 160K160N 1. 12 155E155M 2. 91 160K160A 1. 14 156G156S 1. 04 160K160R 1. 15 156G156K 1. 11 160K160D 1. 19 156G156E 1. 14 160K160C 1. 29 156G156R 1. 21 160K160Q 1. 41 156G156A 1. 21 160K160M 1. 47 156G156P 1. 29 160K160P 1. 66 156G156C 1. 37 161T161L 1. 16 156G156N 1. 38 161T161V 1. 24 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 161T161Q 1. 50 165A165R 1. 29 161T161M 1. 72 165A165Q 1. 32 161T161Y 2. 62 165A165T 1. 32 162T162R 1. 23 165A165P 1. 34 162T162G 1. 82 165A165C 1. 42 162T162S 2. 01 165A165L 1. 55 162T162W 2. 04 165A165M 1. 56 162T162I 2. 21 165A165D 1. 69 162T162Q 2. 45 166R166W 1. 08 162T162Y 2. 89 166R166F 1. 10 162T162K 3. 13 166R166K 1. 20 162T162F 3. 23 166R166N 1. 21 162T162M 3. 49 166R166Y 1. 22 162T162C 3. 57 166R166M 1. 29 162T162L 3. 59 166R166I 1. 39 162T162N 3. 84 166R166P 1. 50 162T162H 3. 91 166R166L 1. 50 162T162P 4. 37 166R166A 1. 51 163E163N 1. 00 166R166D 1. 55 163E163C 1. 08 166R166H 1. 56 163E163D 1. 08 167V167I 1. 00 163E163A 1. 79 167V167S 1. 86 163E163Y 1. 89 167V167H 2. 11 163E163L 1. 94 167V167Y 2. 15 164L164Q 1. 01 167V167R 2. 25 164L164V 1. 02 167V167Q 2. 41 164L164S 1. 11 167V167T 2. 47 164L164M 1. 26 167V167L 2. 56 164L164N 1. 31 167V167G 2. 83 164L164R 1. 61 167V167M 3. 84 164L164P 2. 41 167V167A 4. 99 165A165G 1. 07 167V167C 5. 37 165A165V 1. 13 167V167D 5. 54 165A165N 1. 20 167V167P 6. 08 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 168Y168F 5. 17 172A172D 1. 42 168Y168L 5. 39 172A172Y 1. 76 169S169Y 1. 10 173S173T 1. 29 169 S 169A 1. 13 173S173H 1. 49 169 S 169R 1. 19 173 S 173I 2. 22 169S169K 1. 27 173S173F 2. 30 169S169Q 1. 37 173S173R 2. 47 169S169C 1. 38 173S173V 2. 54 169S169M 1. 40 173S173E 2. 65 169S169L 1. 47 173S173P 2. 66 169S169I 1. 53 173S173A 2. 72 170A170C 1. 06 173S173M 3. 01 170A170E 1. 17 173S173K 3. 01 170A170F 1. 17 173S173C 3. 07 170A170N 1. 17 173 S173Y 3. 54 170A170M 1. 28 173S173W 3. 67 170A170D 1. 32 173S173L 3. 86 170A170P 1. 33 174F174H 1. 05 171L171H 1. 07 174F174K 1. 17 171L171G 1. 33 174F174P 1. 46 171L171Y 1. 35 174F174Y 1. 66 171L171T 1. 36 174F174L 1. 83 171L171V 1. 39 174F174A 2. 09 171L171I 1. 42 174F174M 2. 20 171L171K 1. 53 175M175N 1. 02 171L171A 1. 66 175M175E 1. 43 171L171C 1. 73 176K176C 1. 01 171L171S 1. 76 176K176R 1. 03 171L171Q 1. 93 176K176E 1. 08 171L171F 1. 97 176K176W 1. 16 171L171M 2. 22 176K176D 1. 18 171L171N 2. 79 176K176A 1. 19 172A172M 1. 06 176K176F 1. 28 172A172L 1. 22 176K176V 1. 33 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos./Var. PAD PI Pos. WT/Pos./Var. PAD PI 176km 1. 33 184S184Q 1. 16 178P178K 1. 70 184S184I 1. 21 178P178T 2. 28 184S184V 1. 25 178P178V 2. 70 184S184F 1. 27 178P178G 2. 95 184S184K 1. 61 178P178S 3. 06 184S184A 1. 69 178P178Q 3. 64 184S184M 1. 77 178P178M 3. 87 184S184E 1. 86 178P178E 4. 15 184S184N 1. 93 178P178A 4. 39 184S184L 2. 00 178P178D 6. 44 184S184D 2. 24 178P178Y 6. 91 184S184C 2. 39 178 P178L 7. 15 185 V185F 1. 20 179F179G 1. 16 185V185Q 1. 41 179F179V 1. 17 185V185M 1. 46 179fly 1. 47 186I186L 1. 14 179F179E 1. 80 186im 1. 38 179F179L 1. 89 186I186A 1. 79 180F180W 1. 81 186I186D 4. 29 180F180C 1. 94 187S187K 1. 16 180F180I 2. 11 187S187D 1. 40 180F180L 2. 13 187S187G 1. 46 180F180A 2. 70 187S187L 1. 46 180F180Y 2. 99 187S187H 1. 51 180F180N 3. 05 187S187I 1. 58 180F180V 3. 24 187S187N 1. 59 180F180M 4. 36 187S187C 1. 67 181D181A 1. 23 187S187A 1. 72 183G183P 1. 02 187S187M 1. 87 183G183R 1. 09 188T188N 1. 69 183G183Y 1. 45 188T188E 1. 97 183G183L 1. 50 189D189A 1. 18 183G183C 1. 99 189D189T 1. 21 184S184Y 1. 09 189D189I 1. 27 Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than Wild-Type Than Wild-Type Pos. WT/Pos. Nar. PAD PI Pos. WT/Pos./Var. PAD PI 189D189L 1. 30 197T197A 1. 42 190G190C 1. 17 197T197M 2. 38 190G190Y 1. 39 198E198T 1. 16 190G190P 1. 86 198E198S 1. 18 190G190D 2. 02 198E198F 1. 21 190G190H 2. 92 198E198V 1. 44 190G190A 3. 42 198E198Q 1. 46 190G190M 5. 54 198E198A 1. 46 191 V191T 1. 03 198E198I 1. 48 191 V191R 1. 91 198E198L 1. 54 191 V191K 2. 17 198E198N 1. 67 191 V191F 2. 75 198E198P 1. 72 19. IV19lc 2. 81 198EI98Y 1. 77 191 V191Y 4. 34 198E198W 1. 78 191 V191L 4. 69 198E198C 1. 83 191V191A 5. 06 198E198M 1. 86 191 V191E 5. 46 198E198R 1. 88 191 V191Q 5. 83 199A199F 1. 15 191 V191D 6. 03 199A199H 1. 15 191 V19lM 7. 34 199A199R 1. 17 193 G193S 1. 60 199A199T 1. 22 193G193E 3. 15 199A199E 1. 31 193 G193Q 4. 29 199A199D 1. 33 193 G193V 5. 21 199A199V 1. 45 195H195P 1. 16 199A199K 1. 53 195 H195M 1. 28 199A199Y 1. 59 195H195K 1. 33 199A199L 1. 65 195H195Y 1. 49 199A199C 2. 45 195H195E 1. 70 201N201D 1. 64 195H195D 1. 93 202R202M 1. 76 196fi 1. 12 202R202G 1. 82 196F196L 1. 17 202R202S 1. 84 196F196C 1. 18 202R202C 1. 93 197T197H 1. 24 202R202A 1. 97 Table 10-6. Variants with Peracid Degradation Greater Than Wild-Type Pos. WT/Pos./Var.PAD PI 202R202I 1.99 202R202E 2.05 202R202L 2.05 202R202T 2.06 202R202H 2.09 202R202F 2.16 202R202W 2.52 203 D203Q 1.03 203 D203S 1.13 203 D203I 1.19 203 D203N 1.28 203 D203G 1.33 203 D203F 1.34 203 D203H 1.54 203 D203P 1.71 203 D203R 1.77 203 D203A 1.96 203 D203L 2.08 203 D203C 2.09 The following Table provides variants that exhibited peracid degradation that was less than wild-type.

Table 10-7. Variants with Peracid Degradation Results Less than Wild-Type Pos WT/Pos./Var. PAD PI 1 M001 V 0.94 2A002Y 0.46 2 A002N 0.59 2 A002V 0.60 2 A002I 0.61 2A002T 0.61 Table 10-7. Variants with Peracid Degradation Results Less than Wild-Type Pos WT/Pos./Var. PAD PI 2 A002S 0.66 2 A002G 0. 84 2 A002F 0.93 3 K003V 0.84 4R004L 0.01 4 R004V 0.08 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 4R004I'0. 15 8F008S 0. 01 4R004W 0. 48 8F008R 0. 46 4R004G 0. 79 8F008H 0. 64 4R004S 0. 91 8F008G 0. 65 4R004E 0. 97 8F008T 0. 77 4R004Y 0. 98 8F008K 0. 83 4R004H 0. 99 8F008P 0. 83 4R004Q 0. 99 8F008V 0. 85 4R004T 1. 00 8F008Y 0. 90 5 I005G 0. 01 8 F008N 0. 96 5 IOOSN 0. 01 9 G009H 0. 01 5IOOSP 0. 01 9G009T 0. 01 5 I005R 0. 01 10DOlOW 0. 01 51005F 0. 15 lODOlOK 0. 01 S IOOSS 0. 37 lODOlOY 0. 01 51005H 0. 63 lODOlOT 0. 01 5 IOOST 0. 72 lODOlOI 0. 01 5IOOSV 0. 92 lODOlOV 0. 01 6L006S 0. 01 10 DOl OS 0. 01 6L006K 0. 01 10DOlOG 0. 01 6L006G 0. 01 10 DO 1 OR 0. 01 6L006H 0. 01 IODOIOA 0. 01 6L006R 0. 01 10DO10M 0. 01 6L006W 0. 01 10DOlON 0. 01 6L006E 0. 01 lODOlOP 0. 01 6L006Q 0. 01 10DO10E 0. 15 6L006V 0. 35 11SO11T 0. 01 6L006T 0. 35 11 SOllV 0. 01 6L006I 0. 82 11 SOl 1 D 0. 01 7 C007S 0. 01 11SO11E 0. 01 7 C007R 0. 01 11 SO11F 0. 01 7C007Y 0. 54 11 SOllG 0. 01 7C007M 0. 68 11 SO11L 0. 01 7C007G 0. 69 11 SO11Q 0. 01 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos. Nar. PAD PI Pos WT/Pos./Var. PAD PI 11 SOUR 0. 01 14W014E 0. 15 11 SO1 1H 0. 33 14W014F 0. 22 11 SOUK 0. 40 14W014A 0. 27 11 SO1 1A 0. 53 14W014Y 0. 66 11 SO11I 0. 56 15 G015C 0. 01 12L012V 0. 01 15G015N 0. 01 12L012S 0. 01 15G015D 0. 01 12L012G 0. 01 15GO15E 0. 01 12L012R 0. 01 15G015P 0. 01 12L012D 0. 01 15G015A 0. 61 12L012P 0. 01 lSG015S 0. 63 12L012W 0. 02 16W016S 0. 01 12L012T 0. 06 16W016G 0. 01 12L012A 0. 07 16W016H 0. 01 12L012K 0. 13 16W016T 0. 01 12L012H 0. 16 16W016R 0. 01 12L012F 0. 17 16W016N 0. 01 12L012Q 0. 22 16W016P 0. 15 12L012C 0. 22 16W016Q 0. 31 12L012N 0. 66 16W016M 0. 37 13T013Q 0. 51 16W016A 0. 55 13T013V 0. 63 16W016D 0. 57 13T013S 0. 68 16W016E 0. 65 13T013G 0. 77 16W016V 0. 88 14 W014I 0. 01 17V017A 0. 68 14 W014S 0. 01 17V017E 0. 75 14W014G 0. 01 17V017G 0. 84 14W014K 0. 01 17V017K 0. 84 14 W014V 0. 01 17V017F 0. 85 14 W014L 0. 01 17V017T 0. 86 14 W014T 0. 01 17V017Y 0. 88 14W014R 0. 01 17V017R 0. 94 14 W014N 0. 01 17V017P 0. 96 14 W014P 0. 01 17V017I 0. 99 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 17V017L 1. 00 24P024T 0. 66 18P018S 0. 07 24P024A 0. 68 19vol9p 0. 01 24P024G 0. 76 19V019M 0. 12 24P024I 0. 85 19V019R 0. 34 24P024R 0. 91 19V019Q 0. 40 24P024H 0. 97 19VO19A 0. 55 25T025P 0. 01 19V019G 0. 56 25T025H 0. 01 19V019S 0. 57 25T025L 0. 01 19V019E 0. 62 25T025R 0. 01 19 VOl 9Y 0. 70 25T025M 0. 01 19V019D 0. 79 25T025E 0. 01 19 VOl 9L 0. 91 25T025D 0. 01 l9V019K 0. 97 25T025K 0. 13 20E020L 0. 73 25T025W 0. 14 20E020G 0. 78 25T025I 0. 35 21D021P 0. 86 25T025G 0. 43 22G022K 0. 01 25T025C 0. 51 22G022W 0. 23 25T025V 0. 51 22G022R 0. 56 25T025S 0. 58 22G022V 0. 85 25T025A 0. 86 22G022S 0. 98 26E026S 0. 28 23 A023R 0. 28 26 E026T 0. 40 23A023S 0. 34 26E026W 0. 47 23 A023G 0. 35 26 E026N 0. 48 23A023F 0. 44 26E026R 0. 81 23 A023V 0. 60 26 E026G 0. 87 23 A023Q 0. 73 26 E026C 0. 94 23A023P 0. 73 26E026V 0. 97 23A023W 0. 80 26E026P 0. 99 23 A023M 0. 95 27 R027W 0. 01 23A023Y 0. 96 27R027T 0. 01 24P024S 0. 61 27R027P 0. 48 24P024Q 0. 65 27R027C 0. 58 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 27R027S 0. 69 35T035Q 0. 01 27R027G 0. 84 35T035N 0. 01 27R027E 0. 93 35T035R 0. 01 27R027V 0. 94 35T035V 0. 34 28 F028G 0. 01 36 G036S 0. 26 28F028P 0. 39 36G036T 0. 33 28F028V 0. 53 36G036V 0. 38 28F028S 0. 70 36G036M 0. 54 29A029V 0. 44 36G036N 0. 56 29A029T 0. 47 36G036W 0. 68 29A029S 0. 55 36G036Q 0. 71 29A029Y 0. 59 36G036R 0. 90 29A029P 0. 62 37V037T 0. 81 29A029R 0. 73 37V037H 0. 96 29A029W 0. 74 37V037W 0. 98 29A029M 0. 77 38lek 0. 01 29A029G 0. 80 38L038G 0. 01 29A029E 0. 84 38L038E 0. 01 29A029D 1. 00 38L038P 0. 01 30P030M 0. 79 38L038Q 0. 01 30P030Q 0. 91 38L038R 0. 01 30P030A 0. 92 38L038D 0. 12 31D031E 0. 88 38L038S 0. 29 32V032P 0. 01 38L038A 0. 63 32V032R 0. 72 38L038C 0. 72 33R033V 0. 94 39A039S 0. 01 34W034R 0. 01 39A039G 0. 30 34W034E 0. 01 39A039N 0. 43 34W034Q 0. 04 39A039R 0. 64 34W034S 0. 08 39A039I 0. 71 34W034T 0. 15 39A039P 0. 74 34W034V 0. 73 39A039T 0. 79 34W034G 0. 88 39A039M 0. 81 34W034I 0. 94 39A039E 0. 83 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 39A039C 0. 92 44A044R 0. 01 39A039K 0. 96 44A044E 0. 03 39A039L 0. 97 44A044V 0. 50 39A039V 0. 98 44A044F 0. 80 40Q040P 0. 01 44A044W 0. 85 41Q041V 0. 01 44A044M 0. 98 41Q041S 0. 22 44A044L 0. 99 41Q041P 0. 66 45D045S 0. 38 41Q041Y 0. 70 45D045T 0. 44 41Q041W 0. 88 45D045R 0. 49 42L042W 0. 01 45D045V 0. 50 42L042H 0. 01 45D045P 0. 53 42 L042T 0. 01 45 D045Q 0. 57 42L042Q 0. 28 45D045W 0. 58 42L042S 0. 45 45D045H 0. 78 42L042R 0. 64 45D045L 0. 78 42L042I 0. 66 45D045M 0. 78 42L042V 0. 73 45D045G 0. 84 42L042M 0. 74 45D045A 0. 84 42L042G 0. 76 45D045C 0. 84 43G043S 0. 23 45D045K 0. 87 43G043P 0. 31 46F046T 0. 43 43G043V 0. 33 46F046W 0. 63 43G043Q 0. 48 46F046S 0. 66 43G043R 0. 59 46F046V 0. 79 43 G043C 0. 73 46F046I 0. 88 43G043I 0. 77 46F046G 0. 94 43G043K 0. 86 47E047P 0. 36 43 G043M 0. 88 47E047R 0. 62 43 G043Y 0. 94 47 E047N 0. 63 43G043H 0. 96 47E047S 0. 63 44A044S 0. 01 47E047M 0. 70 44A044Y 0. 01 47E047A 0. 76 44A044T 0. 01 47E047F 0. 76 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 47E047C 0. 77 52G052F 0. 01 47E047T 0. 84 52G052I 0. 07 47E047D 0. 98 52G052P 0. 24 47E047H 0. 99 52G052L 0. 24 48V048R 0. 01 52G052Q 0. 28 48V048S 0. 42 52G052R 0. 35 48V048G 0. 87 52G052E 0. 55 48V048N 0. 98 52G052A 0. 79 48V048E 0. 99 53L053R 0. 01 49I049P 0. 16 53 L053W 0. 01 49I049R 0. 29 53 L053P 0. 01 49I049W 0. 68 53L053D 0. 01 49I049H 0. 74 53L053E 0. 19 49I049S 0. 79 53L053K 0. 24 49I049E 0. 88 53L053S 0. 26 49I049V 0. 97 53L053G 0. 33 50E050R 0. 01 53 L053V 0. 65 50E050W 0. 14 53 L053I 0. 66 50E050V 0. 43 53L053Q 0. 72 50E050I 0. 58 53L053T 0. 84 50E050S 0. 65 54S054F 0. 01 50E050Q 0. 91 54S054W 0. 01 50E050L 0. 97 54S054H 0. 01 51E051R 0. 01 54S054K 0. 08 51 E05 11 0. 04 54 S054I 0. 12 51EO51W 0. 17 54S054Y 0. 12 51EO51V 0. 37 54S054G 0. 17 51E051Q 0. 76 54S054L 0. 26 51EO51L 0. 93 54S054V 0. 29 52G052H 0. 01 54S054E 0. 30 52G052S 0. 01 54S054T 0. 33 52G052V 0. 01 54S054R 0. 35 52G052T 0. 01 54S054M 0. 48 52G052M 0. 01 54S054Q 0. 53 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 54S054D 0. 65 58T058V 0. 96 54S054C 0. 88 58T058S 0. 96 55AO55V 0. 01 59N059R 0. 01 55A055I 0. 01 59N059M 0. 01 55 A055P 0. 01 59N059P 0. 01 55A055W 0. 01 60I060P 0. 32 55ASS 0. 18 60I060D 0. 66 55A055R 0. 25 60I060C 0. 67 55A055T 0. 42 60I060M 0. 68 55ARG 0. 73 60I060A 0. 79 55AO55L 0. 87 60I060R 0. 81 SSAOSSS 0.'87 60I060L 0. 91 55AO55H 0. 92 60I060E 0. 92 56R056C 0. 01 60I060K 0. 96 56R056G 0. 01 60I060S 1. 00 56R056T 0. 01 61 D061F 0. 70 56R056E 0. 01 61D061A 0. 71 56R056Q 0. 01 61D061C 0. 85 56R056S 0. 12 61D061Y 0. 95 56R056L 0. 24 61D061V 0. 97 56R056N 0. 27 61D061N 1. 00 56R056A 0. 69 62D062T 0. 01 57T057R 0. 01 62D062I 0. 01 57T057P 0. 01 62D062V 0. 01 57T057N 0. 25 62D062H 0. 01 57T057C 0. 40 62D062W 0. 01 57T057Y 0. 55 62D062S 0. 01 57T057H 0. 61 62D062L 0. 01 57T057A 0. 65 62D062G 0. 01 57T057L 0. 76 62D062R 0. 01 57T057V 0. 87 62D062M 0. 01 57T057I 0. 87 62D062P 0. 01 58T058M 0. 03 62D062Q 0. 01 58T058A 0. 36 62D062A 0. 11 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 62D062C 0. 49 66P066F 0. 67 62D062E 0. 60 66P066Y 0. 70 63P063A 0. 60 66P066D 0. 72 63 P063R 0. 80 66P066I 0. 84 63 P063S 0. 90 66P066V 0. 89 63P063M 0. 91 66P066H 0. 95 63P063F 0. 93 66P066L 0. 99 63 P063Y 0. 95 67R067F 0. 01 64T064R 0. 11 67R067W 0. 02 64T064D 0. 64 67R067P 0. 04 64T064W 0. 69 67R067E 0. 11 64T064Q 0. 87 67R067V 0. 12 64T064C 0. 88 67R067Q 0. 13 64T064P 0. 94 67R067L 0. 16 64T064H 0. 96 67R067A 0. 22 64T064N 0. 98 67R067T 0. 32 64T064S 0. 99 67R067N 0. 33 65D065V 0. 20 67R067G 0. 41 65D065R 0. 22 67R067K 0. 99 65D065H 0. 40 68L068G 0. 01 65D065Y 0. 42 68L068A 0. 01 65D065P 0. 42 68L068M 0. 03 65D065S 0. 47 68L068C 0. 06 65D065W 0. 50 68L068S 0. 07 65D065T 0. 50 68L068N 0. 10 65D065G 0. 52 68L068E 0. 13 65D065I 0. 62 68L068H 0. 22 65D065A 0. 72 68L068Q 0. 25 66P066N 0. 38 68L068F 0. 25 66P066Q 0. 42 68L068T 0. 32 66P066G 0. 44 68L068P 0. 35 66P066R 0. 51 68L068D 0. 44 66P066C 0. 52 68L068Y 0. 45 66P066A 0. 56 68L068R 0. 47 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 68L068V 0. 51 71A071C 0. 99 68L068W 0. 56 72S072Y 0. 07 68L068I 0. 73 72S072W 0. 34 69N069Y 0. 17 72S072P 0. 56 69N069W 0. 55 72S072Q 0. 66 69N069P 0. 59 72 S072L 0. 70 69N069R 0. 83 72 S072R 0. 74 69N069G 0. 98 72S072D 0. 80 70G070M 0. 01 72S072V 0. 83 70 G070T 0. 01 72 S072E 0. 93 70G070P 0. 01 72S072T 0. 97 70G070V 0. 01 73Y073P 0. 01 70G070C 0. 01 73Y073R 0. 26 70G070R 0. 01 73 Y073L 0. 50 70G070Y 0. 01 73Y073G 0. 51 70G070K 0. 01 73Y073H 0. 52 70G070N 0. 01 73Y073I 0. 64 70G070Q 0. 01 73Y073S 0. 68 70G070F 0. 01 73Y073V 0. 74 70G070I 0. 27 73Y073N 0. 76 70G070E 0. 33 73Y073D 0. 80 70G070S 0. 64 73Y073Q 0. 87 71 A071P 0. 01 73 Y073K 0. 94 71 A071N 0. 61 74L074S 0. 01 71 A071D 0. 65 74L074G 0. 57 71 A071G 0. 68 74L074V 0. 61 71 A071 S 0. 69 74L074I 0. 64 71A071R 0. 77 74L074W 0. 67 71 A071H 0. 78 74L074Y 0. 86 71 A071I 0. 79 75P075M 0. 30 71A071T 0. 79 75P075R 0. 46 71 A071E 0. 81 75P075Q 0. 61 71A071L 0. 84 75P075S 0. 63 71A071F 0. 99 75P075T 0. 69 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos. Nar. PAD PI 75P075I 0. 74 79A079I 0. 67 75P075H 0. 86 79A079S 0. 78 75P075K 0. 88 79A079G 0. 92 75P075G 0. 93 79A079P 0. 94 76S076W 0. 01 79A079L 0. 96 76S076Y 0. 18 80T080W 0. 01 76S076F 0. 46 80T080L 0. 01 76S076Q 0. 90 80T080K 0. 01 77C077Y 0. 01 80T080R 0. 01 77C077R 0. 01 80T080E 0. 01 77C077W 0. 01 80T080P 0. 01 77C077F 0. 01 80T080H 0. 05 77C077G 0. 18 80T080Y 0. 11 77C077L 0. 73 80T080I 0. 15 77C077S 0. 76 80T080N 0. 53 77C077V 0. 80 81H081R 0. 01 77C077A 0. 91 81H081Y 0. 14 78L078E 0. 01 81H081K 0. 56 78L078N 0. 01 81H081S 0. 69 78L078M 0. 48 81H081V 0. 71 78L078Q 0. 52 81H081P 0. 72 78L078C 0. 78 81H081Q 0. 75 78L078Y 0. 81 81H081G 0. 80 78L078V 0. 83 81 H081F 0. 90 79A079H 0. 01 82L082R 0. 01 79A079F 0. 01 82L082S 0. 01 79A079C 0. 03 82L082W 0. 01 79A079Q 0. 27 82L082V 0. 19 79A079E 0. 27 82L082G 0. 31 79A079N 0. 28 82L082T 0. 38 79A079M 0. 28 82L082H 0. 47 79A079R 0. 32 82L082I 0. 51 79A079W 0. 53 82L082K 0. 51 79A079T 0. 60 82L082P 0. 52 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 82L082A 0. 98 86L086H 0. 01 -83P083T 0. 01 86L086S 0. 01 83P083V 0. 19 86L086R 0. 01 83P083L 0. 21 86L086E 0. 01 83P083H 0. 61 86L086Q 0. 01 83P083W 0. 62 86L086W 0. 08 83P083G 0. 68 86L086V 0. 12 83P083S 0. 79 86L086T 0. 28 83P083Q 0. 82 86L086G 0. 70 83 P083D 0. 83 86 L086Y 0. 82 83 P083F 0. 99 86 L086P 0. 99 84L084W 0. 01 87V087S 0. 01 84L084V 0. 42 87V087G 0. 01 84L084P 0. 43 87V087Y 0. 01 84L084T 0. 44 87V087R 0. 01 84L084A 0. 45 87V087K 0. 01 84L084Q 0. 52 87V087D 0. 01 84L084S 0. 55 87V087F 0. 10 84L084R 0. 57 87V087T 0. 15 84L084N 0. 67 87V087A 0. 17 84L084K 0. 79 87V087M 0. 75 84led 0. 85 88I088H 0. 01 84L084I 0. 87 88I088T 0. 01 84L084H 0. 99 88I088G 0. 01 85D085I 0. 10 88I088N 0. 01 85D085L 0. 24 88I088Q 0. 01 85D085V 0. 25 89I089H 0. 01 85D085W 0. 34 89I089S 0. 01 85D085P 0. 54 89I089G 0. 01 85D085Y 0. 55 89I089W 0. 01 85D085S 0. 68 89I089Q 0. 01 85D085T 0. 71 89I089E 0. 01 85D085N 0. 78 89I089F 0. 75 85D085Q 0. 99 89I089V 0. 82 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 89I089T 0. 90 94N094M 0. 03 90M090S 0. 01 94N094C 0. 07 90M090W 0. 01 94N094Y 0. 12 90M09OG 0. 01 94N094G 0. 53 90M090P 0. 01 94N094A 0. 74 90M090V 0. 08 94N094P 0. 79 90M090T 0. 15 94N094S 0. 88 90M09OR 0. 36 95D095E 0. 75 90M090I 0. 66 96T096I 0. 01 90M090Q 0. 77 96T096W 0. 01 90M090L 0. 98 96T096Y 0. 01 91 L091G 0. 01 96T096R 0. 14 91 L091T 0. 01 96T096V 0. 59 91 L091Q 0. 01 96T096S 0. 79 91 L091E 0. 01 96T096P 0. 89 91 L091 S 0. 43 97K097Q 0. 01 91 L091V 0. 79 97K097G 0. 01 91 L091M 0. 88 97K097I 0. 01 92G092V 0. 01 97K097W 0. 01 92G092S 0. 01 97K097L 0. 01 92G092E 0. 01 97K097V 0. 01 92G092F 0. 01 97K097Y 0. 01 93T093Q 0. 01 97K097S 0. 01 93T093Y 0. 03 97K097T 0. 01 93 T093D 0. 23 97 K097M 0. 22 93T093S 0. 49 97K097A 0. 23 93 T093F 0. 54 97K097P 0. 27 93 T093C 0. 95 97 K097R 0. 59 94N094L 0. 01 98A098T 0. 27 94N094T 0. 01 98A098G 0. 56 94N094V 0. 01 98A098S 0. 65 94N094H 0. 01 98A098I 0. 65 94N094R 0. 01 98A098H 0. 92 94N094W 0. 01 99Y099R 0. 29 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/PosJVar. PAD PI Pos WT/Pos./Var. PAD PI 99Y099V 0. 31 103 T103Y 0. 01 99Y099S 0. 37 103T103G 0. 01 99Y099W 0. 57 103 T103K 0. 01 99Y099H 0. 59 103 T103I 0. 01 99Y099I 0. 61 103 T103L 0. 01 99Y099G 0. 70 103 T103H 0. 01 99Y099P 0. 81 103 Tl 03A 0. 01 99Y099A 0. 82 103 T103V 0. 01 99Y099L 0. 86 103T103S 0. 01 100few 0. 01 103 T103C 0. 01 100F100K 0. 01 103T103R 0. 01 100F100D 0. 01 103T103N 0. 01 100F100E 0. 15 103T103F 0. 01 10OF100S 0. 85 103 T103P 0. 01 101 RlOlW 0. 01 104P104R 0. 01 101 R101K 0. 07 104P104W 0. 23 101 R101Q 0. 11 104P104T 0. 33 101 R101V 0. 44 104P104S 0. 53 101 R101D 0. 80 104P104Q 0. 85 101 RlOlY 0. 80 104P104F 0. 86 101 R101P 0. 86 104P104G 0. 98 101 R101N 0. 92 lOSL105V 0. 01 101 R101C 0. 95 105L105E 0. 53 101 R101I 0. 96 105L105S 0. 61 101 RlOlF 0. 97 105L105Y 0. 62 102R102W 0. 01 105L105T 0. 64 102R102F 0. 23 105L105P 0. 90 102R102G 0. 27 106D106R 0. 56 102R102C 0. 36 106D106Q 0. 62 102R102V 0. 61 106D106P 0. 63 102R102D 0. 68 106D106N 0. 64 102R102P 0. 89 106D106M 0. 86 102R102S 0. 96 106D106I 0. 92 103T103W 0. 01 106D106L 1. 00 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 107I107E 0. 01 110 G 1 l OP 0. 22 107I107G 0. 01 IIOGIIOI 0. 23 107I107F 0. 01 110 G1 l OS 0. 30 107I107Q 0. 01 110G110Q 0. 34 107I107R 0. 01 110gar 0. 48 107I107P 0. 32 11OG110H 0. 73 107I107Y 0. 52 11OG11ON 0. 77 107I107A 0. 80 110 ATOM 0. 82 107I107N 0. 93 111 Ml 11R 0. 01 107I107V 0. 97 111 Mil IS 0. 14 108A108E 0. 61 111M111H 0. 19 108 A108Q 0. 73 111M111G 0. 32 108AlO8T 0. 87 111 Ml 11P 0. 57 108A108V 0. 95 111 M111E 0. 67 109L109W 0. 01 111M111L 0. 67 109L109D 0. 11 lllMlllK 0. 71 109 L109I 0. 14 111 Mil IT 0. 76 109L109E 0. 19 IIIMIIIF 0. 78 109L109R 0. 21 111 Mil ID 0. 79 109L109H 0. 22 111 Mil IV 0. 93 109LI09Q 0. 22 112S112Y 0. 01 109L109F 0. 32 112S112R 0. 01 109L109A 0. 32 112S112P 0. 01 109L109S 0. 38 112S112H 0. 38 109L109P 0. 43 112S112V 0. 48 109L109G 0. 51 112S112M 0. 56 109L109V 0. 54 112S112W 0. 58 109L109M 0. 63 112S112K 0. 68 109L109N 0. 66 112S112T 0. 72 109 Ll 09T 0. 79 112S112N 0. 85 109L109Y 0. 83 112S112F 0. 88 llOGllOT 0. 01 112S112A 0. 94 110G110W 0. 01 113V113S 0. 57 110G110Y 0. 01 113V113G 0. 58 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 113V113K 0. 72 118V118K 0. 01 113V113H 0. 76 118V118W 0. 01 113V113W 0. 80 118V118E 0. 01 113V113L 0. 85 118V118R 0. 07 113V113T 0. 86 118V118P 0. 22 113V113D 0. 87 118V118D 0. 40 113V113E 0. 94 118V118I 0. 55 113V113C 0. 94 118V118G 0. 56 113V113F 0. 96 118V118S 0. 82 113V113Y 0. 98 118V118A 0. 85 114L114H 0. 01 118V118T 0. 92 114L114E 0. 01 118V118M 0. 93 114L114Q 0. 12 118V118F 1. 00 114L114P 0. 28 119L119G 0. 01 114L114S 0. 55 119L119S 0. 01 114L1 14V 0. 60 1 l9Ll l9F 0. 01 114L114N 0. 77 119L119R 0. 01 115VI15I 0. 99 119L119P 0. 01 116T116Y 0. 47 ll9L119T 0. 10 116T1 16V 0. 57 119L119N 0. 11 116T116R 0. 62 119 Ll 19V 0. 15 116tel 0. 68 1 l9Ll l9W 0. 20 116T1 16W 0. 75 119 Ll 19C 0. 24 116T116I 0. 76 119L119D 0. 28 116T116Q 0. 77 119L119E 0. 32 116T116P 0. 84 119L119I 0. 43 116T116G 0. 90 119 Ll 19H 0. 46 116T116E 0. 91 119 Ll 19Y 0. 56 116T116A 0. 95 120T120P 0. 01 116T116S 0. 96 120T120H 0. 50 117Q117W 0. 71 120T120R 0. 60 117Q117V 0. 76 120T120A 0. 66 117Q117G 0. 79 120T120Q 0. 78 117Q117S 0. 87 120T120C 0. 92 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos NW/Pos. Nar. PAD PI Pos WT/Pos. Nar. PAD PI 121S121P, 0. 38 124G124M 0. 01 121S121R 0. 70 124G124W 0. 01 121S121W 0. 77 124G124P 0. 01 121S121K 0. 78 124G124A 0. 03 121S121G 0. 99 124G124Q 0. 21 122A122G 0. 01 124G124T 0. 32 122A122D 0. 06 124G124V 0. 33 122A122F 0. 15 124G124R 0. 41 122A122H 0. 17 124G124L 0. 54 122A122R 0. 40 124G124S 0. 56 122A122S 0. 43 124G124Y 0. 56 122A122K 0. 45 124G124N 0. 60 122A122E 0. 47 124G124D 0. 64 122A122T 0. 52 124G124C 0. 67 122A122P 0. 55 124G124F 0. 95 122A122I 0. 65 125V125W 0. 25 122A122N 0. 70 125V125E 0. 39 122A122Q 0. 74 125V125R 0. 47 122A122W 0. 86 125V125C 0. 54 122A122V 0. 89 125V125D 0. 54 122A122M 0. 94 125V125P 0. 62 123G123C 0. 30 125V125F 0. 63 123G123Q 0. 31 125V125S 0. 79 123G123T 0. 54 125V125Y 0. 81 123G123E 0. 56 125V125A 0. 93 123G123V 0. 59 125V125I 0. 94 123G123R 0. 60 126G126I 0. 01 123G123N 0. 71 126G126V 0. 18 123G123H 0. 74 126G126Y 0. 23 123G123F 0. 80 126G126L 0. 54 123G123P 0. 81 126G126A 0. 55 123G123D 0. 84 126G126E 0. 60 124G124I 0. 01 126G126P 0. 67 124G124H 0. 01 126G126T 0. 74 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 126G126R 0. 76 130P130G 0. 01 126G126N 0. 85 130P130S 0. 01 126G126S 0. 90 130P130L 0. 09 126G126C 0. 98 130P130E 0. 22 127T127L 0. 01 130P130W 0. 28 127T127E 0. 01 130P130V 0. 37 127T127Q 0. 15 130P130I 0. 41 127T127I 0. 20 130P130A 0. 44 127T127H 0. 60 130P130F 0. 48 127T127D 0. 62 130P130R 0. 53 127T127M 0. 64 130P130K 0. 55 127T127C 0. 65 130P130C 0. 64 127T127V 0. 68 130P130M 0. 76 127T127G 0. 71 131 A131W 0. 01 127T127P 0. 77 131A131D 0. 40 127T127S 0. 83 131A131Y 0. 48 128T128D 0. 66 131A131L 0. 59 129Y129W 0. 01 131A131S 0. 68 129Y129G 0. 01 131A131P 0. 71 129Y129K 0. 01 131A131Q 0. 74 129Y129V 0. 01 131A131V 0. 78 129Y129T 0. 14 131A131H 0. 82 129Y129A 0. 17 131A131G 0. 87 129Y129R 0. 18 131 A131E 0. 97 129Y129M 0. 21 132P132V 0. 01 129Y129D 0. 23 132P132T 0. 01 129Y129L 0. 27 132P132W 0. 01 129Y129N 0. 53 132P132F 0. 01 129Y129P 0. 59 132PI 0. 01 129Y129C 0. 61 132P132H 0. 01 129Y129S 0. 69 132P132R 0. 01 129Y129F 0. 71 132P132D 0. 01 130P130T 0. 01 133K133C 0. 01 130P130H 0. 01 133K133A 0. 10 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 133K133V 0. 23 137 VI 371 0. 70 133K133G 0. 31 137V137T 0. 93 133K133H 0. 31 138S138I 0. 35 133K133M 0. 33 138S138V 0. 69 133K133T 0. 39 139P139S 0. 01 133K133I 0. 45 139P139G 0. 01 133K133Q 0. 52 139P139R 0. 01 133 K133S 0. 58 139P139C 0. 01 133K133F 0. 59 139P139D 0. 01 133K133P 0. 71 139P139E 0. 01 133 K133E 0. 76 139P139F 0. 01 133K133R 0. 83 139P139H 0. 01 133K133W 0. 99 139P139I 0. 01 134V134Q 0. 79 139P139K 0. 01 134V134T 0. 86 139P139N 0. 01 134V134I 0. 89 139P139Q 0. 01 135L135T 0. 01 139P139T 0. 01 135L135W 0. 01 139P139V 0. 01 135L135K 0. 01 140P140T 0. 01 135L135S 0. 01 140P140S 0. 01 135L135F 0. 01 140P140V 0. 01 135L135G 0. 01 140P140W 0. 01 135L135R 0. 01 140P140I 0. 01 135L135P 0. 01 140P140Y 0. 01 135L135Q 0. 17 140P140Q 0. 01 135L135V 0. 43 140P140R 0. 01 135L135E 0. 63 141P141R 0. 01 135L135M 0. 78 141P141G 0. 01 136V136P 0. 01 141P141S 0. 02 136V136E 0. 20 141P141T 0. 12 136V136N 0. 40 141P141V 0. 16 137V137N 0. 01 141 P141Q 0. 37 137V137G 0. 26 141 P141I 0. 38 137V137S 0. 29 141P141L 0. 65 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results- Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 141P141H 0. 79 145M145F 0. 77 141P141N 0. 97 145M145P 0. 78 142L142W 0. 01 145M145S 0. 78 142L142I 0. 28 145M145T 0. 79 142L142S 0. 31 145M145A 0. 79 142L142Q 0. 33 145M145Y 0. 82 142L142V 0. 33 145M145C 0. 93 142L142P 0. 44 146P146W 0. 68 142L142F 0. 54 146P146T 0. 76 142L142A 0. 56 146P146V 0. 77 142L142K 0. 66 146pus 0. 96 142L142C 0. 70 147H147S 0. 75 143A143W 0. 01 147H147T 0. 84 143A143P 0. 39 147H147I 0. 92 143A143G 0. 42 147H147V 0. 92 143A143S 0. 63 147H147R 0. 94 143A143F 0. 68 147H147A 0. 98 143A143Q 0. 81 148P148Q 0. 98 143A143N 0. 82 149W149R 0. 01 143A143T 0. 97 149 W149E 0. 01 143A143R 0. 99 149W149P 0. 01 143A143V 0. 99 149W149C 0. 12 144P144G 0. 62 149W149I 0. 24 144P144A 0. 79 149W149A 0. 31 144P144T 0. 81 149W149S 0. 33 144P144S 0. 92 149W149Q 0. 40 145M145W 0. 01 149W149T 0. 44 145M145G 0. 26 149W149G 0. 45 145M145E 0. 48 149W149M 0. 49 145M145I 0. 53 149W149F 0. 50 145M145Q 0. 57 149W149L 0. 64 145M145L 0. 61 149W149Y 0. 75 145M145V 0. 63 150F150P 0. 32 145M145R 0. 69 150F15ON 0. 36 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 150F150G 0. 46 155E155V 0. 47 150F150V 0. 51 155E155I 0. 65 150F150A 0. 54 155E155Q 0. 69 150F150T 0. 58 156G156I 0. 01 150 F 1 SOW 0. 62 156G156F 0. 73 150F150M 0. 63 156G156W 0. 90 150 F 1 SOE 0. 73 156G156L 0. 94 150F150C 0. 78 156G156V 0. 97 150F150I 0. 78 157G157R 0. 01 150F150K 0. 85 157G157P 0. 01 151 Q151L 0. 01 157G157S 0. 19 151Q151V 0. 01 157G157V. 0. 40 151Q151F 0. 01 157G157C 0. 61 151 Q151I 0. 01 N 157G157E 0. 84 151 Q151W 0. 32 157G157M 0. 85 152L152I 0. 61 157G157A 0. 87 152L152P 0. 61 157G157D 0. 94 152L152T 0. 69 157G157T 0. 99 152L152Q 0. 76 158E158V 0. 89 152L152G 0. 77 158E158D 0. 89 152L152S 0. 84 158E158T 0. 91 152L152D 0. 86 158E158I 0. 94 152L152V 0. 88 159Q159A 0. 28 152L152R 0. 91 159Q159C 0. 31 152L152K 0. 91 159Q159P 0. 49 152L152H 0. 92 159Q159D 0. 63 153I153N 0. 89 159Q159L 0. 70 154F154T 0. 01 159Q159G 0. 72 154F154G 0. 01 159Q159S 0. 73 154F154V 0. 01 159Q159R 0. 74 154F154S 0. 29 159 Q 159M 0. 84 154F154Q 0. 97 159Q159E 0. 97 155E155R 0. 01 160K160W 0. 01 155El55F 0. 23 160K160G 0. 30 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos. Nar. PAD PI Pos WT/Pos./Var. PAD PI 160K160H 0. 57 166R166T 0. 74 160K160S 0. 70 166R166V 0. 76 160K160L 0. 95 166R166G 0. 91 160K160I 1. 00 166R166S 0. 95 161T161R 0. 01 168Y168G 0. 01 161T161H 0. 01 168Y168T 0. 01 161T161W 0. 01 168Y168V 0. 01 161T161N 0. 01 168Y168I 0. 01 161T161G 0. 43 168Y168C 0. 01 161T161C 0. 56 168Y168Q 0. 01 161T161S 0. 57 169S169P 0. 89 161T161I 0. 98 169S169T 0. 97 163 E163F 0. 27 170A170I 0. 44 163 E163R 0. 49 170A170S 0. 47 163E163V 0. 55 170A170G 0. 62 163E163P 0. 77 170A170T 0. 72 163E163G 0. 80 170A170V 0. 74 163E163H 0. 82 170A170K 0. 83 163E163S 0. 85 170A170W 0. 83 163E163W 0. 98 170A170L 0. 85 164L164Y 0. 01 170A170Q 0. 89 164L164A 0. 01 170A170Y 0. 89 164L164D 0. 01 171L171R 0. 01 164L164E 0. 01 172A172K 0. 01 164L164G 0. 01 172A172R 0. 01 164L164H 0. 12 172A172E 0. 01 164L164F 0. 86 172A172Q 0. 18 164L164C 0. 91 172A172V 0. 39 164L164T 0. 99 172A172W 0. 45 165A165I 0. 59 172A172P 0. 58 165A165K 0. 82 172A172I 0. 58 165A165Y 0. 84 172A172T 0. 71 165A165S 0. 94 172A172N 0. 76 165A165F 1. 00 172A172G 0. 84 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 172A172S 0. 85 180F180K 0. 01 172A172C 0. 86 180F180T 0. 01 174F174W 0. 01 180F180R 0. 01 174F174Q 0. 46 180F180S 0. 01 174F174C 0. 48 180F180G 0. 01 174F174R 0. 52 180F180Q 0. 01 174F174S 0. 61 181D181Y 0. 01 174F174T 0. 64 181D181W 0. 01 174F174V 0. 67 181D181L 0. 01 174F174G 0. 91 181D181T 0. 01 175M175P 0. 08 181D181V 0. 01 175M175A 0. 66 181D181R 0. 22 175M175Y 0. 72 181D181K 0. 47 175M175G 0. 75 181D181G 0. 52 175M175W 0. 76 181D181S 0. 55 175M175V 0. 81 181D181Q 0. 60 175M175Q 0. 83 181D181P 0. 66 175M175L 0. 86 181D181E 0. 72 175M175R 0. 86 181D181C 0. 85 175M175T 0. 90 182A182I 0. 01 176K176S 0. 72 182A182R 0. 01 176K176G 0. 73 182A182Q 0. 01 176K176P 0. 78 182A182P 0. 01 176K176L 0. 92 182A182T 0. 11 176K176Y 0. 93 182A182N 0. 53 176K176N 0. 94 182A182S 0. 85 176K176T 0. 97 182AG 0. 94 176K176Q 0. 97 182A182C 0. 99 178P178W 0. 02 183G183S 0. 01 179F179Q 0. 01 183G183Q 0. 01 179F179S 0. 34 183G183V 0. 01 179F179W 0. 86 183G183F 0. 19 179F179H 0. 93 183G183H 0. 95 179F179N 0. 95 183G183D 0. 99 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 184S184T 0. 60 188T188F 0. 01 184S184H 0. 74 188T188Y 0. 09 184S184G 0. 82 188T188I 0. 10 184S184P 0. 85 188T188V 0. 15 185V185W 0. 01 188T188L 0. 42 185V185H 0. 01 188T188M 0. 75 185V185G 0. 01 188T188G 0. 79 185V185D 0. 01 188T188C 0. 87 185V185S 0. 53 188T188S 0. 91 185V185Y 0. 58 188T188A 0. 95 185V185I 0. 63 189D189F 0. 37 185V185R 0. 79 189D189R 0. 39 185V185K 0. 79 189D189N 0. 57 185V185C.'0. 83 189D189V 0. 71 185V185E 0. 88 189D189W 0. 76 185V185T 0. 91 189D189E 0. 77 185V185L 0. 93 189D189G 0. 80 186I186G 0. 01 189D189S 0. 81 186I186S 0. 01 189D189M 0. 88 186I186R 0. 01 189D189C 0. 94 186I186P 0. 01 189D189H 0. 95 186I186T 0. 23 189D189P 0. 97 186I186V 0. 48 190G190V 0. 01 186I186F 0. 76 190G190S 0. 01 187S187P 0. 01 190G190Q 0. 29 187 S1 87T 0. 23 190 G19OW 0. 41 187S187Q 0. 35 190G19OR 0. 51 187S187W 0. 52 190G190K 0. 57 187S187R 0. 55 190G190L 0. 82 187S187V 0. 58 191 V191H 0. 01 187S187F 0. 65 191 V191W 0. 01 187sly 0. 80 191 V19lS 0. 01 188T188H 0. 01 191 V191G 0. 01 188T188R 0. 01 191 V191N 0. 01 Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than Wild-Type Less than Wild-Type Pos WT/Pos./Var. PAD PI Pos WT/Pos./Var. PAD PI 191 V191I 0. 02 195H195V 0. 60 192D192S 0. 01 195H195Q 0. 96 192D192P 0. 01 195H195A 0. 98 192D192F 0. 01 196F196H 0. 01 192dz 0. 01 196F196G 0. 01 192D192I 0. 01 196F196S 0. 01 192dz 0. 01 196F196Q 0. 01 192D192R 0. 01 196F196W 0. 38 192dz 0. 01 196F196P 0. 39 192D192V 0. 01 196F196V 0. 68 192D192W 0. 01 196F196M 0. 71 192D192N 0. 15 196F196Y 0. 97 192D192C 0. 56 197T197R 0. 01 193G193H 0. 01 197T197L 0. 65 193 G193C 0. 01 197T197S 0. 75 193G193T 0. 01 197T197G 0. 81 193G193N 0. 01 197T197I 0. 84 194I194S 0. 01 197T197C 0. 86 194I194A 0. 01 197T197V 0. 89 194I194C 0. 01 197T197N 0. 91 194I194P 0. 01 199A199M 0. 93 194I194F 0. 01 199A199S 0. 99 194I194W 0. 01 199A199G 0. 99 194I194R 0. 01 201N201Y 0. 01 194I194Y 0. 01 201 N201T 0. 01 194I194G 0. 04 201 N201 V 0. 01 194I194L 0. 58 201N201R 0. 01 194I194V 0. 78 201 N201 S 0. 06 195H195S 0. 08 201N201H 0. 10 195H195C 0. 10 201 N201 G 0. 30 195H195L 0. 18 201N201L 0. 35 195Hl95N 0. 22 201N201F 0. 67 195H195R 0. 24 201N201E 0. 72 195H195F 0. 40 203D203V 0. 50 Table 10-7. Variants with Peracid Degradation Results Less than Wild-Type Pos WT/Pos./Var. PAD PI 203 D203W 0.52 203 D203E 0.90 The following Table provides variants that have protein performance indices ("Prot. PIs) better than wild-type. Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos Vff/PosJVar. Prot. PI 2A002Y 1. 61 17V017A 1. 21 2A002N 1. 30 17V017E 1. 11 2A002I 1. 25 17V017F 1. 09 2A002V 1. 18 17V017I 1. 08 2A002T 1. 17 17V017K 1. 06 2A002S 1. 15 17V017T 1. 03 5 I005M 1. 29 18P018C 2. 56 7 C007A 1. 22 18 PO1 8H 2. 50 7C007G 1. 07 18P018L 2. 50 7C007M 1. 03 18P018E 2. 47 8F008N 1. 23 18P018G 2. 47 8F008M 1. 05 18P018N 2. 35 8 F008G 1. 03 18 PO18V 2. 30 8F008P 1. 01 18P018Q 2. 13 11 SO11H 1. 06 18P018R 2. 01 11 SO 11 A 1. 04 18P018Y 1. 68 11 SO11D 1. 03 18P018S 1. 05 11 SO1 lE 1. 01 19V019G 1. 39 11 SO11Q 1. 01 19V019A 1. 23 12L012N 1. 06 19V019E 1. 10 12L012Q 1. 05 19V019Q 1. 07 13T013V 1. 17 19V019K 1. 03 14W014Y 1. 02 19V019M 1. 00 16 W016Y 1. 02 20E020G 1. 11 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos WT/Pos./Var. Prot. PI 20E020P 1. 08 30P030H 1. 05 20E020A 1. 08 30P030Y 1. 04 20E020N 1. 01 32V032M 1. 11 20E020V 1. 01 32V032A 1. 10 22G022A 1. 07 32V032I 1. 08 22G022I 1. 03 32V032Q 1. 03 23A023F 1. 03 32V032L 1. 01 24P024T 1. 43 35T035C 1. 16 24P024G 1. 34 36G036C 1. 09 24P024S 1. 31 36G036N 1. 08 24P024H 1. 15 36G036Q 1. 07 24P024I 1. 11 36G036S 1. 06 24P024L 1. 06 36G036A 1. 00 25T025C 1. 37 37V037N 1. 09 25T025V 1. 30 39A039V 1. 18 25T025G 1. 27 39A039E 1. 03 25T025A 1. 23 46F046A 1. 05 25T025I 1. 19 46F046C 1. 01 25T025P 1. 10 47E047I 1. 02 25T025M 1. 04 54S054A 1. 33 29A029G 1. 22 54S054C 1. 21 29A029P 1. 07 54S054E 1. 16 29A029M 1. 06 54S054D 1. 08 29A029D 1. 06 54S054H 1. 06 29A029V 1. 05 54S054N 1. 01 29A029S 1. 05 54S054M 1. 01 29A029T 1. 02 55A055N 1. 12 29A029E 1. 02 55A055S 1. 08 30P030E 1. 20 56R056Q 1. 02 30P030A 1. 15 58T058V 1. 13 30P030S 1. 12 60I060A 1. 20 30P030L 1. 07 60I060M 1. 14 30P030Q 1. 06 60I060V 1. 06 30P030K 1. 06 60I060L 1. 02 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos WT/Pos./Var. Prot. PI 61D061A 1. 41 67R067A 1. 39 61 D061N 1. 12 67R067V 1. 24 61D061V 1. 10 67R067P 1. 04 61 D061Y 1. 03 67R067F 1. 01 61D061Q 1. 02 68L068A 1. 07 61D061L 1. 00 68L068V 1. 01 62 D062A 1. 06 68 L068G 1. 00 62D062M 1. 06 69N069C 1. 18 63P063S 1. 17 69N069G 1. 06 63P063Y 1. 12 69N069D 1. 05 63 P063M 1. 09 69N069S 1. 03 63P063Q 1. 08 70G070A 1. 08 63 P063A 1. 06 72S072L 1. 07 63 P063V 1. 06 72 S072A 1. 06 63 P063R 1. 02 72 S072Y 1. 03 63 P063T 1. 02 73 Y073N 1. 25 64T064Q 1. 13 73Y073Q 1. 20 64T064M 1. 07 73Y073C 1. 18 64T064R 1. 05 73 Y073D 1. 09 64T064C 1. 05 73Y073V 1. 08 64T064S 1. 03 73Y073M 1. 05 66P066Q 1. 91 73Y073L 1. 03 66P066G 1. 78 74L074I 1. 45 66P066N 1. 62 74L074Y 1. 19 66P066C 1. 51 74L074V 1. 18 66P066I 1. 51 74L074A 1. 01 66P066R 1. 26 75P075M 1. 22 66P066H 1. 23 75P075S 1. 18 66P066V 1. 12 75P075T 1. 10 66P066Y 1. 08 75P075Y 1. 08 66P066A 1. 03 75P075C 1. 06 66P066F 1. 02 75P075Q 1. 04 67R067Q 1. 60 75P075L 1. 02 67R067L 1. 46 75P075E 1. 00 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos NW/Pos./Var. Prot. PI 76 S076W 1. 06 96T096G 1. 03 77C077L 1. 44 97K097A 1. 11 77C077V 1. 33 97K097R 1. 02 77C077A 1. 20 98ASS 1. 17 77C077S 1. 19 98A098T 1. 03 77C077T 1. 18 98A098N 1. 01 78L078I 1. 06 99Y099S 1. 45 78L078V 1. 04 99Y099L 1. 39 79A079C 1. 16 99Y099H 1. 30 79A079E 1. 12 99Y099A 1. 29 79A079S 1. 09 99Y099V 1. 28 79A079Q 1. 05 99Y099G 1. 23 79A079M 1. 04 99Y099W 1. 20 79A079R 1. 02 99Y099I 1. 11 80T080S 1. 12 10OF100M 1. 20 80T080E 1. 02 100 F 1 OON 1. 12 80T080Q 1. 02 100 F 1 OOW 1. 06 82L082G 1. 24 10OF100S 1. 02 82L082R 1. 15 101 RlOIL 1. 33 82L082V 1. 14 101R101N 1. 11 82L082S 1. 13 101 R101Q 1. 03 82L082P 1. 11 101R101D 1. 02 82L082M 1-. 07 102R102Q 1. 09 82L082K 1. 03 103T103G 1. 20 82 L082A 1. 00 103 T103S 1. 14 83P083G 1. 01 103T103H 1. 14 84L084V 1. 23 103T103N 1. 07 86L086Q 3. 66 103T103K 1. 05 89I089V 1. 09 103T103P 1. 01 89I089L 1. 07 104P104S 1. 44 93T093Q 2. 03 104P104V 1. 40 96T096A 1. 32 104P104E 1. 37 96T096V 1. 12 104P104C 1. 34 96T096S 1. 05 104P104N 1. 32 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos WT/Pos./Var. Prot. PI 104P104T 1. 29 113V113N 1. 01 104P104G 1. 25 114L114C 1. 10 104P104Q 1. 24 114L114A 1. 03 104P104H 1. 11 114L114M 1. 00 104P104I 1. 07 115V115I 1. 14 104P104M 1. 01 115V115C 1. 14 105L105Y 1. 18 115V115A 1. 11 105L105H 1. 07 115V115M 1. 05 105 L1OSG 1. 07 115 Vl 15L 1. 02 105L105C 1. 05 116T116N 1. 68 105 Ll O5Q 1. 03 116T116H 1. 48 105L105T 1. 00 116T116G 1. 44 . 105LI05P 1. 00 116T116C 1. 30 106D106E 1. 02 116T116E 1. 29 107I107S 1. 05 116T116Q 1. 29 107I107V 1. 04 116T116M 1. 28 107I107C 1. 00 116T116S 1. 24 108A108G 1. 15 116T116Y 1. 09 108A108S 1. 14 116T116A 1. 08 108A108T 1. 08 116T116R 1. 03 109L109E 1. 24 116tel 1. 03 109L109I 1. 21 117Q117S 1. 13 109L109D 1. 15 117Q117H 1. 12 109L109N 1. 13 117Q117E 1. 10 109 Ll 09F 1. 11 117Q117T 1. 06 109L109Q 1. 08 117 Q1 17A 1. 03 109L109A 1. 07 118V1 18C 1. 28 109LlO9H 1. 06 118V118A 1. 20 109 Ll 09V 1. 06 118V118I 1. 01 109L109M 1. 00 ll9L119C 1. 18 110G110S 1. 01 119L119A 1. 18 112S112N 1. 09 ll9L119N 1. 14 112S112E 1. 05 ll9L119I 1. 06 113V113C 1. 06 ll9L119S 1. 05 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos WT/Pos./Var. Prot. PI 119 Ll 19V 1. 04 124G124C 1. 07 119L119E 1. 04 124G124Q 1. 02 ll9L119R 1. 00 125V125I 1. 05 120T120S 1. 35 126G126N 1. 04 120T120E 1. 19 126G126E 1. 02 120T120C 1. 14 126G126A 1. 02 120T120K 1. 12 127T127A 1. 10 120T120N 1. 10 127T127S 1. 08 120T120A 1. 09 127T127V 1. 06 120T120H 1. 07 127T127C 1. 04 120T120Q 1. 05 127T127G 1. 04 120T120Y 1. 01 127T127D 1. 03 120T120L 1. 00 127T127E 1. 03 121 S121N 1. 17 127T127M 1. 02 121S121L 1. 12 128T128N 1. 29 121S121A 1. 10 128T128M 1. 28 121S121C 1. 09 128T128Q 1. 24 121S121G 1. 07 128T128A 1. 23 121S121R 1. 06 128T128H 1. 19 121S121K 1. 04 128T128P 1. 18 121S121E 1. 01 128T128D 1. 14 121S121Q 1. 01 128T128K 1. 10 122A122N 1. 11 128T128S 1. 07 122A122L 1. 07 128T128V 1. 05 122A122P 1. 07 128T128R 1. 03 122A122M 1. 06 128T128F 1. 01 122A122V 1. 05 129Y129F 1. 44 122A122S 1. 05 129Y129C 1. 42 122A122E 1. 04 129Y129A 1. 39 122A122I 1. 04 129Y129D 1. 35 122A122Q 1. 02 129Y129M 1. 28 124G124M 1. 36 129Y129N 1. 24 124G124A 1. 20 129Y129L 1. 22 124G124N 1. 18 129Y129P 1. 11 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos WT/Pos./Var. Prot. PI 129Y129G 1. 10 149W149L 1. 06 129Y129S 1. 08 150F150A 1. 70 129Y129W 1. 01 150F150M 1. 69 129Y129V 1. 00 150F150N 1. 52 130P130G 1. 11 150F150C 1. 41 130P130E 1. 08 150F150P 1. 38 130P130K 1. 05 150F1SOK 1. 33 130P130A 1. 03 150F1SOE 1. 32 130P130M 1. 03 150F150T 1. 27 133K133Q 1. 13 150F150V 1. 26 133 K133S 1. 02 1SOF150W 1. 26 133K133A 1. 01 150F150Y 1. 24 133K133R 1. 01 150F150I 1. 19 133K133E 1. 01 150F150L 1. 14 135L135M 1. 01 150F150G 1. 13 136V136L 1803 lSOF150H 1. 09 138S138A 1. 44 151 Q151K 1. 04 138S138C 1. 17 153I153N 1. 04 138S138G 1. 09 157G157A 1. 00 141 P141A 1. 13 159Q159E 1. 14 141 P141G 1. 02 159Q159A 1. 13 142L142I 1. 05 159 Q 159G 1. 03 143A143G 1. 17 161T161C 1. 01 145M145I 1. 16 162T162C 1. 17 145M145L 1. 07 162T162I 1. 16 147H147L 1. 09 162T162H 1. 08 147H147C 1. 04 162T162L 1. 05 149W149G 1. 39 162T162F 1. 05 149W149A 1. 35 162T162Y 1. 03 149W149M 1. 32 164L164M 1. 09 149W149S 1. 28 164L164V 1. 08 149W149F 1. 27 165A165G 1. 14 149W149Y 1. 15 165A165Q 1. 05 149W149Q 1. 10 165A165S 1. 05 Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than Wild-PI Values Better Than Wild- Type Type Pos WT/Pos./Var. Prot. PI Pos WT/Pos./Var. Prot. PI 166R166M 1. 26 184S184G 1. 15 166R166K 1. 19 184S184D 1. 15 166R166G 1. 19 184S184C 1. 14 166R166N 1. 16 184S184Q 1. 09 166R166D 1. 16 184S184H 1. 07 166R166A 1. 12 184S184N 1. 03 166R166L 1. 08 184S184V 1. 03 166R166T 1. 04 184S184K 1. 02 167V167L 1. 13 185V185I 1. 03 167V167H 1. 12 186I186M 1. 11 167V167G 1. 08 188T188C 2. 04 167V167M 1. 04 188T188I 1. 85 167V167I 1. 04 188T188L 1 ; 76 167V167S 1. 04 188T188M 1. 60 167V167C 1. 01 188T188V 1. 53 168Y168F 1. 28 188T188S 1. 52 168Y168L 1. 27 188T188R 1. 41 170A170C 1. 02 188T188A 1. 40 171L171I 1. 16 188T188G 1. 32 172A172C 1. 09 188T188N 1. 24 172A172G 1. 07 191 V191C 1. 04 175M175Y 1. 35 194I194L 1. 32 175M175L 1. 19 194I194C 1. 17 175M175W 1. 14 194I194A 1. 15 175M175N 1. 11 194I194W 1. 12 175M175R 1. 02 194I194V 1. 03 176K176R 1. 06 194I194Y 1. 01 176K176Q 1. 02 196F196L 1. 09 178P178E 1. 05 201N201H 1. 49 182A182C 1. 03 183 G183S 1. 08 184S184E 1. 39 184sa 1. 31 184S184M 1. 25

The following Table provides variants that have a PAD PI that is greater than 1.5, a PAF that is greater than or equal to 0.1, and a protein PI that is greater than or equal to 0.1 Table 10-9. PAD PI > 1. 5 Table 10-9. PAD PI > 1. 5 with PAF > 0. 1 and with PAF > 0. 1 and protem PI > 0. 1 protein PI >0. 1 Wild-Wild- Type Type Amino Amino. Acid/Variant Acid/Variant min i Pos. Amino Acid Pos. Amino Acid M1 L G36 K K3 A, C, H, I, L D, G, K, S, T, R4 A Q40 W, Y IS A, C, E, L Q41 A, K, L L6 A G43 E, L C7 K A44 C T13 A, C F46 L C, E, Gi H, L, V48 A, C, L, M, P P18 Q, R, V, Y I49 A E20 C, Q E51 A D21 A, G, K, L, Y L53 H G22 A A, C, D, E, F, P24 L G, K, L, Q, S, E26 L N59 T, V, W, Y R27 A, K, L D61 I, K, R F28 D, L N69 H, I, K, V P30 T, V A, C, G, H, M, D31 L, N S72 N A, D, E, G, I, K, D, G, K, S, T, V32 L, M, N, Q, W P75 W, Y R33 C, G, K, L S76 D, E, G, M T35 A, C, I, M T80 G Table 10-9. PAD PI > 1. 5 Table 10-9. PAD PI > 1. 5 with PAF > 0. 1 and with PAF > 0. 1 and prote nPI >0. 1 protei lPI >0. 1 Wild-Wild- Type Type Amino. Amino Acid/Variant Acid/Variant Pos. Amino Acid P H81 M A, C, D, E, H, P83 A, M Q151 K, P, R, S, T, Y D85 F, G L152 W L86 C I153 F, H, K, P, S, T V87 C, L F154 Y I89 A A, L, M,, N, P, T96 A, C, L, M E155 Y A98 D G156 D, M, T F100 A, M G157 H R102 A, L F, K, L, M, N, P104 C, E, I, M E158 Y L105 C, F, W T161 M, Q D106 V C, F, G, H, I, K, I1Q7 T L, M, N, P, Q, G110 E, L T162 S, W, Y VI 15 G E163 A, L, Y Q117 A, M 165 D, L, M VI 18 Q R166 A, D, H, L T120 E, I, Y A, C, D, G, H, S121 A, C, V L, M, P, Q, R, T128 F, K, L, R, S169 16 F, L A, C, E, G, L, Y8 F, L P132 Q, S, Y S169 I K133 L A, C, F, K, M, V134 A, M L171 N, Q, S 134 A, M L171 N, I, K, A A L, M, P, R, V, S173 W Y L, M, P, R, V, 140 A P 144 H, Y S 173 W, Y 146 C, F, H, L F174 A, L, M, Y A, D, E, G, K, 14. S. T. P17 L. M, O. S, T. Table 10-9. PAD PI > 1. 5 with PAF 0. 1 and protein PI > 0. 1 Wild- Type Amino Acid/Variant A' Pos. Amino Acid V, Y F179 L G190 A, H, M A, C, D, E, F, K, L, M, Q, R, V191 Y G193 S, V T197 M C, L, M, N, P, E198 R, W, Y A199 C, K, L, Y A, C, E, F, G, H, I, L, M, S, T, R202 W D203 A, C, H, L, R G205 A C, E, F, G, H, K, L, M, N, P, V206 R A209 E, L E210 D, K Q211 M, N, P A, C, D, F, G, I, K, L, R, T, V, S214 W, L215 E, M, T, V, Y GC821-2 The following Table provides variants with a PAD PI that is less than 0.5, a PAF that is greater than or equal to 0.1, and a protein PI that is greater than or equal to 0.1. Table 10-10. PAD PI < 0. 5 with Table 10-10. PAD PI < 0. 5 with PAF >0, 1. a d Protein PI 0. 1 PAF >0. 1. and Protein PI >0. 1 Wild-Type Amino Acid Wild-Type Amino Acid Residue/Pos. Variant Re i E47 4 I L V V48 S 15S MSP. R L6 IS, T. V E50 V __ 8 E1 IV D10 G G52 H. L. S. V A, C, F, G, K, Q, R, L53 E. G. KR. S 12 T. V F, G, I, K, L, R, T, F, G, I, K, L, R, S, S54 V. W. Y W14 T. V A55 I. R. T. V G15 C. N R56 C. G. S. T P18 S T57 C ! N Vl9 M O. R T58 A. M G22 9 R I60 G, H, I, K, L, M, P, C, G, H, I, L, R, S, T25 R. W D62 T. V. W E26 S T W 64 R27 P. T. W D65 H. R. S. V ! Y 65 R S V Y F28G P66G. N. O A29 T, V E, F, G, L, N, P, Q, T35 N. Q. V R67 F. V. w G36 S. T A, C, E, F, G, H, M, L38 G. S L68 P R S T Y 041 S. V N69 Y LA2 S T G70 C. T G43 P. S V S72 Y D45 R, S, T Y73 L. R F46 T P75 M. R Table 10-10. PAD PI < 0. 5 with Table 10-10. PAD PI < 0. 5 with PAli >0. 1, al d Protein PI >0. 1 PAF >0. 1* al d Protein PI >0. 1 . 1 n Protein I PAF 1 in Wild-Type Amino Acid Wild-Type Amino Acid Residue/Pos. Variant (s ! Residue/Pps. Variant (s) S76 F. W. Y R. S. W C77 F. W. Y G110O. S. T L78 Mill G H. R. S A79 C. E. H.. N. Q. R S112-H. R, V. Y T80 H. I. L. W. Y LI 14Q H81 R. Y Tl 16 y 82 G H R S T V W V118 P. R. W P83 T. V C, D, E, F, G, H, I, L84 V W L11 R T V W D85 1. L. V. W T120 H L86 S. T. V. W S121 P V87 A. F. G. S. T. Y D, E, F, G, H, K, R, I88 V 22 S I89 S G123 C M90 S. T. V, H, I, M, Q, R, T, L91 T. V G124 V. w T93 S Y V125 E. R. W N94 H. L. T. V. G126 I. V. Y 6 I R W Y T127 E L aTa IX. G, I, L, P, G, H, I ; L, 9W 99 S V P130 S T V W F100 E K W 131 D W Y R101 V W P132 F H I T V R102 C G I133 V R102C. G A, C, G, H, I, M, T, , C, F, G, H, I, K, L135 F S T V L, N, P, R, S, V, W, T103 : V137 S P104 R. T S138 I L105 V P139 s 1107 P 40 S L109 A. D. E. F. H. I. O. P141 G I R S T V Table 10-10. PAD PI < 0. 5 with PAF 1 n te'n PI Wild-Type Amino Acid R sidu V ri L142 V ~143 G. P, W M145 E. G. W A, C, F, 0, I, M, Q, W149 S. T F150 G. N. P. W E1SS F. R. V G156 I G157 R. S. V Q159 A. C. P K160 G 161 H R W E163 F. R Y168 C. L V 170 I S ATOM V 174 C W 1 S FI, S. V W V191 G. H. I. N. S. W 93 C H T 1194 A, C. G. S F196 G. Q. W T197 R G, H, L, R, S, T, V, N201Y D203 V L208 S. V. Y V212 G L215 A, C. G. KP. R L216 G I T

In addition to the assay results described above, various mutations were found to result in unstable protein such that perhydrolase protein was not expressed. Thus, in contrast to the substitutions that resulted in enhanced expression as compared to wild- type, there were some substitutions that are not as favorable, at least under the conditions used herein. However, it is not intended that the present invention exclude these substitutions, as it is contemplated that these substitutions, taken alone or in combination will find use in alternative embodiments of the present invention. Table 10-11. Mutations that Table 10-11. Mutations that Produced Unstable Protein Produced UnstaMe Protein Wild-Wild- V ian t , E, F, G, K, N, P, R, 48 A, E, F, G, K, N, P, R, V48 W mi S, T. W E51 15 W R56 H. KP. W*Y C7 L. T. W T57 W G9 C E L P R V T58 E. G. K. P. R. W. Y T13 F. R. W L74 D. H. P. 0. R. T G15 H. KL. R. Y C77 N. P P18 A L78 A. P. R. S D21 V A79 V F28 I R L86 F R33 D. E, H. P.'W rss R. Y W34 K rss D R r35 L. P. W, Y L91 H, K. P, R, W, Y G36 P A, D, L, M, P, R, T, W, V37 0. R G92 L38 w T93 P. R. V. W A39 F A, D, G, H, K, L, N, Q, L42 D D95 R. S, T. V. W. Y A44 D. H. P K97 D F46 H P104 A L Table 10-11. Mutations that Produced Un table Protein Wild- Type/Pos. Variant Ammo Acid L105 A. M 1107 H. W A108 D. F. H. I. N. P. R G110 L L114 F. K. R. W. Y VI 15 H. K. VI 34 D. K. R. W. Y V136 R. W V137 D. E. F. P. R. W S138 E. F. H. L. M. 0. R. W. Y P139 L. W. Y P140 D. K. L. M L142 D. G. M. N. R. T H147 G F154 E L. P. T161 D. E. P Y168 E H K N P R S W L171 D F179 A. P. R F180 E D181 H I. M. N A182 H. K. L. M. W. Y 1186 K. W. Y 18 D I P W F196 A Ke N, R

The following Table provides performance indices obtained in PAF and PAD assays for various variants, as well as the protein performance index. Table 1-12. Performs nce Ind ices Table10-12. P rforman In' Wild-Type Wild-Type ResJ PAF PAD Prot. Res./PAF PAD Prot. Po u P P I Pos. Mut. P ! PI PI Pos. Mut. PI Pt PI M1 A-0. 12-0. 12-0. 01 K3 K, l. OC 1. 00 I. OC M1 E-O. 12-0. 12-0. 01 K3 L 1. 04 1. 84 0. 50 1-0. 12-0. 12-0. 01 K3 M 0. 8s 1. 44 0. 71 0. 12 M1 G-0. 12-0. 12-0. 01 K3 P 0. 8C 1. 45 0. 55 M1 [0. 96 1. 19 0. 31 K3 00. 87 1. 19 0. 69 1-0. 12-0. 12-0. 01 K3 R 0. 87 1. 29 0. 46 1 0. 75. 2. 11 0. 30 0. 94 1. 1 0. 44 moi m 1. 00 1. 00 1. 00 K3 T 1. 01 1. 03 0. 71 M1 N-0. 12-0. 12-0. K3 v 0. 81 0. 84 0. 33 M1 P-0. 12-0. 12-0. 01 K3 Y 1. 0f 1. 39 0. 8e mi R-0. 12-0. 12-0. 01 R4 A 0. 41 1. 64 0. 2S M1 S-0. 12-0. 12-0. 01 R4 C 0. 71 1, 34 0. 35 M1 r-0. 12-0. 12-0. 01 R4 D 0, 27 1. 18 0. 3Z M1 V 0. 87 0. 94 0. 52 R4 E 0. 32 0. 97 0. 2 . 1-0. 12-0 O 1 4 0 1 4 0 . 1. 12 4 1 0 0. 8 0 4 0. 0 ... . 12-0. 1 41 A2 A 1. 00 1. 0 1 00 0. 0. 99 0. 59 130 105 0. 77 4 0. 24 1. 8 A2 E 0. 61 1. 38 0. 52 R4 L 0. 21-0. 03 0, 18 A2 F 1. 24 0. 93 0. 89, R4 P 0. 14 1. 44 0. 13 A2 G 1. 15 0. 84 0. 95 R4 1 0 0 0 70 A2 1 1. 18 0. 61 1. 25 R4 1. 1 0 A2 N 0. 93 0. 59 1. 30 R4 S 0. 91 0. 64 A2 P 0. 52 1. 17 0. 68 R4 T 0. 80 1. 00 0. 69 A2Q0. 81 1. 29 0. 65 R4 V 0. 29 0. 08 0. 22 0. 90 1. 17 0. 70 R4 W 0. 04 0. 48 0. 12 A2 g LO1 0. 66 1. 15 R4 Y 0. 63 0. 98 0. 39 A2 T 0. 98 0. 61 1. 17 15 A 0. 60 1. 88 0. 62 A2 v 0. 89 0. 60 1. 18 15 C 0. 44 2. 47 0. 54 1. 75 1. 17 0. 53 15 D-0. 13 3. 11 0. 06 A2 0. 84 0. 46 1. 61 15 E 0. 69 1. 59 0. 33 0. 86 2. 14 0. 48 15 F-0. 13 0. 15 0. 06 K3 c 0. 81 1. 52 0. 67 15 G 0. 05-3. 88 0. 10 K3 E 0. 12 3. 51 0. 11 IS 0. 55 0. 63 0. 18 K3 G 0. 72 3. 74 0. 08 IS I 1. 00 1. 00 1. 00 H 1. 01 1. 89 030 IS 0. 80 1. 63 0. 96 K3 1. 05 2. 44 0. 16 IS 0. 63 1. 09 1. 29 Table 10-12. Performance Indice T I 10-12 P rf rman In's Wild-Type Wild-Type ResJ PAF PAD Prot. ResJ PAF PAD Prot. Mut PI P PI P 1 v Pos. Mut. PF PI PI Pos. Mut. PI PI PI [5 N-0. 13-2. I5 0. 1, C7 Y 2. 05 0. 5'0. 69 [5 P-0. 13-0. 86 0. 08 F8 A 0. 5'1. 3 : 0. 96 5-0. 1-6. 48 0. 08 8-0. 11 4. 01 0. 1 [5 s 1. 02 0@3, 0 $3< F8 F l. OC 1. 0 {l wOC 15 2 0. 7 0. 9. 0 1. 00 1. 00 00"0 [5 V 0. 94 O. 9Z 0. 5'F8 _ 1. 02 0. 6'0. 9/ 5 1. 2 0. 72 0. 5 G 10 0 5 0. 94 0. 92 0. 54 8 1. 02 0. 64 0. 9 L6 13-0. 44-0. 01 8 0. 81 0. 8 0. 95 . 87 1. 99 0. 26 8 0 13 0. 9 6 C. 85 1. 22 0. 55. 6. 1 1 L6. H 0. 21-1. 0E O. OS F8 R 1. 43 0. 4 (0. 73 L6E-0. 20-0. 59 0. 09 F8-0. 11 0. 96 1. 23 0 4 0 1 0 i. 1 0.. 0. 9 1. 4. 4 0 . O1 0. 4. 1 g . 00 1. 00 1. 00 L6 L l OC 1. OC 1. 0 (F8 V 1. 1E 0. 8'0. 88 L6 m 0. 9 1. 44 0. 63 6 0 9 LgQ-0. 20-1. 63 0. 12 e§-0. 15-0. 18-0. 01 L6 R 0. 0e-1. 59 0. 12 G9 C. 15-0. 1-0. 01 0-1. 26. 23 9. 15. 1-0. 1 L6 r 1. 0 (, 0. 35 0. 4C G9 G l OC l. OC l. oa L6 V 1. 0S, 0. 35 0. 40 G 10 i. 100 10 1- 0 06-2. 97 0. 09 G9 K-0. 15-0. 18-0. 01 C A 1. 42 1. 03 1. 22 G9 L-0. 15-0. 18-0. 01 C 1. 00 1. 00 1. 0 9-0 1-0. 1-0 Ol o7,-0. 26 1. 63 0. 20 G9 Q-0. 15-0. 18-0. 01 C70139 0. 69 1. 07 G9 R-0. 15-0. 18-0. 01 C7 H 1. 73 1. 37 0. 41 G9 T 0. 21-2. 561 0. 12 C7 _,'1. 76 1. 48 031 09V-0. 15-0. 18-0. 01 C7 K 2. 69 2. 95 0. 21 10-0. 29-14. 24 0. 02 C7 L-0. 26-0. 16-0. 01 D10 1. 00 1. 00 1. 00 C7 1. 13 0. 68 1. 03 D0 E 0. 01 0. 15 0. 72 C7-0. 26-0. 16-0. 01 10 G 0. 41-0. 92 0. 17 C7 R 0. 22-1. 04 0. 15 D10 11. 28-6. 86 0. 04 v7 ! 0. 62-2. 83 0. 10 D0 K 2. 13-5. 30 0. 02 C7-0. 26-0. 16-0. 01 10 L 3. 97 2. 04 0. 02 C7-0. 26-0. 16-0. 01 D10 M-0. 29-5. 94 0. 04 Table 11 Performance Indices-12. P rformance In ices Wild-Type Wild-Type Res/PAF PAI) Prot. Res/PAF PAD Prot. P 1V I PI'PI Pos, Mut. PI PI PI Pos. Mut. PI PI PI D10 N-0429-2. 2'0. 0, L12 V 09E-0s0X 045] 1-0. 29-4. 16 0. 05 L12. 0 0. 0 10 22-43 0. 6 1 0. 2 1. 0 RIO 0. 79.-0. 8 0. 06 1 0 10 T1. 47-0. 45 0. 06 T13 E-0. 10 1. 09 0. 44 dito V0. 98-4. 22 0. 06 T13 F-0. 10-0. 11-0. 02 0 dito W3. 18-3. 70 0. 02 T13 G 0. 32 0. 77 0. 57 ri D10 Y1. 51-4. 97 0. 03 ri3 I0. 12 1. 05 0. 69 Sil A 0. 25 0. 53 1. 04 T13 L 0. 5 1. 4, 0. 7e Sil D-0. 25-0. 22 1. 03 T13 M 0, 1, 1. 4'0. 94 Sil 025-02 1. 0 T13 N-0. 10 2. 61 0. 27 Sil F-0. 25-0. 0 6 113E. 0. 10 2. 73 0. 17 Sll a-0, 25-0. 05 0. 8 (T13 Q 0. 01 0. 5] 0. 9E SD8-0. 25-0. 09 0. 86 1Q0. 01 0. 51 0. 98 1-0. 1 S-0. 25 0. 6 063 S 0 0. 68 Sil 1-0, 25. 6 3 T13 s 0. 73. 0. 681 0. 88 Sil K-0. 25 0. 40 0. 62.- 1 0 0 0 . Sil L-0. 25-0. 22 0. 6 0. 19 0. 63 1 Sil 0-0. 25-0. 26 1. 01-0. 10-0. 11-0. 02 Sll R-0. 25-0. 08 0. 69 W14 A-0. 23 0. 27 0. 94 Six 3'1. 1. 00 1. 00 W14 20. 06 0. 15 sa S11 0. 04-03. 87 14 0 2 0 2 1 sit V0. 03-0. 15 0. 59 W14 G0. 30-0. 97 0. 70 12 1. 10 0. 07 0. 71 14 0. 3-0 42 0 6 L12 n 2. 29 0. 22 0. 81 W14 K0. 29-0. 17 0. 71 L12 0. 04 0. 00 039 W14 0. 25-0. 36 0. 82 L12 F 0. 13 0. 17 0. 60 W14 N-0. 23-0. 12 0. 81 L12 G 0. 44-0. 06 0. 60 W14 P-0. 23-0. 29 0. 34 L12 H 0. 02 0. 16 0. 77 W14 0. 23-0. 40 0. 66 L12 K 0. 18 0. 13 0. 40 W14 S 0. 31-0. 95 0. 69 12 L 1. 00 1. 00 1. 00 14 T 0. 24-0. 77 0. 64 L12 N 0. 53 0. 66 1. 06 W14 V0. 26-0. 49 0. 58 L12 p 0. 03-0. 16 0. 31 14 1. 00 1. 00 1. 00 L12 2. 65 0. 22 1. 05 W14 y 0. 31 0. 66 1. 02 L12 R 0. 23-0. 02 0. 34 G15 A 1. 54 0. 0. 87 L12 S 0. 54-0. 07 0. 80 G15 C 0. 71-0. 27 0. 66 L12T0. 68 0. 06 0. 89 G15 D-0. 18 0. 01 0. 26 Table 10-12. P rformanc Indic T 1 1-2. P rman Indi Wild-Type Wild-Type Res t PAI PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI G15 E-0. 18-1. 42 0. 11. V17 R 1. 10 0. 94 0. 76 G15 G 1. 00 1. 00 1. 00 V17 S 0. 96 1. 04. 89 G15 H-0. 18-0. 14-0. 01 V17 F0. 93 0. 86 1. 03 G15 K-0. 18-014-0. 01 V17 V 1. 00 1. 00 1. 00 G15 L-0. 18-0. 14-0. 01 V17 Y0. 91 0. 88 0. 99 G15 N 0. 46-0. 63 0. 71 P18 A-0. 28-0. 94-0. 03 Gl-0. 18-5. 42 0. 09 18 C 1. 26 4. 16 2. 56 1-0. 18-0. 14-0. 01 1 E 1. 22 4. 87 2. 47 G 15 3 1. 05 0. 63 0. 76 P 18 G 1. 07 4. 96 2, 4 ; G15 tu-0. 18-0. 14-0. 01 P18 H 1. 12 6. 05 2. 5t W16 ~ 0. 12 0. 5$ 0. 50 pl8 L 0. 93 7, 40 2. 5 ( 16. 12 0 5. 0 1 0. 4. 0 0 2. 57 0 2 1 14 1 0. 6. 65 0. 46 1. 0 00-00 0 8 W16 0. 03-0 02 0. 8 1 1. 9 O1 W16 H 0. 03-0. 02 0. 5'P18 R 1, 16 3. 97 2. 01 W16 ? 0. 03-0. 02 0. 55 &18R1. 16 3. 97 2. 01 0. 02. 0. 74 P18 S 0. 11 0. 07 1. 05 W16 K 0. 01 1. 0'0. 71 P18 V 1. 19 4. 851 2. 30 16-0. 48 1. 16 0. 76 P18 Y133 4. 17 1. 68 W16 W 0. 04 0. 0. 19. 1. 5 1. 1. 1 0. 0. 0 0. 43 19 W16 0. 0 0. 1 0. 7 19 0 4 2. 0 W16 P 0. 03 0. 1'0. 3', Vl9 E, 0. 74 0. 62 t. la W16 00. 05 0. 31 0. 47 19 1. 32 0. 56 1 9 W16 0. 03. 41 0. 30 19 0 96 0. 7. 0 W16 S 0 09-0. 17 0. 39 19 1. 00 0. 1 0. 90 W16 T 0. 03-0. 31 0. 41 1 0. 33 0. 12 1. 00 W16 _V 0. 01 0. 88 0. 76 Vl9 P 0. 00-0. 41 0. 76 W16 W 1. 00 1. 00 1. 00 V19 00. 93 0. 40 1. 07 W16 Y 0. 22 1. 09 1. 02 V19 1. 0 0. 34 0. 82 V17 A 1. 01 0. 68 1. 21 V19 s 1. 241 0. 57 0. sa V17 E 0. 82 0. 75 1. 11 V19 V 1. 00 1. 00 1. 00 V17 F 0. 92 0. 85 1. 09 Vl9 Y 0. 94 0. 70 0. 92 V17 G 1. 17 0. 84 0. 93 E20 A 1. 29 1. 28 1. 08 V17 1 0. 95 0. 99 1. 08 E20 C 1. 57 176 0. 71 V17 K 0. 94 0. 84 1. 06 E20 0. 87 1. 14 0. 97 V17 L 0. 90 1. 00 0. 76 E20 E 1. 00 1. 00 1. 00 V17 P 0. 77 0. 96 0. 97 E20 G236 0. 78 1. 11 Tabl 10-12. Performance Indices Table0-12. Perform nce Indices Wild-Type Wild-Type ResJ PAF PAD Prot. ResJ PAF PAD Prot. P P P t 2 2. 1 1. 20 0. 92 0. 45 0 0. E20 H2. 17 1. 20 0. 92 A23 G0. 45 0. 35 0. 93 2. 20 0. 3 0 2 3 0. 16 104 3 E20 L 2. 2 (0. 71 0. 92 A23 H 0. 1 (1. 04 0. 9' 0 1. 40 1. 4 1 O1 3 0 30 13 0 75 E20 p 1. 00 1. 43 1. 08 A23 m 0. 85 0. 95 0. 90 E20 1. 27 1. 56 0. 99 A23'-0. 11 0. 73 0. 82 E20 2. 01 1. 18 0. 1. 23. 7 0. 91 2 2. 2 1. 25 0. 94 A23 0. 11 0. 28 0. 80 2 2. 11 1. 27 1. 01 3 0. 69 0. 34 0. 87 0 2. 4 1. 3. 0. 9 0 0. 60 0. 73 D21 G 1. 4f, 1. 74. 0. 84 A23 W 0. 25 0. 8C 0, 7] D21 D l. OC 1, OC 1, OC A23 Y 0. 2C 0. 9e 0. 7 D2 1 E. 0. 84 1. 3S 0. 85 P24 A 0. 54 0. 6E 0. 8E 21 1. 46 1. 75 84 0. 29. 80 0 1 1. 0 1 00 0. 9. 7 0 : 84 1 0. 85 4.. 8 D2 1 0 1. 41. 81 4. 4 0. 2 7. 76 0. 93 4 0 9 1 8 1 80. 74 4 0 4 1 D21 L1. 46. 57 0. 82 P24 I0. 42 0. 85 1. 11 D2 1 L 1. 46 1. 57 0, 82 P24 t 0, 42 0. 85 1, 11 D21 p 0. 81 0. 86 0. 74 P24 K 0. 52 1. 36 0. 71 D21 S1. 24 1. 11 0. 73 P24 L 0. 58 1 1. 06 D21 V-0. 17-0. 12-0. 02 P24 1. 0 1 0. 0 D21 w 1. 5 1. 44 0. 61 P24Q0. 50 0. 65 0. 93 D21 Y 1. 3C 2. 01 0. 42 P24 R 0. 58 0. 1 0. 85 G22 ~ 1. 55 1. 66 1. 07 P24 S 0. S3 0. 61 1. 31 G22 0. 15 1. 19 0. 56 P24 T 0. 44 0. 66 1. 43 G22 1. 00 1. 00 1. 00 5 133 0. 86 1. 23 G22 1037 1. 03 1. 03 T25 Q 0. 67 0. 51 1. 37 G22 K 0. 23-0. 22 0. 78 T25 D 0. 03-0. 07 0. 87 G22 L 0. 38 1. 35 0. 84 T25 E 0. 08-0. 29 0. 98 G22 0. 28 1. 36 0. 80 T25 G 1. 86 0. 43 1. 27 G22 00. 35 1. 44 0. 96 T25 H 0. 42-0. 02 0. 94 G22 R 0. 11 0. 56 0. 73 T25 1 1. 02 0. 35 1. 19 G22 S 1. 02 0. 98 0. 94 T25 K 0. 36 0. 13 0. 87 G22 r 1. 03 1. 16 0. 80 T25 L 0. 40-0. 04 0. 95 G22 0. 40 0. 85 0. 89 T25 91 0. 29 O. 1C 1. 04 G22 0. 25 0. 23 0. 58 T25 0. 97-0. 05 1. 10 A23 1. 00 1. 00 1. 00 25 0. 32-0. 06 0. 94 A23 0. 05 0. 44 1. 03 T25 S 1. 60 0. 5 0. 95 Table 11 Performance Indic T bl 1-1. Performan Ind'e Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI Pos. Pos. Mut. Pi T25 T 1. OC 1. 00. 00 28 10 00 1. 0 T25 V 0. 91 0. 51 1. 30 2 0 4-15 0. T25 W 0. 33 0. 14 0. 86 F28 H-0. 20-O. lS-0. 01 E26 A 1. 93 1. 4 0. 79 F281-0. 20-0. 19-0. 01 E26 1. 40 0. 94 0. 82 F28 L1. 09 2. 02 0. 51 E26 D 0. 65 139 0. 90 2 1 3 1. 37. 70 E26 E1. 00 1. 00 1. 00 F28 P 0. 02 0. 39 0. 42 E26 G 1. 28 0. 87 0. 82 F28R-0. 20-0. 19-0. 01 E26 1 3 1. 19 71 2. 0 0 7. 2 E26 K 1. 46 1. 47 0. 77 0 0. 0. 53 0. 85 E26 L 1. 3 1. 71 0, 7 F28 w 1. 16 1. 17 2 0. 10 0. 8 p 1 37. 8,. 1 1. 00 0 43 0. 99. 6 2 1. 4 1 0 77 9 1. 0 1. 7 p. 9 1. 4 102 26 1. 44 0. 40 0. 2 9 1.. 8U 1 2 1E26 N 1. 37 0. 4E 0. 88... A29 ~ 1. 00 1. 00 1. OC E26 P 0. 43 0,. 9S 0. 63 A29 f X ap8 1. 15 0. 76 E26 R 1. 48 0. 81 0, 77 A29 D 0. 87 1. oa l PQÇ E26 S 1. 27 0w2N, 092 A29 E 1. 12 0. 84 1. 02 E26 T 1. 44 0. 4 (0. 82 A29 J 1. 60 oßa 1. 22 E26 V1. 39 0. 97 0. 85 A29 0 67 0. 77 1. 06 E26 1. 25 0. 4 0. 68 A29, 0. 78 0. 62 1. 07 R27 A 0. 45 27 0 7 9 17 7 81 . 3 5 0 0. 4 1 R27 C0. 35 0. 58 0. 50 A22§1. 49 0. 55 1. 05 R27 E 0. 58 O. 95 0. 46 A29 r 1. 42 0. 47 1. 02 R27 G0. 42 0. 84 0. 24 A29 V1. 80 0. 44 1. 05 R2710. 72 1. 41 0. 70 A29W1. 91 0. 74 0. 82 R27 1. 22 1. 55 0. 69 A29 Y 1. 70 0. 59 0. 96 R27 L 0. 48 2. 60 0. 51 P30 A 1. 05 0. 92 1. 15 R27 P 0. 93 0. 48 0. 46 P30 E1. 01 1. 24 1. 20 7 R 1. 00 1. 00 1. 00 P30 G 0. 90 1. 09 0. 99 R27 S 0. 53 0. 69 0. 56 P30 1. 01 1. 08 1. 05 R27 T 0. 41 0. 01 0. 74 P30 : 0. 97 1. 38 0. 95 R27 V 0. 71 0. 94 0. 85 P30 1. 21 1. 39 1. 06 R27 W0. 21-0. 59 0. 33 P30 L 0. 96 1. 17 1. 07 F28 A 1. 27 1. 48 0. 92 30 0. 96 0. 79 0. 94 28 C 0. 93 1. 21 0. 87 30 P 1. 00 1. 00 1. 00 F28 D 0. 67 2. 07 0. 40 30 1. 01 0. 91 1. 06 F28 E 0. 51 1. 04 0. 85 P30 R 1. 16 1. 14 0. 94 Table 10-12. Performance Indi Ta le 0-12 P rformanc In' Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. N PI PI 30 S 1. 03 1. 49 1. 12 R33 G 0. 64 2. 63 0. 47 P30 T1. 05 1. 64 1. 00 R33 H-0. 16-0. 30-0. 02 P30 V1. 06 1. 74 0. 99 R33 0. 85. 2 0. 1 30 0. 79 1. 31 1. 04 R33 L 0. 34 2. 90 0. 7 D31 A1. 24 1. 18 0. 80 R33N0. 90 1. 30 0. 92 1 1. 00 1. 00 1. 00 R33 P-0. 1 6-0. 3 (-0. 02 D31 E 1. 13 0. 88 0. 93 R33 1. 00 1. 00. 00 D3 1 F 1 44 1. 35 0. 65 S 1. 0 1. 0 0. 79 D31 1 Q 144 1. 16 0. 79 0.. 4 0. 9 0. D31 1. 81 161 0. 5-0. 1-0.-0. 2 D31 N 1. 55 0. 62 W34 A-0. 15 2. 29 0. 41 B3jQ1. 07 1. 13 0. 74 W34 C-0. 15 1. 49 0. 52 1. 22 1. 49 0. 50 W34 E-0. 15-1. 86 0. 17 m a o s 4 31 1. 11 76 4 I 0 1 4 0. 7 ) 31 T. L45 1. 11 0. 76 W34 [0. 18 0'94 0. 74 D31 V1. 28 1. 08 0. 50 4-0. 1. 1 02 1. 83 1. 14 0. 60 W34 M 0. 16 1. 22 0. 91 V32 A 0. 43 3. 64 1. 10 W34 P-0. 15 1. 2 0. 26 0. 45 4. 19 0. 95 W34 00. 02 0. 04 0. 25 V32 E 0. 57 3 92 1. 00 W3 0. 22-0. 33 0. 16 V32 G 0. 58 2. 65 0. 98 W34 S 0. 47 0 0. 29 3. 91 3. 51 1. 08 W34 IT 0. 36 0. 15 0. 29 V32 K 1. 09 4. 73 0. 75 W34 V 0. 24 0. 73 0. 71 V32 0. 96 4. 72 1. 01 W34 W1. 00 1. 00 1. 00 V32 M 0. 64 3. 41 1. 11 T35 0. 45 3. 85 0. 98 V32 N 0. 54 1. 61 0. 99 T35 C 0. 55 4. 72 1. 16 V32 P 0. 01-1. 17 0. 31 T35 E 30 5. 73 0. 26 {32 O 0. 64 1. 74 1. 03 T35 1 0. 63 5. 38 0. 45 V32 R 1. 05 0. 72 0. 51 T35 K-0. 13-0. 54-0. 01 V32 S 0. 77 1. 09 0. 85 T35 L-0. 13-0. 54-0. 01 V32 V1. 00 1. 00 1. 00 T35 W 0. 17 2. 72 0. 40 V32 _ 0. 94 1. 71 0. 70 T35 N0. 20-2. 29 0. 43 R33 A 0. 20 132 0. 52 35-0. 13-0. 54-0. 01 3 C 0. 44 1. 73 0. 95 T35 0. 57-2. 07 0. 52 3-0. 16-0. 30-0. 02 T35 0. 18-11. 34 0. 23 3 E-0. 16-0. 30-0. 02 35 T 1. 00 1. 00 1. 00 Tabl10-12. erformanc Indices Table 1-12 Performanc Indices Wild-Type Wild-Type ResJ PAF PAD Prot. Res./PAF PAD Prot. Pos. PI I Pos. Mut. I PI P T35 0. 71 0. 34 0. 81 L38 c 0. 64 0. 72 0. 89 1 T35 W-0. 13-0. 54-0. 01 L38 D-O. ls 0. 12 0. 24 T35. 3-0. 54-0. 01 L38 E-0, 15-0. 61 0. 2 d36 A 0. 63 1. 07 1. 00 L38 G0. 15-0. 72 0. 32 3 0 3 1. 06 1. 09 L38 K0. 63-0. 22 0. 16 G36 D-0. 12 2. 50 0. 28 L38 L 1. 00 1. 00 1. 00 G36 cil-0. 12-0. 10-002 L38 p-0. 15-0. 78 0. 28 0. 7 1. 10 0. 98 38-0. 02 0. 47 1. 32 1. 81 0. 31 L38 R-0. 1'-0. 96 0. 34 336 K 1. 2S 1. 71 0. 84 L38 S 0. 3E 0. 25 0. 48 1. 7 1. 24 0. 84 0. 3S L38 0. 29 0. 48 l. 8 6 0 5. 4 0. S. 1-0. . 4. 5 1 8 1 0. 00 1. 2-0. 1 02. 9. 50 . 5 0 1. 0 9 1. 9 3 G36 p-0. 12.-0. 10-0. 02 A39 c 0. 63. 9. 50 336 O 0. 56 0. 71 1. 07 X39 E I. OS 0. 83 1. 03 . 9 0. 90 0. -0. 1-0. 1 0 G36 P-0. 12-0. 10-0. 02 A39 C0. 63 0. 92 0. 50 G36 00. 56 0. 71 1. 07 A39 E1. 09 0. 83 1. 03 G36 R0. 99 0. 90 0. 85 A39 ?-0. 17-0. 11-0. 02 6 s 0. 78 0. 26 1. 06 A39 1. 17 0. 0 0 92 G36 T 0. 76 0. 33 0. 83 A39 I 1. 2f 0. 71 0. 91 36 V0. 95-0. 38 0. 42 A39 K 1. 36 0. 9e 0. 90 3 0 91 0. 68 0. 57 L. 4 0 0 V37 A 1. 25 2. 00 0. 63 0. 52 0. 81 0. 46 V37 C1. 09 1. 63 0. 68 A39 Sot0. 51 0. 43 0. 45 V37 H 1. 21 0. 96 0. 78 A39 P0. 69 0. 74 0. 45 V37 1 1. 26 1. 04 0. 77 9 R 1. 17 0. 94 V37 L 1. 16 1. 16 0. 71 ~39 S 0 45 4. 31 0. 16 V37 N 0. 90 1. 52 1. 09 9 T 1. 26 0. 79 0. 92 V37 P 0. 53 2. 10 0. 73 A39 V1. 21 0. 98 1. 18 V37 Q-0. 11-0. 14-0. 02 A39 W1. 23 1. 02 0. 94 V37 R-0. 11-0. 14-0. 02 A39 Y 1. 36 1. 13 0. 90 V37 S 1. 4C 1. 49 0. 81 40 1. 16 1. 59 0. 69 V37 T 1. 05 0. 81 0. 63 40 E 1. 08 1. 28 0. 81 Q40 G 1. 79 2. 17 0. 93 0. 112 0. 1441 040 1 2. 58 1. 10 0. 49 V37 V S 2-0. 02 340 K 2. 61 3. 64 0. 52 37 0. 92 0. 98 0. 62 40 L 2. 14 1. 49 0. 53 L38 A 0. 55 0. 63 0. 78 40 1. 53 1. 00 0. 78 Table 10-12. Performance In ice Table 1-12 rf rman I i Wild-Type Wild-Type Res./PAF PAD Prot. ResJ PAF PAD Pro PI PI s M Pos. Mt, PI PI Ft Pos. Mat. PI PI H Q40P0. 45-0. 19 0. 24 G43E1. 48 0. 73 036 040 P 00 1. 00 1. 00 43 125 1 8 0 6 040 Q l. OC l. OC l. Ot d43 E 1. 25 1. 8t 0. 66 040 R 1. 89 1. 48 0. 61 G43 1. 0 1 1, 0 040 S 1. 57 1. 65 0. 8 G43 H 1. 1 0. 9 0. 63 2. 01 1. 81 0. 75 G43 1 0. 9 0. 7 0. 4 239 2. 59 0. 54 543 K 1. 42 0. 86 0. 65 040 Y 1. 83 2. 02 0. 65 G43 1. 22 1. 82 0. 42 041 A 1. 03 2. 58 G43 1. 37 0. 88 041 G0. 97 1. 09 0. 77 G43 P1. 08 031 0. 65 V 041, H 1. 12 1. 14 0. 85 343 O 0. 91 0. 4E, 0. 61 041 K 1. 3E 1. 61 0. 7C 343 R 1. 22 0. 55 0. 59 041 L l oa 1. 92 0. 75 343 S l. la, 0. 2 0. 79 041 P 0. 21 0, 6e 0. 44 343 V 0. 93 0. 3 : 0. 44 04,. X Q 1. 00 l. OC l. OC 343 Y 1. 26 0. 94, 0. 3e 041 R 1. 19 1. 27 0, 74 ~44 A l. oa l. Ot l OC 1. 12 1. 14 0. 92 4 0. 91 0. 4 . 8 1 61 0 70 4, 2 l 6 070 19-79 1. 1.. 0 9 .1.9 1. 2 1 0 0 Po 041 V 1. 07-0. 05 0. 90 ~44 D-0. 17-0. 11-0. 01 Q41 W 1. 14 0. 88 0. 71 A44E-0. 17 0. 0 0. 10 109. 70 0. A44 F 2. 84 0. 80 0, 9 J42 c 0. 7 1. 43 0. 68 44-0-0-0 O1 L42 D-0. 14-0. 17-0. 02 s44 L 1. 61 0. 99 0. 871 L42 F 1. 07 1. 02 0. 48 A44 m 1, 2 0, 98 0. 71 L42 G 1. 17 0. 76 0. 5C ~44 P-0. 17-0. 11-0. 01 L42 H 1. 92-0. 33 0. 15 A44 R 0. 29-2. 17 0. 08 L42 I 0. 97 0. 66 0. 83 A44 S. 52-0. 92 0. 16 L42 K 2. 46 1. 41 0. 13 A44 T030-1. 11 0. 14 L42 L 1. 00 1. 00 1. 00 44 V 2. 13 0. 50 0. 94 L42 M 0. 78 0. 74 0. 95 A44 1. 40 0. 85 0. 61 L42 P 0. 71 1. 34 0. 23 A44 Y 0. 3a-0. 23 0. 10 L42 0. 57 0. 28 0. 40 D45 A 1. 04 0. 84 0. 99 I, 42 138 0. 64 0. 15 D45 v 0. 83 0. 84 0. 48 2 S 0. 97 0. 45 0. 46 D45 D100 100 100 L42 T 1. 08-0. 04 0. 41 D45 F 1. 11 1. 04 0. 66 L42 V 0. 91 0. 73 0. 74 D45 G 1. 13 0. 84 0. 94 LA. 2 2. 06-0. 70 0. 14 45 1. 13 0. 78 0. 70 G43 1. 49 1. 07 0. 45 45 K 1. 34 0. 87 0. 86 Table 10-12. Performan In ices Ta 1 0-12. Perf rmanc Wild-Type Wild-Type Res./PAF PAD Prot. ResJ PAF PAD Prot. Pos. Mut. PI PI'I 1VI D45 L1. 05 0. 78 0. 55 E4. 91 0. 63 0. 99 D45 0. 86 0. 78 0. 88 1 6. 6 4 D45 P 0. 75 0. 53 0. 72 E47 2. 45 0. 62 0. 75 1. 04 0. 57 0. 81 E47. 1. 2. 6 D45 R. 16 0. 49 0. 72 E47 r 1. 96 0. 84 0. 98 D45 s 1. 13 0. 38 0. 95 V48 60 1. 63 0. 47 D45 1. 27 0. 44 0. 86 4 0 83. 2. 91 D45 V 1. 05 0. 50 0. 70 V48 30. 02 0. 0. 18 D45 W 1. 15 0. 58 0. 54 V48 ? 0. 67 1. 42 0. 57 F46 A 0. 92 1. 25 1. 5 4 0. 61 0. 8 0. 25 4. 4 1 1. O1 4 0 1 F46 C 0. 84 1. 1 (1. 01 V4g L 0. 92 2. 2S 0. 91 46 1 25 1. 4 2. F46 D1. 17 1. 39 0. 54 V48 M0. 85 1. 79 0. 71 F46 E 1. 25 1. 3] 0. 38 V48 S-Q. 15 o. sa 0. 23 F4Ç F 1. 00 1. 0 (1. 00 V48 D 0, 21 3sQk 0. 34 F46 10. 94 0. 61 4 0. 9 1. 3 F46 H-0. 13-0. 1 :-0. 01 V48 R 0. 76-1. 1 S 0. 15 1460. 90 0. 88 0. 91 V48 g 0. 65 0. 42 0. 4 F46 1. 00 1. 46 0. 48 V48 V 1. 00 1. 00 1. 00 4 0. 78 1. 54 0. 74 V48 W-0. 15-O. 1S-0. 02 F46 78 1. 42 0. 81 149 0. 92 8 0. 58 F46 0 64. 0 0. 26 4 1. 02 0. 8 0. 75 F46 g 0. 73 0. 66 0. 72 1. 34 1. 12 0. 28 F46 T0. 86 0. 43 0. 79 149 H 1. 27 0. 74 0. 77 F46 v 0. 82 0. 79 0. 89 49 1. 00 1. 00 1. 0 F46 W 0. 94 0. 63 0. 91 149 K 1. 23 1. 26 0. 72 E47 A 0. 95 0. 76 0. 84 149 L 1. 14 1. 03 0. 93 E47 C 0. 83 0. 77 0. 99 149 m 1. 01 1. 02 0. 69 E47 D 0. 99 0. 98 0. 97 149-P 0. 47 0. 16 0. 29 E47 E 1. 00 1. 00 1. 00 149 t 1. 05 0. 29 0. 56 E47 F 1. 09 0. 76 0. 96 149-S 1. 24 0. 79 0. 70 E47 G 1. 20 1. 10 0. 76 149 V 120 0. 97 0. 94 E47 H 1. 27 0. 99 0. 93 149 W 0. 70 0. 68 0. 64 E47 1 1. 03 1. 15 1. 02 149 y 1. 07 1. 02 0. 82 E47 K 1. 19 1. 06 0. 89 E50 A 1. 12 1. 23 0. 58 E47 L 1. 00 1. 02 0. 96 E50 D 0. 78 1. 22 0. 80 E47 0. 90 0. 70 0. 84 E50 E 1. 00 1. 00 1. 00 Table 10-12. Performance Indices Table 1 (-12. Performance Indices Wild-Type Wild-Type ResJ PAF PAD Prot. Res./PAF PAD Prot. Pos. P P P P I ES0 0. 93 1. 11 0. 60 0. 10. 17 0. 76 E50 0. 84 0. 58 0. 67 G52 V0. 10. 0. 16 0. 86 E50 L1. 19 0. 97 0. 41 G52 W0. 92 2. 47 0. 13 ESO M 1. 1E 1. 0 0. 3E L53 D 0. 0] 0. 0] 0. 72 E50 1. 18 1. 04 0 38 5 0. 7 E50 O 0. 9E 0. 9 0. 7 (L53 G 1. 3,, 0. 3'o. sa 50'0. 85 1. 02 0. 1 0. 0. 77 ESO S 0. 8, 0. 6. 0. 7f L53 _ O. S'0. 6 (o. sa E50 00. 98 0. 91 0. 70 53 1 2 0. 80 0. 98 E50 W 0. 7'0. 14 0. 15 L53 L 1. 0 (1. 0 (, l. OC S 0. 46-0. 77 0. 20 3 5. 5 170 0. E50 1 D 0. 65 0. 76 5 0. 5 0. 6 0. 88 ES I E 1. 0 (1. 01 1. 0 (L53 R Q. 2 (-Q. O, 0. 6e E50 1. 001 0. 43 0. 81 L53 K 0. 89 0. 24 0. 70 0. 7 0 14 0. 19 5 1. 00 00 . 2 7 0 74 1 . 6. 2 0. 91 1. 48 0 0. 8 1 0 1. 00 1. 0 0 . 9 Tua. 14 E51 K 0. 38 2. 00 0. 36 V0. 52 0. 65 0. 88 E51 1 1. 11 0. 93 0. 57 L. 0-0. 0 1 0. 40 1. 2 0. 84 S54 A 3. 4f 1. 41 1. 33 E51'-0. 12-0. 39-0. 02 S54 1 0. 121 7 E51 00. 98 0. 76 0. 84 S54 3-0. 17 0. 65 1. 08 E51 R 0. 35-0, 97 0. 29 S54 E-0. 1. 16 ES1 r 1. 1E 1. 1'0. 4E S54 F 0. 74-0. 14 0. 91 1. 47 0. 37 0. 70 54 1. 43 0. 17 0. 93 0. 44 0. 1 0. 22 S54-0. 17 0. 00 1. 06 4 78M2 G52 A0. 54 0. 79 0. 0 S 4 I 4. 78 0. 12 0. 94 G52 E-0. 12 0. 55 0. 41 S54 K 1. 44 0. 08 0. 78 G52 F-0. 12-0. 0X 0. 52 S54 L 2. 02 0. 26 0. 59 G52 G 1. 00 1. 00 1. 00 S54 M 0. 01 0. 48 1. 01 G52 H 0. 18-0. 60 0. 49 S54 N 0. 29 1. 29 1. 01 G520. 10 0. 07 0. 80 S54 P5. 20 130 0. 98 G52 L 0. 1, 092 0+5E S54 1. 03 0. 53 0. 99 G52 0. 05-0. 64 0. 56 S54 R 3. 3E, 0. 3. 0. 84 G52 P-0. 12 0. 24 0. 76 S54 S 1. 00 1. 00 1. 00 G52-0. 12 0. 28 0. 52 S54 T 1. 46 0. 33 0. 88 G52-0. 12 0. 35 S54 v 4. 72 0. 29 0. 95 G52 S 0. 13-0. 1 0. 83 S54 0. 11-0. 07 0. 83 Table 10-12. Perf r an e Indi s T bl 10-2 Pe rman I Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. M Pos. Mut. PI PI PI Pos. Mut PI PI PI 554 Y 0+37 0. 12 085 T57 L 063 °. ? f o. s< - 0. 11-0. 15-0. O1 7 0. 8 0. 2 0. 6 A55 C 0. 14 1. 26 0. 9E T57 P 0. 33-0. 8', 0. 1 ASS G 1. 69 0. 73 0. 9E T57 R 1. 61.-0. 6 (0. 14 A55 H 0. 04 0. 93 57. 3 1. 01 0. 88 A55 [0. 34-0. 43 0. 80 7E1. 00 1. 00 1. 00 ASS K 0. 2 1. 08 0. 68 T57 1. 28 0. 87 0. 84 A55 L 0. 11 0. 7 0. 81 5-0. 08-0. 10-0. 01 A55 N 0. 34 1. 05 1. 12 rs7 Y 0. 52 0. 5s 0. 4 A55 p-0. 11-0. 01 0. 84 T58 0.. 6 . 25 0. 99 5 0 0 u , t7 1. S . 1 . 4. 91 8 i. 4 0 9 6 0 0 0 8 0. 94 5 0 0. 0 0. 0 0 ASS s 0. 7. 87 1. 08 T58 G. 0. 19-I-o. 02 A55 T 1. 69 0. 42 0. 91 T58 H 0. 89 1. 0 74 A55S : 0. 00-0. 05 0. 88 T58 JL0. 88 1. 12 0. 78 A55 Y0. 00 0. 18 0. 94 r58 M 0. 56 0. 03 0. 50 A55 0. 2. 69 0. 8. 1-0. 1-0 0 ASS r 1. 69 0. 42 o. sl rss H 0. 8S 1. 45 0. 74 ~55 V 0. 49 t0. 5 1 0. 96 T58 K-O. l';-O. 1C-O. OZ Gss w o. oo-0. 05 o. sa rss IL 0. 8E 1. 1¢ 0. 7E A55 Y 0. 00 0. 18 0. 94 T58 M 0R5f 0. 01 0. 5C 6 C 0. 45-0. 02 0. 93 T58R-0. 19-0. 10-0. 02 6-0. 12 4 0 6 S 0. 9 0 0. 0-0. 9 0. 56 158 1 1. 00 R56 3 0. 30-0. 59 0. 56 rss T l. OC l. OC l OC R56 H-0. 12-0. 2 0. 9 13 R56 K-0. 12-0. 37-0. 02 T58 W-0. 19-0. 10-0. 02 0. 87 R56 L 0. 05 0. 24 0. 87 5. 19. 1-0. 02 R56 N 0. 18 0. 27 0. 31 N59 A 0. 35 10. 44 0. 73 R56 P-0. 12-0. 37-0. 02 N59 C 0. 40 11. 23 0. 78 R56 0. 01-0. 01 1. 02 N59 D 0. 52 11. 72 0. 67 R56 R 1. 00 1. 00 1. 00 N59 E 0. 6e s. sa 0. 38 R56 Is 39 0. 12 0. 55 N59 F 0. 82 10. 23 0. 57 R56 T 0. 10-0. 37 0. 85 N59 G0. 88 10. 00 0. 66 R56 W-0. 12-0. 37-0. 02 N59 0. 89 8. 21 0. 31 R56 Y-0. 12-0. 37-0. 02 N59 L 0. 8E 14. 74 0. 32 T57 A 0. 60 0. 65 0. 59 N59 M0. 42-1. 42 0. 72 T57 c 0. 60 0. 40 0. 85 N59 N1. 00 1. 00 1. 00 rs7 J 0. 92 1. 05 0. 53 N59 P0. 12-55. 11 0. 14 T57 H0. 83 0. 61 0. 23 N59 1. 02 1. 86 0. 73 7 1. 19 0. 87 0. 65 N59 R l. OS-11. 2E 0. 39 Table 10-12. Performance Indices Table1-12. erformanced' Wild-Type Wild-Type ResJ PAF PAD Prot. Resl PAF PAD Prot. Pos. Mu. PI I s. Mu P 9 S 1. 06 732 0. 74 D62 s-0. 24 0. 11 1. 06 N59 T1. 07 5. 63 0. 56 D62 C 0 2 0. 49 0. 96 NS9/0. 81 9. 97 0. 96 62. 02 0. 60 0. 93 9 1. 3 2. 8 059 02-02 0. 0. 80 11. 14 0. 61 D62 H 0. 61-0. 01 0. 89 6 A 0. 81 0. 79 1. 20 2 0. 2 2 0. 92 0 0. 69 0. 67 0. 97 2 L 0. 51. 7 0. 95 6 0 8 0. 66. 56 62-0. 24 1. 06 I60 0. 0. 92 0. 8o D62,-0. 24-0. 55 0. 69 160 00 1. 04 0. 86 6-0. 24 0. 6 1 1 06 0 1.. 0 1 0 0. 5 1 0. 160 0 0. 96 0. 73 7. 41 0. 62 6 0 0 91. 02 62 p, g . 8 1. 14 6 0. 160 0. 0. 2 0. 31 1. 3 6 1 0 1. 0. 81 0. 79 p g S ! L0. 95 0. 91 1. 02 D62 V0. 62-0. 26 0. 87 166M0. 96 0. 68 1. 14 DM W0. 58-0. 45 0. 79 [6QE0. 23 0. 32 0. 31 P63A1. 35 0. 60 1. 06 160E1. 00 0. 81 0. 79 P63 F1. 25 0. 93 0. 97 60 S 0. 78 1. 00 0. 92 63 G 1. 71 1. 22. 00 160, 0. 2 : 0. 32 0. 31 P63 t 1. 35, 0. 6C 1. 06 6 0. 87. 6. 06 6 1. 40 l. 0. 99 0 78. 19. 9 1. 84 D6jA0. 70 0. 71 1. 41 P63M1. 46 0. 91 1. 09 160 {0. 8', 1. 0f 1. 0f P63 K l. 4a 1. 02'0. 99 1 C 0 79 0. 85 0. 92 6. 0 OS 1 8 D61 31. 00 1. 00 1. 00'SR1. 31 0. 80 1. 02 D61 n °. ? s 0. 85 0. 92 P63 D 1. 09 1. 05,, l, os D61) 1 OC 1 oa l. OC P63 _ R 1. 31 0. 8C 1. 02 61. 1 0. 70 0. 61 P63 s 1. 42 0. 90 1. 17 61 G 0. 81 1. 25 0. 84'63r1. 50 1. 32 1. 02 D6 1 S 1. 44 1. 67 0. 97 P63 V 1. 31 1. 04 1. 06 D61. l. oa 1. 66 0. 9E P63 W 1. 35 1. 11 0. 86 61 0. 92 1. 72 0. 97 P63 Y 1. 35 0. 95 1. 12 D61 L 0. 80 1. 20 1. 00 T64 ~ 0. 96 1. 20 0. 97 D61 N 0. 79 1. 00 1. 12 T64 v 0. 78 0. 88 1. 05 MlP0. 83 1. 13 0. 97 T64 D 0. 87 0. 64 0. 81 D61 00. 89 1. 16 1. 02 T64 G 1. 23 1. 08 1. 00 D61 R 1. 11 1. 59 0. 69 T64 H 0. 89 0. 96 0. 90 D61 s 1. 2f 1. 35 0. 97 T64 L 0. 63 1. 22 0. 93 D61 0. 95 0. 97 1. 10 T64 0. 68 1. 09 1. 07 D61 Y 0. 84 0. 95 1. 03 T64 0. 69 0. 98 0. 91 Table 10-12 Perf r ance In ice Tabl-12. Perf rman e Indic s Wild-Type Wild-Type Res. PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos PI T64 P 0. 76 0. 94 0. 61 F 1. 21 0. 01 1. 01 T64 00. 76 0. 87 1. 13 R67 G139 0. 41 0. 81 . 1 0 11 1. 0. 1 0. 99 0. 76 T64 1. 1 0. 99 1. 03 R67 L 1, 2C 0. 1 1 1. 46 T64 T l. OC 1. 0 (1. 00 R67 1 5 0. 33 1. 00 T64 W 0. 71 0. 69 0. 72 6 1. 0 0. 04 1. 04 D65 A 1. 1 0. 72 0. 72 R67 Q 1. 16. 13 1. 60 D65 D 1. 00 1. 00 1. 00 R67 R 1. 00 1. 0 1. 00 D65 0. 8 0. 52 0. 88 67 1. 28. 3 6 6 1. 10. 40 0. 71 7 0 9 0. 1 1 4 0. 53 0. 4 1 7 - 0 42 0. 0 0 59-0. 11 1. 0 D65 R 0. 41 0. 221 0. 84 L68 C0. 76 0. 06 0. 85. D65 S1. 17 0. 47 0. 76 L68 3-0. 16 0. 44 0. 55 D65 R 0. 41 0. 2, 0. 84 t68 C 0. 7f. 0. 0d 0. 85 6S 0. 0. 5 0. 68 L68 E 1. 44 0. 0. 87 D65 T O. 9C 0. 5 (0. 68 L68 E 1. 44 0. 13 0. 87 D65 0. 88 0. 2 (0. 64 L68 F 0. 7C 0. 25 1. 00 D65 0. 7 0. 50 0. 65 L68 a 1. 09-0, 0E 1. 00 D65 Y 0. 83 0. 42 0. 64 L68 H1. 05 0. 22 0. 89 6 0 5 0. 56 1. 03 L68 1. 1 0. 0. 6 P66 c 0. 51 0. 52 1. 51 L68 L 1. 00 1. 00 1. 00 . 00 0 7 0 6 0 66 0.. 67 1. 02 8 0. 1 0. 10 0. 99 ? 66 _ O. 9S 0. 6,. 1. 02 L68 N 0. 51 O. 1C 0. 95 P66 G1. 50 0. 44 1. 78 L68 0. 29 0. 35 0. 82 ? 66 H 1. 59 0. 95 1. 23 0. 25 0 90 P66 1 1. 591 0. 84 1. 51 L68 R 0. 19 0. 47 0. 75 66 L 1. 14 0. 99 0. 92 L68 S 0. 99 0. 07 0. 93 P66 N 1. 12 038 1. 62 L68 T1. 03 0. 32 0. 92 66-0. 09-0. 11-0. 01 L68 V 1. 09 0. 51 1. 01 P66 01. 46 0. 42 1. 91 L68 W 1. 21 0. 56 0. 88 P66 R 1. 85 0. 51 1. 26 L68 Y 0. 71 0. 45 0. 97 P66 S 1. 39 1. 02 0. 98 N69 A 0. 92 1. 13 0. 93 P66 T 1. 41 1. 10 0. 72 69 C 1. OS 1. 20 1. 18 P66 V. 83 0. 89 1. 12 N69 D 0. 90 1. 11 1. 05 66 1. 33 0. 70 1. 08 N69 G 1. 20 0. 98 1. 06 R67 A-0. 20 0. 22 1. 39 N69 H 1. 36 1. 52 0. 73 R67 E 1. 04 0. 11 0. 85 N69 11. 47 1. 75 0. 69 Ta le 1-12 Performance In i, T ble 0-12 Perf rman n li Wild-Type Wild-Type ResJ PAF PAD Prot. ResJ PAF PAD Prot. M P p Pos. Mut. Pt PI PI Pos. Mut. PI PI PI N69 (1. 72 l. St 0. 8'A71 [1, 5] Q-7S 0. 81 N69 130 120 0. 1 1. 44 1. 01 0. 76 N69 N. 00 1. 00 1. 00 A71 L1. 23 0. 84 0. 85 N69 1. 00 9 0. 66 7 0 8 1 0. 1 N69 07. 14. 74 71 123 1 0. 77 N69 R. 49 0. 83 0. 84 8'A71 P 0. 14-0. 0 ; 0. 46 N62§. 21 1. 42 1. 03 71 1. 40 0. 0. 1. N69 1. 35 1. 43 0. 87 1. 75 0. 6 0. 8 N69 1. 99 1 73 0. 87 71 1. 70 0. 79 0. 83 N69 w 1. 05 0. 55 0. 36. S72 A 0. 5 3. 521 1. 06 1 44 p 1. 41 108 0. 4 0-0. 90 0. 4 0. 1 0 99 2 0 94 1.. 8 . 6 21 1 0 G70 1. 0 00 1. 00 S7 121 2. 48 0. 80 G70, 0. 1,-0. 9 (0. 4 (S72 E 0. 61 0, 93 0, 99 , G70, _ q-O. lC 0. 3. 0. 2t S72 F 0 94 1. 15 0. 80 570 i O ; OC-0. 3d 0. 2] S72 a 1. 2C 1. 76 0. 8 7 G70 J 1 rOC 1. 0 (1. 0 (S72 H 1. 2] 2. 42 0. 82 G70 H 0. 04 1. 90 0. 26 S7 1. 26. 0. 0 70 [0. 04 0. 27 0. 33 S72 M 0. 36 2. 13 0. 94 G70 0. 0 0 0. 26 72 2. 8 0. 9 1V o . o. 3 1. 01 0. 3 S72-0. 25 0. 56 0. 63 G70 M0. 62-0. 72 0. 29 §2200. 62 0. 66 0. 98 G70 N 002-0. 76 0. 37 S72 0. 86. 74 0. 87 370 P 0. 16-0. 5E 0. 29 S72 Is 1. 00 1. 00 1. 00 0 0. 02-0. 83 0 36 S72 1. 10. 97 0. 88 G70 0. 08-1. 84 0. 25 S72 1. 08 0. 83 0. 90 G70 S 0. 69 0. 64 0. 88 S72 W0. 98 0. 34 0. 92 G70 T 0. 27-0. 10 0. 45 S72 Y1. 07 0. 07 1. 03 G70 V 0. 16-0. 52 0. 34 Y73 A-0. 10 1. 40 0. 82 G70 0. 08-0. 33 0. 38 Y73 C-0. 10 1. 20 1. 18 A71 A1. 00 1. 00 1. 00 Y73 D0. 13 0. 80 1. 09 A71 c 1. 01 0. 99 0. 85 73 G 0. 71 0. 51 0. 95 71 0. 70 0. 65 0. 68 Y73 H 0. 67 0. 52 0. 96 A71 E 1. 45 0. 81 0. 83 Y73 1 0. 82 0. 64 0. 97 A71 F 1. 13 0. 99 0. 75 Y73 1. 07 0. 94 0. 95 71 G 1. 59 0. 68 0. 85 73 L 0. 98 0. 50 1. 03 A71 H 1. 7C 0. 72 0. 75 Y73 M-0. 10 1. 13 1. 05 Ta 1 10-12. P rf rmanc ndic T b 0-12 Perf rmanc n i Wild-Type Wild-Type Resl PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI s. Y73 N0. 56 0. 76 1. 25 P75 1. 28 0. 69 1. 1 Y73 0. 64-0. 54 0. 42 P75 v 0. 93 1. 39 0. 90 3 1. 2. 87 1. 20 1 04 131 0. 84 Y73 R 1. 2 0. 2 96 P75 Y 0. 69 1. 32 1. 08 S 1 0. 68 0. 77 0 38 1. 11 0. 60 Y73 V 0. 88 0. 74 1. 08 S76 C0. 39. 06 0. 67 Y73 Y-0. 10-0. 10-0. 02 S76 D 0. 41 1. 94 0. 49 L74 A 0. 0 2. 90 1. 01 S76 0. 4 2. 09 0. 58 74-0 8. 1-0. 0 . 44 0. 4 0. 68 L74 q 0. 99 1. 1'0. 5E S76 G 0. 64 2. 1'0. 69 4 0. 99 1. 13 0. 58 S76 0. 6 4. 57 0. 1 0 1 L74 H-0. 18-0. 18-0. 03 S76 K 0. 59 1. 53 0. 32 08-0-03 L74 0. 86 0. 64 14 76 4 4. 7. 2 L74L1. 00 1. 00 1. 00 S76M0. 49 1. 61 0. 45 L74 15 1. 21 0. S 1. 3. 0. 7 4. 8-0. 18-0. 3 S7. 84 0. 9. 88 . 74 b-0. 18-0. 18-0. 03 S76 S100 1. 00 1. 00 L74 R-0. 18-0. 18-0. 03 S76 0. 75 1. 11 0. 80 _74 g 2. 72-1. 52 0. 25 S7 0. 67 1. 35 0. 78 L74 T 18-0. 18-0. 03 S76 W0. 57-0. 25 1. 06 8 8 I. 6 0. 0 0 61 i. l 7 1, 7 J74 13 0. 67 0 0 C7 0. 8 0. 1. 20 74 0. 90 0. 86 1. 19 C7 C 1. 00 1. 00 1. 00 , 74 {0. 90 0. 8e 1. 19 C77 Q l. OC l. oa l oa 75 n 0. 54 1. 42 1. 06 C77 D 0. 92 1. 05 0. 45 75 0. 67 2. 09 0. 86 C77 0. 25-0. 61 0. 75 P75 E 0. 83 1. 19 1. 00 C77 G 1. 01 0. 18 0. 53 75 G 1. 16 0. 93 0. 81 C77 L 0. 98 0. 73 1. 44 P75 H 1. 05 0. 86 0. 89 C77 N-0. 13-0. 06-0. 04 P75 1 0. 69 0. 74 0. 78 C77 P-0. 13-0. 06-0. 04 P75 K 0. 60 0. 88 0. 91 C77 R 0. 70-1. 02 0. 34 75 L 0. 44 1. 19 1. 02 C77 S 0. 95 0. 76 1. 19 P75 0. 36 0. 30 1. 22 C77 T 1. 12 1. 03 1. 18 75 1. 00 1. 00 1. 00 C77 V1. 05 0. 80 1. 33 P75 1. 21 0. 61 1. 04 C77 W 039-0. 24 0. 73 P75 R 1. 60 0. 46 0. 89 C77 0. 95-0. 01 0. 66 P75 S1. 39 0. 63 1. 18 L78 A-0. 11-0. 14-0. 01 Tabl 1-12 Performance Indi T b 10-1 P rfo man Wild-Type Wild-Type Res./PAF PAD Prot. ResJ PAF PAD Prot. P I PI P P u P m Pos, C0. 92 0. 78 0. 91 T80 H 0. 21 0. 05 0. 66 L78 C 0. 92 0. 7E 0. 91 T80 H 0. 2] o, os 0. 66 L78 E 3. 01-1. 14 0. 16 80 1 0. 50 0. 15 0. 78 L78 G 4. 98 1. 38 0. 12 T80 0. 15. 2. 74 L78 H 4. 82 1. 57 0. 25 80 0. 1-0. 1. 6 L78 I 1. 43. 1. 11 1 06 T80 N 0. 53 0. 5 0. 97 L78 L 1. 00 1. 00 1. 00 T80 P-0. 11-0. 0'0. 55 L78 M 0. 52 0. 48 0. 75 T80 Q 0. 9 1 1. 07 1. 02 78 2. 6-0. 4 0. 22 T80-R 0. 08-2 0. 78 00 L78 P-0. 11-0. 14-0. 01 T80 S. 0. 6. _ 140. 12 L78 ° 1. ? 3 0-52 0. 46 T80 T 1 sOC 1. 0 (1. 00 L78 1. 7 0. 52. 46 8 1. 00 1. 0 . IM4 7-0. 1-0. 01 3. 46 . 1.. 1 0. 4 0. 1 0 9 . 3 83 1. 04 1 4 L78 1 3 0. 81 0. 46 1 1 0 A79 A-0. 15-0. 13-0. 02 H81 p 1. 10 0. 90 0. 87 A79 C 0. 97 0. 03 1, 16 H81 b1. 17 0. 80 0. 94 A79 E 1. 12 0. 27 1. 12 H81 1 1. 00 1. 0 1. 00 A79 F-0. 15-2. 02. 17 81 1. 2 6. 1 A79 2 0. 92 0. 99 H8 L 1. 23 0. 93 A'. 93-0. 09 0. 85 H81 0. 94. 54. 2 A29L-. 59 0. 67 0. 87 H81 17 LOO 0. 82 A79 L 80 0. 96 0. 88 81-0. 10 0. 72 0. 42 A79 M 1. 50 0. 28 1. 04 H81 _ O 0. 8s 0. 75 1. 00 A79 N 1. 48 0. 28 0. 97 H81 R 0. 34-0. 29 0. 85 A79 P 0. 70 0. 94 0. 81 H81 S 1. 04 0. 69 0. 94 A79 1. 47 0. 27 1. 05 H81 V 1. 10 0. 71 0. 89 A79 R 1. 47 0. 32 1. 02 81 1. 13 1. 09 0. 90 A79 s, 0. 82 0. 78 1. 09 H81 Y 0. 77 0. 14 0. 76 A79 r 1. 17 0. 60 0. 90 L82 A 0. 62 0. 98 1. 00 A79 V-0. 15-0. 13-0. 02 L82 G 1. 38 0. 31 1. 24 A79 W 1. 27 0. 53 0. 46 L82 H 1. 33 0. 47 0. 95 T80 A 1. 00 1. 11 0. 90 L82 I 1. 17 0. 51 0. 58 T80 C 1. 31 1. 15 0. 91 L82 K 1. 19 0. 51 1. 03 T80 E 0. 07-0. 16 1. 02 L82 L 1. OC 1. 00 1. 00 T80 G 1. 16 1. 50 0. 81 L82 0. 65 1. 06 1. 07 Table 10-12. Performance In'e T ble 10-1. Per ormanc Ind' Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos, Mut. PI PI PI L82 p 1. 461 0. 52 1. 11 L84 3 75 0. 55 0. 93 L82 R1. 34-0. 18 1. 15 L84 T0. 86 0. 44 0. 9 L82 1. 15 0. 0 1, 13 L84 V 0. 79 0. 42 1. 23 L82 T 1. 18 0. 3 0. 97 84 036. 2 0. 91 L rr L82 1. 02 0. 19 1. 14 D85 A0. 79 1. 09 0. 63 L82 W0. 27-0. 46 0. 93 D85 0. 8E 150 0. 6 P83 A 0. 3 2. 3 0. 66 D85 100 1. 0. 00 P83 C0. 53 1. 01 0. 81 D85 E 1. 12 1. 25 0. 97 P83 D 0. 75 0. 8'0. 92 D85 F 1. 01 1. 98 0. 52 P83 E0. 84 1. 26 0. 92 8 14 1. 60 0. 69 8. 76 0. 9 0. 9 8 1. 31 0. 6 1. 01 8 0. 10 0. 76 P83 a 1. 31 0. 6t 1. o] D85 [0. 55 0. 1 (0. 46 P83 H 1. 27 0. 6 0. 93 0 0. 4 0 52 1. 7. 1. 8 1 4 0. 2. 6 P83 L0. 04 0. 21 0. 1 D85 P 0. 97 0. 5 0, 63 P83 m 0. 58 1. 88 0. 71 D85 2 3. 0S O. 9S 0. 82 8 0. 0 1. 10. 90 5 2. 38 1. 0 0. 66 P83 1. 00 1. 00 1. 00 D85 is 2. 28 0. 68 0. 93 P83 10 0. 73 0. 82 0. 95 1 3 71 0 77 P83 R 1. 19 1. 9. 78 85 0 1 0 2. 65 l'17 P83 s 1. 17 0. 75 0. 85 D85 W 0. 87 0. 34 0. 72 P83 T 0. 86-0. 02 0. 62 D85 98 0. 55 0. 78 P83 V 0. 78 0. 19 0. 72 L86 A 1. 38 3. 32 0. 40 P83 w 0. 98 0. 62 0. 69 L86 1. 16 2. 44 0. 85 L84 A 0. 45 0. 45 0. 76 L86 E0. 06-0. 92 0. 46 L84 D 0. 19 0. 85 0. 48 L86 F-0. 15-0. 26-0. 02 L84 0. 72 1. 01 0. 74 L86 G 1. 15 0. 70 0. 83 L84 G 0. 77 1. 01 0. 53 L86 H0. 88-0. 72 0. 57 L84 H 1. 01 0. 99 0. 66 L86 L 1. 00 1. 00 1. 00 L84 1 0. 901 0. 87 0. 99 L86 P-0. 15 0. 99 0. 22 L84 K 1. 101 0. 79 0. 59 L86 0-0. 15-2. 60 3. 66 L84 L 1. 00 1. 00 1. 00 L86 R 0. 43-4. 46 0. 26 L84 N 0. 54 0. 67 0. 86 L86 S 0. 78-036 0. 78 L84 _-0. 12 0. 4'0. 5E L86 T 0. 9e 0. 28 0. 75 L84 in 0. 41 0. 52 0. 93 IL86 0. 92 0. 12 0. 93 L84 R 0. 56 0. 57 0. 71 L86 0. 67 0. 08 0. 78 Table 10-12. Performance Indices Table 1-12. Performance Indices Wild-Type Wild-Type Res./PAF PAD Prot. ResJ PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI L86 0. 5 0. 82 0. 92 I89 5 0. 7-1. 66 0. 49 V87 A 0. 65 0. 17 0. 88 I89 94 0. 90 0. 60 V87 n 0. 6 n 2. 22 0. 93 V 0. 91 0. 82 1. 09 7-0. 09-2. 53. 2 8 p V87 F 0. 60. 0. 10 0. 56 M90 _A 0, 78 1. 41 0. 67 87 0. 4-2. 95 0. 54 M90 C0. 79 1. 09 0. 83 V87 K0. 04-8. 34 0. 26 M90, _ D-0. 2'2. 8E 0. 15 8 L 0. 71 4. 30 0. 4 0. 24 1. 1 0. 29 V87 0. 3 0. 5 0. 86 90 7-122 0. 7 0. 0 1. 64 9 90 1 6 4 0. 7-1. 0 44 0. S-0. 0 0. 7 1. 0 0 7 1 0 0. 0 0 0 V87 Y 0. 33-1. 24 0. 42 M90 R-0. 2 0. 36 0. 23 . 188 oui-2. 63 0. 27 90 S 1. 06-O i. 6 _6 25 V§2V1. 00 1. 00 1. 00 M90 00. 68 0. 77 0. 71 V87Y0. 33-1. 24 0. 42 M90 R.-0. 24 0. 36 0. 23 188a1. 01-2. 63 0. 27 M90 S1. 06-0. 17 0. 56 188 S 1. 0-6 25. 21 1 27 0 0. 59 88 1. 00 1. 00 1. 00 90 1. 08 0. 08 0. 62 188 vu 0. 24 1. 09 0. 86 M90 W 0. 7S-4. 04 0. 21 18-0. 14-0. 5 0. 29 9 A 0 1. 45 0. 81 188, _,-0. 14 3. 51 0. 18 L91 C 0. 1. 27 0. 87 [88Q0. 01-1. 10 0. 36 91-0. 12 1. 47 0. 12 188-R-0. 1-0. 3-0. 02 L91 E.-0. 12-0. 51 0. 13 [88 r 1. 03-0. 1 0. 52 9. 21-0. 58 0. 17 18-0. 14-0. 32-0. 02 L91 H-0. 12-0. 13-0. 01 189 A 0. 55 1. 83 0. 63 L91 : 0. 9E 1. 05 0. 89 I89-0. 10-0. 14-0. 02 L91 K-0. 12-0. 13-0. 01 I89-0. 10-2. 05 0. 24 91 L 1. 00 1. 00 1. 00 189 F 0. 68 0. 75 0. 90 L91 M 0. 28 0. 88 0. 80 189 G 0. 64-3. 84 0. 2S L91,-0. 12-0. 13-0. 01 189H1. 00-1. 01 0. 33 L91 00. 05-0. 14 0. 18 9 I 1. 00 1. 00 1. 00 L91 R-0. 12-0. 13-0. 01 189 L 0. 87 1. 22 1. 07 L91 S 0. 92 0. 43 0. 24 rss, 38 1. 91 0. 30 L91 T 1. 06-0. 11 0. 36 189Q0. 25-0. 30 0. 32 91V0. 94 0. 79 0. 72 I89-0. 10-0. 14-0. 02 L91 W-0. 12-0. 13-0. 01 Table 1-12. P rformance In ices-Table It 1-12. P rformance In *ces Wild-Type Wad-Type ResJ PAF PAD Prot. ResJ PAF PAD Prot. PI I Pos. Mut. Pi Pi Pi-Pos, Mut. PI Pi Pi L91 y-0. 12-0, 13-0. 01 N94 R 0. 10---8. 20 0. 19 G92-0. 1-0. 1-0. 02 94 S 0. 10 84 G92-0. 1 2. O5 0. 18 94 0. 25-1. 4. 6 G92 n o la 2. 04 0. 1E N94 T 0. 25-1. 43 0. 6e G92 D-0. 10. 1-0. 02 N94 V 0. 15-0. 32 0. 64 9-0. 10-2. 31 0. 21 N94 W0. 10-1. 20 0. 69 G92 «-O. 10-3. 24 0. 17 N94 0 08. 12 0. 6 92 1. 00 1. 00 1. 00 5-0. 14-0. 14-0. 01 G9-0. 10-0. 18-0. 02 D95 C-0. 14-0. 14-0. 01 92-0. 1-0. 18-0. 02 D95 31. 00 1. 0 1. 0 9-0. 10-0. 18-0. 02 D95 E2. 04 0. 75 0. 66 G92 0. la-0. 1E-0. 02 D95 G-0. 14-0. 14-0. 01 G92 g 1. 26-2. 96 0. 21 D95 H-0. 14-0. 14-0. 01 . G92 T-0. 10-0. 18-0. 02 D95 K-0. 14-0. 14-0. 01 -0 *98 4 4 G92 W-0. 10-0. 18-0. 02 D95 N-0. 14-0. 14-0. 01 . 2-0. 1-0 8-0. 02 95 14. 14. 1 r93 A138 1. 05 0. 50 D95 R-0. 14-0. 14-0. 01 93 C 1. 08 0. 95 0. 64 95-0. 14-0. 14-0. 01 T93 n I. 08 o. gs 0. 64 D95 S O. 14-0. 14-0. 01 T93)-0. 18 0. 23 0. 22 D95 r-0. 14-0e14-0. 01 T93 1 3. 52 0. 54, 0. 63 D95 V-0, 14-0. 14-0. 01 - 18-0. 19-0. 02 395 W-0. 14-0. 14-0. 01 .. T93 0-0. 18-6. 75 2. 03 D95 Y-0. 14-0. 14-0. 01 T93 R-0. 18-0-19-0. 021 T96-A 0. 3 4. 20 1. 32 9 _ ; 0. 89 0. 49 0. 89 rs6, 0. 44 3. 76 0. 79 93 T 1. 00 1. 00 1. 00 r96 F 0. 53 1. 24 0. 69 T933-0. 18-0. 19-0. 02 96 G 0. 78 1. 28 1. 03 93-0. 18-0. 19-0. 02 T96 1 0. 95.-0. 22 0. 88 T93 Y 5. 26 0. 03 0. 77 T96 L 0. 92 1. 93 0. 93 N94 A-0. 45 0. 74 0. 96 T96 0. 39 2. 53 0. 80 N94 « 0. 01 0. 07 0. 94 T96 ?-0. 11 0. 89 0. 35 N94 J 0. 15 0. 53 0. 76 T96 R 0. 17 0. 14 0. 50 N94 0. 11-0. 94 0. 7 í T96 S 1. 04 0. 79 1. 05 N94, 0. 61-0. 18 0. 49 T96 T 1 1. 00 LOO 1. 00 N94 M-0. 45 0. 03 0. 94 T96 V 0. 81 0. 59 1. 12 N94 1. 00 1. 00 1. 00 T96 0. 38-4. 29 0. 51 N94 P-0. 45 0. 79 0. 40 T96 Y0. 38-3. 73 0. 59 Table 1-12. Performance Indices Table 1-12. Performance Indices Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI P. M P K97 A 0. 01 0. 23 1. 111 Y99 w 0. 68 0. 57 1. 20 K97 D-0. 23-0. 17-0. 01 Y999 1. 00 1. 00. 00 K97 G 0. 84-0. 64 0. 100 0. 78 2. 02 0. 93 K97 ! 0. 74-0. 55 0. 47 F100 C0. 73 1. 28 0. 78 K97 K1. 00 1. 00 1. 00 F100 D0. 38-0. 03 0. 33 K97 L 0. 38-0. 28 0. 30 F100 E1. 01 0. 15 0. 83 K97. 0. 02 0. 22 0. S 100 1 0 1. 00 1 0 K97 p 0. 16 0. 27 0. 36 100 0. 65-0. 60 0. 53 K97 O 1. 14 O. OC 0. 7'F100 M 0. 7S. 2. 15 I. 2a K97 01. 14 0. 00 0. 73 100 0. 2. 19 1. 0 K97 S 0. 28-0. 46 0, 5E F100 S 0. 8, 0. 8'1. 02 K97 T 0. 22-0. 42 0. 5] ^ 100 r. O. sfl 1. 42 0. 71 2. 80 0. 59 1. 02 1 0 0. 9. 4 1. 12 2. 46 1 0 1 2 K97 22-0. 42 100. 0 2 0. . 4 0. 10 1. i. 6 . w ru 0 4 O 1 1 71 0 29-0. 6 0. 0 0 0 8 1. 0 w-T K97 1. 00 1. 00 1 00 1 1 0. 4 7 0. 6 A98 A 1. 00 1. 00 l'O R101 F 0. 84 0. 9 0. 6 K97 Y0. 29-0. 65 038 R101 p 0. 85 0. 80 1. 02 A98 A1. 00 1. 00 1. 00 tRIOl F0. 84 0. 97 0. 66 A98 C130 1. 42 1. OC R101 [0. 79 0. 96 0. 68 A98 D 1. 11 2. 19 0. 81 R10 1 K, 1. 24 0. 07 0. 90 A98 G 1. 57. 0. 0. 9 101. 8 1. 12 1. 33 A98 H 2. 09, 0. 92 0. 82 R101 N 0. 72 0. 92 1. 11 2. 05 0. 65 0. 72 R101 P 0. 5C. 0. 8 (0. 75 A98 L 2. 22 1. 47 0. 71 R101 0. 86 0. 11 1. 03 A98 N1. 24 1. 40 1. 01 R101 100 1. 0 1. 00 A98 P 1. 10 1. 26 0. 90 R101 V0. 74 0. 44 0. 90 A98 S 1. 73 0. 65 1. 17 R101 W 0. 9''O. OC 0. 89 A98 T 1. 72 0. 27 1. os R101 Y 0. 74 0. 80 0. 67 A98 Y 2. 02 1. 15 0. 87 R102 G 0. 15 1+75 0e98 Y99 A 0. 66 0. 82 1. 29 R102 C 0. 22 0. 36 0. 78 Y99 0. 83 0. 70 1. 23 R102 D 0. 01 0. 68 0. 26 Y99 H 0. 77 0. 59 1. 30 102 0. 46 0. 23 0. 31 Y99 10. 81 0. 61 1. 11 R102 G 0. 44 0. 27 0. 43 Y99 L 0. 66 0. 86 139 R102 L033 1. 64 0. 95 Y99 P 0. 89 0. 81 1. 00 R102 ?-0. 0, 0. 89 0. 26 Y99 0. 61 0. 29 0. 97 R102 0. 67 1. 19 1. 09 99 S 0. 72 0. 37 1. 45 102 R 1. 00 1. 00 1. 00 - Y99 V 0. 61 0. 31 1. 2E R102 g 0. 4e 0. 9d 0. 98 Table 1-12. Performan Indices ble 10-12 Perform nce I di Wild-Type Wild-Type Res. l PAF PAD Prot. ResJ PAF PAD Prot. Pos. Mut. PI PI PI M. I I P R102 V0. 28 0. 61 0. 80 105-0. 11. 18-0 02 R102. 29-1. 03 0. 34 L105 C 1, 56 1. 92 1. 05 R102 Y 0. 40 1. 29 0. 70 L105 E-0. 11 0. 53 0. 26 T103 A 0. 97-9. 64 0. 8S L105 F 1. 3 (1. 73 0. 95 1 3 C. 90-6. 91 0. 89 L105 roi 1. 08 1. 40 1. 07 T103 0. 74-3. 39 0. 85 L105 H 0. 85 1. 23 1. 07 T103 G1. 11-5. 27 1. 20 L105 L1. 00 1. 00 1. 00 T103 H 0. 99-4. 15 1. 14 L105 M-0. 11-0. 18-0. 02 T103 1. 08-5. 15 0 89 105 1. 1 0. 90. 00 T103 K 1. 09 4. 36 1. 05 L105 D 0. 94 1. 04 1. 03 T103 L 1. 05-1. 86 0. 88 L105 R 0. 99 1. 25 0. 94 T103 N 0. 77-6. 03 1. 09 LlOs g 0. 93 0. 61 0. 95 T103 P 0t69-5. 11 l. Ql LloS r 0. 92 0, 64 l. OC T103 N 0. 7-603 0 L105 s 0. 93 0. 61 0. 95 T103 0 69. 11 1. 1 10. 9 0 1. 0 1 0. 87-6. 9-0 9 1 0. 2-1. 6 4 1, g T103 1. 00 100 1. 00 1 5 72 0. 62 1. 18 T103 V 0. 95-1. 95 0. 9 D106 0. 72 1. 13 0. 6 T103 W1. 26-2. 60 0. 77 D106 C 1. 01 1. 10 0. 80 0. 1-4.. 6. 8 10 1. 00 1. 00 1. 0 . 41-0. 1-0 04 10 1. 09 1. 02 P104 C 1. 95'1. 83 1. 34 D106 d 1. 02 1. 45 0. 34 P104 E 1. 84 1. 97 1. 37 D106 118 1. 45 0. 67 P104 1. 79 0. 86 0. 67 D106 H 1. 09 1. 18 0. 66 P104 G 2. 67 0. 98 1. 25 D106 11. 04 0. 92 0. 45 P104 H2. 84 1. 03 1. 11 D106 1. 28 1. 24 0. 68 P104 12. 43 2. 05 1. 07 106 L 1. 20 1. 00 0. 56 104 L-0. 41-0. 19-0. 04 D106 m 1 0. 73 0. 86 0. 77 P104 W 1. 09 2. 24 1. 01 D106 N 0. 92 0. 64 0. 91 P104 N 1. 62 1. 44 1. 32 D106-0. 17 0. 63 0. 18 P104 P 1. 00 1. 00 1. 00 D106 ? 0. 92 0. 62 0. 94 P104 0. 34 0. 85 1. 24 D106 R 0. 98 0. 56 0. 91 P104 R 1. 62-0. 39 0. 83 D106 g 0. 98 1. 02 0. 81 P104 3 2. 48 0. 53 1. 44 D106 T 1. 06 1. 38 0. 64 104 T 2. 70 0. 33 1. 29 D106 V0. 98 1. 68 0. 61 P104 V 2. 59 1. 02 1. 40 D106 W 0. 78 1. 07 0. 34 P104 W 2. 05 0. 23 0. 59 1107 0. 81 0. 80 0. 83 Tal 1 10-12. Performan Indi e Ta le 10-12. Performance Indi Wild-Type Wild-Type Res./PAF PAD Prot. Res PAF PAD Prot. Pos. Mut. PI PI P Po. M. P PI 107 C 0. 95 1. 41 1. 00 1 9 G 0 2 0. 51 0. 8 Il E 2. 55-0. 28 0. 21 1 0 85 0 22 1. 6 [107 0. 9-0. 02 0. 19 L109 1 1. 0-0. 141 1. 21 I107 1. 76-10. 12. 2 L109 1. 0 1. 1 0 10-0. 07. 20-0. 02 L109 kt 0. 74 0. 63 1 OC 1107 1. 0 1. 00 1. 00 10 1. 2 0. 66 1. 1 0. 6 1. 04 0. 2 1 9 0. 7 0. 43 0. 35 Il N 1. 81 0. 93 0. 56 L109 01. 18 0. 22 1. 08 1107 0. 0. 0. 32 0. 40 L109 R 0. 4 0. 21 0. 95 Il 7 0. 3-0. 02 0. 43 L109 S 79 0. 38 0. 94 0 R 0. 08-2. 75 0. 28 L109 r 0. 63 0. 79 0. 87 1107 T 0. 64 1. 33 1. 05 L109 V 2. 4 10 '1 0. 4 15 0. 95 lp 1 8g 1. 0. 97 1 0 . 0. 0-0. 02 11 0. 91 0. 8 [10 ? r Z. 1 oa 0. 97 1. 04 L109,,, Y 1. Xf 0. 83 0. 7S [107 v-0 07-0. 2a-0. 02 3110 N. 0. 91 1. 01 0. 8E [107 r 0. 49 OS 02 11. 4 0. A108 A-0. 12-0. 07-0. 02 G110 D 76 1. 40 0. 87 A108 D-0. 12-0. 07-0. 02 G110 p. 26 1. 76 0. 46 A108 E0. 14 0. 61 0. 25 G11 0. 04 2. 9 0 0 A108 F-0. 12-0. 07-0. 02 110 G 100 1 1. 0 0. 99 1. 1 1. 15 G110 0. 6 0. 731 0. 46 @108 H-0. 12-0. 07-0. 02 G110 10. 06 0. 23 0. 32 A108 1-0. 12-0. 07-0. 02 1 10 L-0. 20-0. 12-0. 02 A108 K0. 60 2. 97 0. 31 G 10 0. 16 0. 82 0. 34 A108 1. 41 2. 56 0. 20 s110 ç 0. 7a 0. 77 0. 89 A108 N-0. 12-0. 07-0. 02 G110 0. 02 0. 22 0. 50 A108 P-0. 12-0. 07-0. 02 GIIO 0 0. 4 034 0. 77 A108 0. 58 0. 73 0. 98 G110 0. 05 0. 48 0. 45 A108 R-0. 12-0. 07-0. 02 Gl 10 s 0. 79 0. 30 1. 01 t108 ; 0. 94 1. 00 1. 14 G110 0. 45-0. 05 0. 42 A108 r 05 05 0. 8 1. 08 G110 W-0. 20-1. 18 0. 20 t108 z 0. 76 0. 95 0. 99 G110 Y0. 01-0. 88 0. 40 L109 0. 34 0. 32 1. 07 Mill A0. 65 1. 02 0. 89 L109 1. 00 0. 11 1. 15 Mill C 0. 92 1. 01 0. 95 L109 0. 74 0. 19 1. 24 Mill-0. 27 0. 79 0. 37 L109 Mill IE 0. 25 0. 67 0. 56 Table 1 (-12. P rformance Indices Table 1 (-12. P rformance Indices Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI Mill F 1. 47 0. 78 0. 75 V113 H134 0. 76 0. 84 Mill 0. 8 0. 32 0. 44 V113 K 1. 19 0. 7, 0. 9, Mill H0. 98 0. 19 0. 40 11 L 1. 50 0. 85 0. 85 111 1. 95'1. 0 0 91 11 0. 78 1. 06 0. 93 Mill K 1. 98 0. 71 0. 5 (V113 N 0. 8E 1. 22 1. 01 Mill 1. 55 0. 67 0. 93 VI 13 p 0. 72 1. 14 0. 65 Mill M1. 00 1. 00 1. 00 11 1. 03 1. 1 0. 9 Mill N 0. 49 1. 31 0. 79 V113 R 1. 13 1. 11 0. 82 Mil1-0. 27 0. 57 039 11 0. 80 0. 91 Mill 0. 2-0. 99. 34 1 0. 94 0. 89 Mil1. 03 4 2 11 1 1. 0 Mill 1. 4 76 0 11 0 91 0. 8 11 4 0. 9 0. 8 1 1. 11 Mill W0. 96 1. 23 030 L114 A0. 78 1. 07 1. 03 Mill 143 1 06 6 14. 4 1. 0 S112 A 0. 58 0. 94 0. 98 L114 E0. 32-0. 14 0. 42 S112 E 0. 1 1. 16 1. OS 1-0. 11-0. 21-0. 2 S112 0. 37 0. 88 0. 61 114 0. 6 1. 14 0. 78 S 112 1. 00 0. 8. 9 114 0 92-0 55 0. 21 0. 8 S112 K 0. 84 0. 68 0. 92 L114 10. 97 1. 17 0. 86 S112 L1. 03 1. 01 0. 8 (L114 K-0. 1-0. 21-0. 02 S 112 M 0. 4 : 0. 56 0. 98 114 1. 0 1. 00 1. 0 S112 N 0. 52 0. 85 1. 09 L114 4. 3 128 1. 00 S112-0. 19-0. 82 0. 33 L114 N 0. 65 0. 77 0. 9 S112 R0. 20-0. 44 0. 99 L114 030 0 28 0. 42 S112 S 1. 00 1. 00 1. 00 L114 0. 59 0. 12 0. 68 S112 T 0. 95 0. 72 0. 87 L114 R-0. 11-0. 21-0. 02 S112 v 0. 86 0. 48 0. 73 L1 14 S 0. 87 0. 55 0. 72 S112 W 0. 74 0. 58 0. 85 L114 0. 88 1. 05 0. 82 S112 Y0. 68-0. 10 0. 90 L114 V 0. 91 0. 60 0. 84 113 A 0. 71 1. 31 0. 70 L114 W-0. 11-0. 21-0. 02 Vl 13 c 0. 87 0. 94 1. 06 L114 Y-0. 11-0. 21-0. 02 V113 D 0. 7E 0. 87 0. 97 vi 15 A 0. 60 1. 19 1. 11 V113 E0 fA0. 9 1 0. 94 0. 99 VllS C 0. 73 1. 08 1. 14 VI 13 F 1. 05 0. 96 0. 80 Vl 15 D-0. 15 2. 21 0. 19 V113 G 0. 9 (0. 5E 0. 89 V115 0. 54 1. 69 0. 32 Table 10-12 Performance Indic T 1 1-1. Perform nce In i Wild-Type Wild-Type Res./PAF PAD Prot. ResJ PAF PAD Prot. Pos. M t. PI I PI Pos t V115 G 1. 0 1. 76 0. 43 0117 M 1. 5= 1. 89 0. 87 VllS H-0. 1'0. 13-0. 02 0117 P-0s24 1. 13 0. 61 115 1. 05 0 99 1. 14 117 1. 0 1. 00 00 V1 K-0. 15-0. 13-0. 02 OH ? R1. 56 1. 05 1. 00 V115 L 1. 12 1. 3C 1. 02 OH S 1. 951 0. 87 1. 13 11 0. 48 1. 2 1. 5 117 2. 2 1. 10 1. 06 V115 P-0. 15 2. 21 0. 26 0117 V 2. 1'0. 76 0. 67 V115 0-0. 15 1. 15 0. 32 0117 W2. 16 0. 71 0. 57 0 10 1. 63 0 21 117. 2 1. 13 0. 76 115 S. 95 1. 14 0. 72 V118 A 0. 84 0. 85 l. 2a V115 T1. 15 1. 28 0. 72 V118 C0. 78 1. 14 1. 28 V115 T 1. 1s 1. 2 0. 118 0. 1. 14 VllS V 1. 0 ('1. 0 (l. OC V118 D-0. 14 0. 40 0. 38 V115 1. 0'1 1. 00 8 0. 4 1 2 2. 0 11. 14 1. 0. 07 11, g 116 1. 01 0. 9 108 118 1. 0. 0 7 T116 A 1. 01 o. gs 1, 0E V118 3 1. Q8 0. 56 0. 67 T116 0. 9 1. 18 I 0. 6 0 S 1 TI 16 c 0. 8 1. 0 1. 3 VI 18 1 0. 96 0. 55 1. 01 T116 E 0. 80 0. 91 1. 29 V118 9 1. 13-2. 50 0. 28 116 G 1. 10 0. 90 1. 44 V118 L0. 93 1. 05 0. 93 T116 t1. 00 1. 08 1. 48 V118 M0. 60 0. 93 0. 90 11 0. 8. 6. 82 1 0. 0. 2. 2 1 0. 7 0. 8 1 03 118 0. 8 1. S rl 16 M 0. 83 1. 3S 1. 28 V118 R. 0. 36 0. 07 0. 46 ril6 N0. 93 1. 05 1. 68 V118 g 0. 95 0. 82 0. 96 rl 16 P 0. 74 0. 84 0. 99 VI 18 r 0. 99 0. 92 0. 90 T116 00. 95 0. 77 1. 29 118 1. 00 1. 00 1. 00 rl 16 R 0. 64 0. 62 1. 03 V118 {V 0. 83-1. 28 0. 42 rl 16 S 0. 8E 0. 96 1. 24 V118 Y 1. 25 134 0. 60 T116 T 1. 00 1. 00 1. 00 L119 A 0. 81 1. 02 1. 18 T116 0. 86 0. 57 0. 85 L119 C 0. 76 0. 24 1. 18 rl 16 W 0. 85 0. 75 0. 96 L119 D 0. 24 0. 28 0. 97 TI 16 y 0. 90 0. 47 1. 09 L119 E 0. 45 0. 32 1. 04 0117 A 2. 0 1. 73 1. 03 L119 0. 56-0. 61 0. 93 Q117 E 1. 1 1. 21 1. 10 L119 G 0. 93-0. 06 0. 97 117 1. 57 1. 02 0. 61 L119 H 0. 91 0. 46 0. 89 0117 G 2. 0E 0. 79 0. 97 Lu 19 1 0. 90 0. 43 1. 06 0117 H233 1. 12 1. 12 L119 L 1. 00 1. 00 1. 00 Table 10-12. Performance In ic T 1 1-P rformanc di Wild-Type Wild-Type ResJ PAF PAD Prot. ResJ PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI Lll9 N 0. 5E 0. 1 : 1. 14 S121 1. 12. 5. 9 L119 P-0. 14-0. 01 0. 71 S121 33 0. 7 0. 91 119 0. 43-0. 66 1. 00 A122 A l. OC 1. OC 1. 00 1 9 S 0.-0. 17 1. 05 1 2 0. 2 0. 7 119 0. 97 0. 1 0. 94 122 E 0. 71 0. 47 1. 04 l i9 0. 89 15 1. 04 A122 F 0. 97 0. 15 0. 87 119 0. 77 0. 20 0. 88 A122 0. 9. 4 5 L119 Y 0. 7 í 0. 51 0. 89 A122 H 1. 14 0. 17 1. 00 T120 0. 25 0. 66 109 122 I 1. 1. 04 T120 C0. 75 0. 92 1 : 14 A122 C1. 08 0. 45 0. 96 T12 0. 58 1. 531 1. 19 A122 L 0. 9 1. 07 0 0. 0. 5 0 1. 4 1. o i i oo a . i i 0. 8. 0 1 1 0. 1 T120 88 0. 26 1 0 2 M0. 81 0. 94 1. 06 0. 05 4 ri20 K0. 87 1. 09 1. 12 A122 P0. 61 0. 55 1. 07 ri20 L0. 80 1. 26 1. 00 A122 00. 69 0. 74 1. 02 120. OS. 2 0 9 1 0. 71 4 4 T120 [0, 91 1, 5< 1. 00 A122 N 0, 81 0, 7C I, 11 T12O K 0. 8, 1. 05 1. 12 A122 P 0. 61 0, 55 1, 0, T120 0. 7 1. 4 1. 10 122. 03 4 105 T120 0. 07-0. 4 0. 82 A122 T1. 08 0. 52 0. 97 T120 __ N 0W3r} 1. 48 1. 1C A122 S 1. 03 O. 43 1, 0 T120 P o Ori-o-4< 0&8-A122 r 10E O s2 o. 9ro T120 0 26 0. 78 1. 05 A122 V1. 04 0. 89 1. 05 T120 0. 24 0. 6 0. 12 0 9 ri20 S1. 09 1. 07 1. 35 G123 0 89 0. 9 120 1. 00 1. 00. 0 G 123 0. 0 0 92 120 0 26 1. 07 0. 93 G123 D 1. 73 0. 84 0. 90 12 Y0. 57 1. 61 1. 01 G123 E1. 13 0. 5 0. 96 S121 A 1. 12 1. 55 1. 1 G123 F 0. 84 0. 80 0. 85 S121 C 1. 18 1. 64 1. 09 G123 G 1. 00 1. 00 1. 00 S121 E 0. 89 1. 04 1. 01 G123 H1. 00 0. 74 0. 84 S121 G 1. 20 0. 99 1. 07 G123 K 0. 97 1. 12 0. 93 S121 K 1. 24 0. 78 1. 04 G123 L 0. 99 1. 38 0. 79 S121 L 1. 35 1. 49 1. 12 G123 M 0. 84 1. 38 0. 85 S121 N 1. 14 1. 06 1. 17 G123 N 0. 85 0. 71 0. 92 S121 P0. 83 0. 38 0. 92 G123 P 1. 32 0. 81 0. 89 S121 00. 92 1. 09 1. O 1 G 123 0. 01 0. 31 0. 37 S121 1. 26 0. 70 1. 06 G123 0. 66 0. 60 0. 83 S121 S 1. 00 1. 00 1. 00 G123 T 1. 06 0. 54 0. 85 S121 T1. 13 1. 26 0. 93 G123 V1. 40 0. 59 0. 89 Table 14-12. P rformance In ices Table 1-12. Performance In'es Wild-Type Wild-Type ResJ PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI'PI PI Pos. PI PI 12 0 9 1. 39 0 77 126 0. 67 0. 60 1. 02 G 2 0. 9 1. 24 0. 87 126 1. 00 1 00 G124 A 0. 84 0. 03 1. 2C G126 I 0. 84 0. 01 0. 81 G124 C 0. 72 67 1. 07 G12 L 1. 17 0.. 9 G124 4 0. 7 0. 9 12 0. 4 1. 7. 2 G124 F 1. 32 0. 95 0. 70 G126 N0. 38 0. 85 1. 04 l G124 G 1. 00 100 1. 00 G126 1. 1 0. 7 2 G124 1. 59. 10 0. 98 G126 R 0. 43 0. 76 0. 8 G124 I1. 85-0. 08 0. 92 G126 S 0. 76 0. 0. 90 G124 L 1. 92 0. 54 0. 98 G126 T 1. 58 0. 74 0. 90 G124 0. 97-0. 05 1. 3 1 G124 N 0. 98 0. 60 1. 1 G126 Y0. 54 0. 23 0. 82 4-0. 11-0. 08. 3 1.. 10 1. 12 0. 1 0. 4 1 4 1 14 41 0. 88 4 1 12 S 1. 27 0. 56 1.. 00 1 7 0. 40-0 1 1--oo o G124 S1. 27 0. 56 1. 00 ri27 E0. 40-0. 01 1. 03 G124 r 1. 64 0. 32 0. 97 T127 G 0. 95 0. 71 1. 04 G124 V 1. 44 0. 33 0. 93 T127 1. 57 0. 60 0. 99 G124 7-031 0. 4 1 1. 06 0. 20 0. 91 G124. 3 0 56. 66 12 9 0. 4 1 1 L V125 1. 69 0. 93 0. 91 1 7 0. 7 0. 64 1. 02 12 C 0. 96 0. 54 0. 67 127. 14 0. 7 0. 95 V125 D 1. 24 0. 54 0. 76 T127 0. 55 0. 15 0. 86 V125 E 0. 81 0. 39 0. 73 127 S 1. 0 0. 1. 8 V125 F 0. 96 0. 63 0. 77 127 T 1. 00 1. 00 1. 00 V125 G 2. 95 1. 09 0. 60 T127 V1. 07 0. 68 1. 06 V125 1. 01 0. 94 1. 05 T128 A 0. 76 131 1. 23 V125 1. 50 0. 62 0. 83 T128 D 0. 78 0. 66 1. 14 V125 R 1. 30 0. 47 0. 82 T128 F 0. 79 1. 71 1. 01 V125 S 1. 94 0. 79 0. 75 T128 H 0. 99 1. 08 1. 19 12 V 1. 00 1. 00 1. 00 T128 K 1. 06 1. 57 1. 10 V125 W0. 37 0. 25 0. 48 T128 L1. 06 1. 72 0. 97 V125 Y1. 08 0. 81 0. 82 T128 M 0. 72 1. 06 1. 28 G126 A 0. 96 0. 55 1. 02 ri28 N0. 70 136 1. 29 G126 C 035 0. 98 0. 96 T128 P 0. 87 1. 16 1. 18 G126 0. 33 1. 22 0. 93 T128 0. 78 1. 341 1. 24 Table 10-12. Performance Indices Tabl 1-12 P rformai es Wild-Type Wild-Type Res./PAF PAD Prot. Res PAF PAD Prot. Pos. Mut PI PI PI Pos. Mut. PI PI PI T128 0. 87 1. 70 1. 03 V 1. 15 0. 3rF 09 T128 S0. 92 1. 27 1. 07 P130 W1. 15 0. 28 0. 80 T128 1 0 1. 00 l. 0 1 1. 00 1. 0. 00 128. 98 1 1 OS 131 1.. 40 T128 0. 92 1. 2 0. 95 1 1. 6 T128 0. 95 1. 81 0. 96 A131 G 1. 66 0 8 Y129 A0. 64 0. 17 1. 39 A131 H1. 72 0. 82 0. 75 Y129 0. 6 0. 6 1. 42 A131 L1. 83 0. 59 0. 73 Y129 9. 35. 2 35 131 1. 0. 4 Y129 F 0. 7] 0. 71 1. 44 A131 Q 1. 29 0. 74 0. 65 Y129 0. 1. 1 1. 44 1 1 0 74 0. G 0.9 6 1. 10. 76 1. 04 0. 61 n29K0. 31-0. 29 1. 00 Am S1. 48 0. 68 0. 87 Y129-L0. 78 0. 27 1. 22 A131 V1. 59 0. 78 0. 89 Y129 1. 2 1 1 1. 4. 8 zozo x v 0. 1-0 4 9 0. 24 1. 4 Y129 0. 1 OS 1. 11 1 2 0. 49 6. 8 0 4 Y129 W Or6t 0. 21 1. 21 A131 W 1. 61-0. 42. Q Çs 1 9 038. 18 1. 00 1 0. 49. 8 0. 94 Y129 s 0. 671 0. 69 1. 08 P132 D-0. 11-7. 16 0. 62 Y129 R 0. 3E 0. 18 l. OC. P132 C 0. 4S 5. 6E 0. 94 Y129 s o. 6r} 0 69 1*0E P132 D-0. 11-7. 1f 0. 6ur Y129 T 0. 46 0. 14 1 OC P132 0. 02 0 80 Y129 V 0. 24-0. 29 l. OC P132 F () 76-1. 33 0. 45 0. 24-0 29. 00 0. 7 0. 49 . 47. 1. 1 2 0 8. 79 Y129 Y1. 00 1. 00 1. 00 P132 0. 50-1. 95 0. 68 P130 A0. 82 0. 44 1. 03 P132 0. 58-3. 19 0. 64 P130 C0. 95 0. 64 0. 93 P132 L 0. 8ru 2. 2 0. 6 P130 1. 00 0. 22 1. 08 132 0. 0 1. 05 0. 83 P130 F 1. 08 0. 48 0. 89 P132 P0. 09 6. 91 1. 03 P130 J 1. 1f-0. 19 1. 11 P132 00. 41 6. 15 0. 91 P130 H 1 0. 01 1. 00 P132 R0. 02-2. 19 0. 65 P130 11. 12 0. 41 0. 94 P132 S 1. 13 5. 05 0. 96 P130 K 1. 16 0. 55 1. 05 P132 r 0. 84-2. 01 0. 75 P130 L 1 12 0. 09 0. 98 P132 0. 85-2. 29 0. 78 P130 0. 66 0. 76 1. 03 P132 0. 77-2. 64 0. 37 P130 1. 00 1. 00 1. 00 P132 1. 57 4. 78 0. 60 P130 R 1. 11 0. 53 0. 95 K133 A 0. 67 0. 10 1. 01 P130 S 1. 16-0. 14 0. 96 133 C 0. 56-0. 11 0. 72 P130 T 1. 191-0. 061 0. 96 E 0. 631 0. 76 1. 01 Table 1-12 Performance Indic s T ble 10-12. Per rmanc Indi s Wild-Type Wild-Type Resl PAF PAD Prot. ResJ PAF PAD Prot. Pos., Mut. PI PI PI Pos. Mut. PI PI PI K133 F 0. 86 0. 59 0. 73 Ll 5 0. 66-1. 2 0. 8 1 3 G 0. 97 031 0. 8 135 1. 00 1. 0. 0 K133 1. 02 0. 31 0. 87 Ll 0. 8 1. 01 K133 10. 89 0. 45 0. 78 L135 P-0. 13-1. 31 0. 22 K133 K1. 00 1. 00 1. 00 L135 00. 34 0. 17 0. 66 K133 L 1. 05 1. 92 0. 76 L135 R0. 06-1. 41 0. 25 1 3 0. 68 0. 33 0. 98 L135 S 0. 50-0. 65 0. 44 K133 P 0. 39 0. 71 0. 89 L135 r0. 73-0. 42 0. 50 1 1 K133 3 0. 69 0. 52 1. 13 L135 0. 8 0. 43 0. 82 K133 R 0. 78 0. 83 1. 01 L135 W 0. 71-0. 42 0. 36 K133 S 0. 84 0. 58 1. 6 0 1 0. 3 0 9 39 0. 7 1 1 0.. K133 T 0. 93 0. 87. 09 0. 2 2 0. 9 0 46 1. 1. 12 144. 0.. 4 0 1 4 0. S 1. 64 0.. 9-0 2 52 . K133 Y 1. 12 1. 44 0. 75 V136 N-0. 05 0. 4 (0. 26 V134 A 0. 75 1. 64 0. 87 V136 P-0. 05-0. 1 : 0. 52 1 4 0. 77 1 7 0. 91 1 6.-0. 0-012-0. 02 V134 D-0. 08-0. 08-0. 02 V136 1. 13 1. 13 0. 68 V134 G 1. 71. 1. 42 0. 45 V136 V1. 00 1. 00 1. 00 v 1 r y 1 1 12 0 8 0. 99 3 0-0. 12-0 02 V134 K-0. 08-0. 08-0. 02 V137 A1. 07 1. 46 0. 64 V134 L 1. 13 1. 45 0. 78 V137 C 0. 9E 1. 4'0. 85 V134 M 0. 82 1. 89 0. 83 V137 D-0. 1-0. 23-0. 01, V134 N 1. 18 2. 80 0. 25 V137 E-0. 17-0. 23-0. 01 V134 P-0. 08 1. 71 0. 43 V137 F-0. 17-0. 23-0. 01 V134 0. 04 0. 79 0. 44 V137 G 1. 02 0. 26 0. 13 V134 R-0. 08-0. 08-0. 02 V137 I0. 98 0. 70 0. 83 V134 S 1. 16 1. 44 0. 62 V137 L 1. 09 1. 27 0. 82 V134 1. 25 0. 86 0. 82 V137 M 1. 22 1. 13 0. 89 V134 V 1. 00 1. 00 1. 00 V137 0. 46-1. 29 0. 15 V134 W-0. 08-0. 08-0. 02 V1 37 P-O. 1,-0. 2'-0. 0 1 V134 Y-0. 08-0. 08-0. 02 V137 R-0. 17-0. 23-0. 01 L135 D-0. 13 2. 90 0. 27 V137 S 0. 96 0. 29 0. 50 L135 E-0. 13 0. 63 0. 39 V 137 T 1. 08 0. 93 0. 73 L135 F 0. 34-0. 03 0. 45 137 V 1. 00 1. 00 1. 00 Ll 5 G 0. 33-1. 71 0. 2E V137 W,-0. 1,-0. 2 :-0. 01 Ta le 10-12. Performanc Indi es Ta 1 0-12. Per orma e L ic, Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI V137 Y-0. 17-0. 23-0. 01 P140 A 1. 90 1. 83 0. 61 S138 A 0. 6S 1. 28 1. 44 P140 C 0. 35 1. 0', 0. 40 S138 C 0. 64 1. 18 1. 17 P140 D-0. 45-0. 23-0. 02 0-23 S138 E-0. 13-0. 19-0. 02 P140 F _0. 4s 2. 9 0. 19 S138 F-0. 13-0. 19-0. 02 P140 0. 3. 1 S 13 G 1. OS 1. 1 109 140 0. 9 2. 25 0. 3 S138 H-0. 13-0. 19-0. 02 P140 L_ 0. 44-1. 03 0. 24 S138 I 1. 15 0. 35 0. 56 40-0. 45-0. 3-0 02 S138 L-0. 13-0. 19-0. 02 P140 L-0. 45-0. 23-0. 02 L v. Vi S138 M-0. 13-0. 19-0. 02 P140 M-0. 45-0. 23-0. 02 1 0 1. 1. 77 14 00 1. 0 00 S138 P0. 54 1. 39 0. 45 P140 0-0. 45-1. 32 0. 32 v v w w S138 0-0. 13-0. 19-0. 02 P140 R-0. 45-2. 74 0. 25 1-0. 19-0. 2. 14 1 0. 4 S138 100 1. 00 1. 00 140 1.-0. S138 V1. 00 0. 69 0. 67 P140 V0. 50-1. 12 0. 34 S138 W-0. 13-0. 19-0. 02'140 W0. 50-0. 97 0. 17 S138 Y-0. 13-0. 19-0. 02 P140 Y 0. 32-1. 90 0. 24 P139 C0. 08-0. 12 0. 18 P141 A1. 10 1. 08 1. 13 P139 D-0. 13-144 O1 141 G. 1. 64-0. 0 1. 02 P139 E-0. 13-5. 11 0. 19 P141 2. 7. 9 P139 F-0. 13-4. 13. 16 141 2. 29 0. 38 0. 90 P139 G. 50-3. 08 0. 23 P141 L 2. 32 0. 65 0. 74 P139 H-0. 13-6. 03 0. 19 P141 N 1. 32 0. 97 0. 96 P139 [-0. 13-3. 71. 0. 21 P141 P1. 00 1. 00 1. 00 P139 K-0. 13 4. 09 0. 12 P141 01. 39 0. 37 0. 88 P139 L-0. 13-0. 17-0. 02 P141 R 1. 65-0. 26 0. 61 P139 N-0. 13-2. 11 0. 16 141 S 1. 70 0. 02 0. 90 P139 P1. 00 1. 00 1. OC P141 r 1. 84 0. 12 0. 82 P139 0-0. 13-0. 32 0. 18 P141 V 1. 96 0. 16 0. 72 P139 R 0. 37-1. 04 0. 23 L142 A 0. 80 0. 56 0. 67 P139 S 0. 88 0 52 0. 43 L142 C 0. 74 0. 70 0. 78 P139 r 0. 01-3. 48 0. 15 L142 D-0. 12-0. 13-0. 01 P139 V-0. 13-1. 70 0. 17 L142 1. 05 0. 54 0. 46 139-0. 13-0. 17-0. 02 L142 G-0. 12-0. 13-0. 01 139-0. 13-0. 17-0. 02 L142 I 0. 64 0. 28 1. 05 Table10-12. Performanc Indices Tabl 10-12. P rformance Indices Wild-Type Wild-Type ResJ PAF PAI ? i Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI L142 1. 60 0. 66 0. 23 1 1. 44. 1. 49 0. 74 142 L 1. 00 1. 00 1. 00 1 1. 00 1. 0 1. 00 142-0. 12-0. 1-0. 01 144 13 1. 80. 7 L142 N-0. 12-0. 13-0. O1 144 1. 76 1. 14 0. 68 L142 P 0. 54, 0. 44 0. 4 144 S 1. 69 0. 92 0. 77 L142 00. 67 0. 0. 49 144 1. 46 0. 81 0. 80 L142 R-0. 12 0. 13-0. 01 P144 Y 2. 34 1. 65 0. 70 L142 s 0. 84 0. 31 0. 65 M145 A 0. 44 0. 79 0. 94 L142 T-0. 12-0. 13-0. 01 M145 c 1. 02 0. 93 0. 94 L142 V 0. 84 0. 33 0. 82 M145 E 0. 28 0. 48 0. 74 .. L142 2. 41 89. 1 145 1. 49 0.. 95 A143 A 1. 10 1. 00 M145 G 0. 4E 0. 2, t 0. 92 A143 c-1. 9 1. 0. 81 4 0 0 1 1 AIN 14 1. 0. 71 1 1 1. 0 A143 1 4 1. 1 1 5 1 1... 00 A143 F 1. 56 0. 68 0. 9 M145 p 0. 6 0. 0. 78 A143 G 1. 48 0. 42. 17 14 0. 6 0. 57 0. 86 A143 H 2. 90 1. 36 0. 70 M145 R 1. 15 0. 69 0. 78 A143 3. 16 1. 37 0. 62 M145 S 0. 64 0. 73 0. 91 A143 L 2. 51 1. 28. 71 1 5 1. 0. 79 91 A143 N1. 30 0. 82 0. 79 M145 V0. 72 0. 63 1. 00 A143 P1. 53 0. 39 0. 63 M145 W1. 15-0. 13 0. 49 A143 qui 1. 74 0. 81 0. 72 M145 Y 0. 94 0. 82 0. 68 A143 2. 15 0. 99 0. 62 P 146 A 0. 2C 136 0. 73 A143 s 1. 77 0. 63 0. 98 P146 C 31 1. 69 0. 62 A143 IT 18 0. 97 0. 74 P146 F 0. 55 1. 53 0. 51 A143 V 2. 45 0. 99 0. 81 P146 G 0. 24 1. 04 0. 51 A14 W 2. 27-0. 21 0. 37 P146 H 0. 50 1. 57 0. 56 P144 A l. OS 0. 79 0. 91 146 L 0. 56 2. 00 0. 53 P144 D1. 45 1. 38 0. 60 P146 0. 39 1. 23 0. 79 P144 F1. 82 1. 08 0. 66 P146 N 0. 37 1. 00 0. 78 P144 G 1. 45 0. 62 0. 78 P146 1. 00 1. 00 1. 00 P144 H 1. 94 1. 60 0. 66 P146 R 0. 36 1. 06 0. 66 P144 K 2. 0S 1. 09 0. 67 P146 S 0 4e 0. 96 0. 82 P144 L 1. 43 1. 15 0. 86 P146 r 0. 3E 0. 76 0. 80 P144 M 1. 24 1. 01 0. 76 146 V 0. 55 0. 77 0. 89 Table 10-12. Performance Indi e T I 10-12. P forman e In' Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut., PI PI PI P146 _. 0. 5f 0. 68 0. 64 W149 C 0. 18 0. 12 P146 0. 35 1. 44 0. 54 W149 E0. 00-0. 04 0. 85 H147 A1. 28 0. 98 0. 96 W149 0. 53 0. 50 1. 27 H147 c 0. 94 1. 17 1. 04 W149 0. 2. 45 1. H147 0. 9 1. 18 1. 00 W149 H 0. 6 1. 01 0. 81 H147 E1. 11 1. 10 0. 96 W149 10. 21 0. 24 0. 831 H147 G-0. 12-0. 15-0. 02 W149 L 0. 3C 0. 64 1. 06 H147 1. 00 1. 00 100 14 0. 3 4 1. 32 H147 0. 89 0. 92 0. 89 149-0. 2-0. 6 2 H147 K 0. 94 1. 06 0. 89 W149 0. 11 0. 40. 10 H147 L 0. 69 1. 29 1. 05 W149 R 0, 04-0, 32 0, 6S H147 144 86 149 0.. 2 H147 0. 4 1. 2 0 98. 44 84. 1 1. 1 0 71 4 1. 0 ) H147 P1. 12 1. 21 0. 71 W149 W1. 00 1. 00 1. 00 H147 Q 0. 71 1. 03 0. 8C W149 Y o. sa 0, 75 1. 1 H147 R 0. 89 0. 94 0. 69 F150 A0. 01 0. 54 1. 70 H147 S 1. 26 0. 75 0. 92 F150 C 0, 43 0. 78 1. 41 14 T 1. 20 0. 84 0. 85 F150 E 1. 23 0, 73 1. 32 H147 V 0. 96 0. 92 0 90 1 1. 00 1. 00 1 H147 W0. 88 1. 05 0. 79 F150 G0. 14 0. 46 1. 13 H147 Y0. 75 1. 12 0. 94 F150 H0. 53 1. 18 1. 09 P148 A 1. 64 1. 06 0. 96 F150 10. 40 0. 78 1. 19 P148 1. 03 1. 34 0. 74 150. 41 0. 85 13 P148 E 1. 42 1. 19 0. 76 F150 L 1. 29 1. 30 1. 14 148 137 1. 50 0. 64 F150 m 0. 80 0. 63 1. 69 P148 G 0. 87 1. 20 0. 70 F150 N 0. 55 0. 36 1. 52 P148 K 1. 79 130 0. 72 F150 P 0. 18 0. 32 1. 3E P148 L 1. 64 139 0. 74 F150 T037 0. 58 1. 27 P148 P 1. 00 1. 00 1. 00 F150 V 0. 22 0. 51 1. 26 P148 0 1. 331 0. 98 0. 81 F150'W0. 19 0. 62 1. 26 P 148 R 1. 51 1. 25 0. 79 F 150 Y 0. 72 1. 07 1. 24 P148 S 1. 46 1. 21 0. 74 OlSl G 1. 29 2. 93 0. 46 P148 T 1. 50 1. 09 0. 79 0151 C1. 05 2. 55 0. 38 P 148 V 2. 43 1. 04 0. 76 151 D 1. 47 2. 81 0. 83 P148 Y 1. 46 1. 37 0. 72 151 E 1. 14 2. 07 0. 99 W149 0. 21 0. 31 1. 35 151 031-8. 08 0. 21 Table 10-12. Performance Indic s T bl-12. Performanc In's Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI 0151 H1. 06 2. 19 0. 94 1153 L 1 o2ri 1. 50 0. 82 0151 0. 08-2. 76 0. 16 1153 s 0. 72 0. 89 1. 04 0151 K1. 07 2. 19 1. 04 153 P 0. 24 1, 8, 0, 31 0151 L0. 40-1. 53 0. 17 1153 s 0 1. 6. 61 0151 M 1. 2, 4 6. 36 0. 24 1153 1. 2 1.. 64 0151 1. 35 1. 91 0. 50 1153 V 0. 9t 1. 15 0. 78 0151 1. 00 1. 00 1. OC F154 D-0. 19-1. 06-0. 02 0151 R136 2. 32 0. 68 F154 E-0. 19-1. 06-0. 02 151 S 1. 05 2. 25 0. 86 F154 F 1. 0 1. 00 1. 0 0151 1 24 2. 37 O F154 G-0. 19-0. 64 0. 17 0151 V0. 36-1. 65 0. 25 F154 L-0. 19-1. 06-0. 02 0151 w 7 0. 32 0. 33 F154 P-0. 19-1. 06-0. 02 0151 101 2 5 0. 41 5 0 9 0. 97 04 L152 A0. 88 1. 29 0. 85 F154 S0. 13 0. 29 035 L152 C1. 00 1. 14 0. 87 F154 T0. 12-1. 76 0. 19 1 L152 D 1. 0 0. 86 0. 81 F154 V-0. 19-14 0, 18 L152 E 1. 08 1. 23 0. 93 F154 y 1. 32 4. 6 0, 92 L152 G1. 08 0. 77 0. 85 E155 G O. 9S 2. 5 0. 83 L152 H 1. 09 0. 92 0. 93 E155 D 1. 0E 1. 24 0. 89 L152 11. 04 0. 61 0. 77 E155 E1. 00 1. 00 1. 00 L152 121. 91 0. 93 E15 1. 07 0 0 L152 L1. 00 1. 00 1. 00 E155 G 1. 17 1. 12 0. 82 L152 0. 9 1. 10 0. 82 E155 1 0. 95 0. 65 0. 61 L152 0. 81 0. 61 0. 54 E155 K 1. 2^ 1. 33 0 83 L152 1. 07 0. 76 0. 84 E1 L 1. 31 2. 07 0. 60 L152 R 1. 20 0. 91 0. 89 E155 0. 73 2. 91 0. 74 L152 S 1. 12 0. 84 0. 84 E155 N 0. 79 1. 79 0. 86 L152 T 1. 12 0. 69 0. 82 E155 p 0. 79 2. 60 0. 65 L152 V 1. 22 0. 88 0. 83 E155 0. 90 0. 69 0. 87 L152 W 1. 18 1. 55 0. 74 E155 R 1. 47-0. 07 0. 71 L152 Y 1. 09 1. 37 0. 89 E155 S1. 08 1. 12 0. 82 1153 A 1. 19 1. 49 0. 76 E155 1. 49 1. 19 0. 76 1153 1. 23 1. 75 0. 47 E155 0. 79 0. 47 0. 63 1153 H 1. 46 2. 00 0. 56 E155 Y 1. 27 2. 65 0. 55 1153 I l. OC 1. 00 1. OC G156 A0. 99 1. 21 0. 88 I153 K 1. 62 2. 44 0. 43 G156 C 1. 07 1. 37 0. 84 Table 10-12. Performance Indi e Ta 1 10-2 Per ormanc di Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. PI Pi Pi G156 D0. 96 1. 62 0. 93 E158 F2. 06 1. 77 0. 46 G156 E0. 94 1. 14 0. 91 158 32. 40 1. 01 0. 59 G156 F 0. 90 0. 73 0. 78 E158 1138 0. 94 0. 76 G156 G 1. 00 1. 0 1. 00 E158 2. 08 1. 0. 2 G156 H1. 04 1. 40 0. 84 E158 L. 59 1. 96 0. 70 G156 li 0. 0-0. 08 0. 44 158 1. 3 1. 73 0. 71 G156 1. 10 1. 11 0. 88 E158 N 1. 41 1. 58 0. 82 G156 0. 90 0. 94 0. 74 58 141 1. 19 0. 85 G156 1. 09 1. 62 0. 80 158 1. 49 1. 24 0. 85 1 6. 07 138 0. 97 E158 R 1. 99 1. 29 0. 62 G156 1. 44 1. 2. 9 8 1. 57 1 2. G156 In 1*04 1. 21 2. 80 1. 45 1. 77 6 S 1 2 1. 4 0. 8 1 1 2. 8 0. 81 1 15 0. 1 0. 88 0. 58 1 1. 77 G1 6 0. 89 0. 90 0. 6 1 9 10 0. 28 113 8 8 . gr G156 Y 0. 9e 1. 40 0. 80 1 9 1. 13 0. 31 0. 79 G157 0. 7 0. 87 1. 00 1 9 1. 09 0. 63 0. 90 0. 96 0. 61 0. 92 59 0. 99 0. 97 1. 14 G157 D 0. 93 0. 94 0. 41 15 0 0. 1 3 G157 E o. sa 0. 84 0. 61 0159 H 0. 96 1t48 O. 9C G157 F 1. 27 1. 42 0. 61 0159 L 1v02 0970 0e83 G157 G 1. 00 1. 00 1. 00 1 9 1. 07 0. 84 0. 83 G1 7 1. 14 1. 57 0. 70 0159 P1. 06 0. 49 0. 81 G157 11. 11 133 0. 36 0159 01. 00 1. 00 1. 00 G157 IK 1. 28 1. 47 0. 46 Q159 R 1. 15 0. 74 0. 76 G157 M 0. 96 0. 85 0. 70 0159 S l. lC 0. 73 0. 81 G157 0. 86 0. 01 031 160 A039 1. 14 0. 86 G157 R 1. 51-O. lC 0. 42 K160 C 0. 4a 1. 29 0. 77 G157 S 1. 30 0. 19 0. 93 K160 D-0. 15 1. 19 0. 40 G157 T 1. 74 0. 99 0. 68 K160 G 0. 91 0. 30 0. 56 G157 V 1. 23 0. 40 0. 59 K160 H o. sa 0. 57 0. 65 E158 A 1. 45 1. 28 0. 91 160 I 0. 97 1. 00 0. 78 E158 C 1. 46 1. 37 0. 67 160 K 1. 00 1. 00 1. 00 E158 1. 35 0. 89 0. 82 K160 L 0. 97 0. 95 0. 77 E158 E1. 00 1. 00 1. 00 K160 M031 1. 47 0. 78 Table 10-12 Perf rmance In i e T 1 10-12 Performance Indi Wild-Type Wild-Type Res./PAF PAD Pro Res./PAF PAD Prot. s. M I I P p ut I K160 N 0. 37 1. 12 0. 65 162 T1. 00 1. 00 1. 00 1-0. 15 1. 66 0. 31 ri62 15 2. 04 0. 85 K160 Q 0. 44 1. 41 0. 75 T162 Y 1. 03 2. 89 1. 03 K160 R 0. 83 1. 15 0. 76 E163 A 1. 11 1. 7 0. 73 K160 0. 85 0. 70 0. 74 E163 C 1. 11 1. 08 0. 67 K160 0. 89-0. 34 0. 21 E163 0. 90 1. 08 82 T161 C 0. 84 0. 56 1. 01 E163 S1. 00 1. 00 1. OC T161 D-0. 14-0. 21-0. 02 E163 F 1. 07 0. 27 0, 4S T161 E-0. 14-0. 21-0. 02 E163 G 1. 25 0. 80 0. 79 T161 G 0 9 0. 43 0. 94 E163 H 1. 32 0. 82 0. 69 T161 H 1. 82-0, 15 0. 42. E163 1. 1. 4 8 161. 40 0. 0. 91 16. 91 0. 7 1 1. 25. 1 0. 1. 2 0. 1. 4. 2 161. 8. 8 0 2 1 3 S. 2 81 T161 M 0, 59 1, 72 0. 83 3163 R 1. 12 0, 49 0. 72 T161 N 0. 8C-0. 86 0. 32 E163 S 1. 12 0. 85 0, 81 61. 14. 21-0. 02 E163 1. 0. 5 0. 69 ri61 01. 04 1. 50 0. 90 E163 W1. 21 0. 98 0. 49 T161 R 3. 61-1. 68 0. 42 E163 Y, 1. 41 1. 89 0. 60 T161 S 0 92 0. 57 0. 98 L164 A-0. 14-0. 85 0. 21 161. 00. 0 100 L164 C 0. 0 0. 6 T161 V1. 27 1. 24 1. 00 L164 D-0. 14-0. 85 0. 12 T161 w 1. 41 0. 00 0. 52 L164 E-0. 14-0. 48 0. 18 T161 2. 40 2. 62 0. 23 164 F 0. 50 0. 86 0. 94 T162 C 0. 95 3. 57. 17 Li64-0. 14. 14 19 T162 F 0. 99 3. 23 1. 05 L164 0. 0 0. 12 0. 16 162 G 1. 00 1. 82 0. 88 L164 L 1. 00 1. 00 1. 00 T162 H 1. 02 3. 91 1. 08 L164 0. 69 1. 26 1. 09 T162 l 0. 99 2. 21 1. 16 L164-0. 14 1. 31 0. 26 162 K 1. 22 3. 13 0. 98 L164-0. 14 2. 41 0. 17 162 L 1. 00 3. 59 1. 05 L164-0. 14 1. 01 0. 24 T162 M 0. 77 3. 49 0. 89 L164 R-0. 14 1. 61 0. 17 T162 N 0. 83 3. 84 0. 98 L164 S 0. 32 1. 11 0. 25 T162 P 0. 96 4. 37 0. 81 L164 T 0. 82 0. 99 0. 52 T162 0. 93 2. 45 0. 89 L164 V 0. 87 1. 02 1. 08 T162 R 1. 19 1. 23 0. 80 L164 Y 0. 43-1. 28 0. 20 T162 S 0. 98 2. 01 0. 97 A165 A 1. 00 1. 00 1. 00 Table 1-12. Performance In ices Table 1-12. Performance In ices Wild-Type Wild-Type ResJ PAF PAD Prot. Res./~ PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI A165 0. 99 1. 42 0. 97 V167 G 0. 99 2. 83 1. 08 A165 D 0. 8S 1. 69 0. 62 V167 1. 3 2. 11. 12 A165 F1. 23 LOO 0. 74 V167 1. 08 100 1. 04 A165 1. 05 1 07'1. 14 V167 L 0. 84 2. 5 1. 1 A1 65 I 1. 1, 0. 55 0. 64 V167 M0. 53 3. 84 1. 04 A165 K 1. 3'0. 82 0. 78 V167 P031 6. 08 0. 85 A165 L1. 08 1. 55 0. 70 67 O 0. 55 2. 41 0. 9 A165 0. 97 1. 6 0. 77 V167 R 0. 78 2. 25 0. 88 A165 N 1. 01 1. 0. 91 16 S. 96 1. 04 A165 1 14 1. 4 0. 91 167 1 1 2. 4 0. 9 1 21 1 2 l. 7 1. 00 A165 7 1. 2. 8. 1. 4 A165 0. 94 1. 05 Y168. C 0. 69-4. 73 0, 5 í 8 1 0. 1 12 1 13 0. 88 1.'1 1. 2 0. 0. 7 1 0.. 1 8 A165 Y 1. 2C 0. 84 0. 67 Y168 F 0. 68 5. l', 1, 2E R166 A 0. 73 1. 51 1. 12 Y168 G 1. 9-40. 74 0. 23 R166 _ 0. 56 1. 55 1. 16 Y168 H-0. 11-1. 981-0. 03 R166 100 1. 10. 85 Y168 1 0. 83-0, 5.-0. 90 R166 G 1. 15 0. 91 1. 19 Y168 K-0. 11-1. 98-0. 03 R166 120 1. 5 0. 97 Y168 L 0. 59 5. 39 1. 27 R166 1 1. 26 1. 39 0. 86 Y168 N-0. 11-1, 98-0. 0 9 R166 11 1. 20 1. 19 168-0. 11-19-0. 0 R166 L 1. 27 150 1. 08 Y168 O 0. 28-8, 2'i. 0. 2 R166 m 0. 65 1. 29 1. 26 Y168 R-0. 11-1. 98-0. 0 R166 N 0. 75 1. 21 1. 16 Y168 S-0. 11-1. 98-0. 03 R166 P 0. 43 1. 50 0. 97 Y168 T 1. 51-22. 96 0. 39 R166 R 1. 00 1. 00 1. 00 Y168 V 1. 19-12. 96 0. 57 R166 S 1. 16 0. 95 0. 98 Y168 W-0. 11-1. 98-0. 03 R166 T 1. 19 0. 74 1. 04 Y168 Y 1. 00 1. 00 1. 00 R166 1. 17 0. 76 0. 94 S169 A 0. 94 1. 13 0. 95 R166 W 1. 25 1. 08 0. 80 S169 C 1. 03 1. 38 0. 78 R166 Y 1. 2S 1. 22 0. 85 S169 I 1. 16 1. 53 0. 66 V167 A 0. 56 4. 99 0. 98 S169 K 1. 21 1. 27 0. 94 167 C 0. 79 537 1. 01 S169 L1. 08 1. 47 0. 82 V167 D0. 56 5. 54 0. 98 S169 M0. 86 1. 40 0. 86 Table 10-12. Performance In ices Table 10-12. Performance Indices Wild-Type Wild-Type Res./PAF PAD Prot. ResJ PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI S169 0. 87 0. 89 0. 6 L171 R 0. 71-0. 20 0. 24 S169 01. 02 1. 37 0. 88 L171 s 1. 43 1. 76 0. 72 S169 R 1. 24 1. 19 0. 77 L171 T 1. 54 1. 3 0. 80 S1 9 1. 00 1. 00 1. 00 71 1. 02 1. 39 0. 92 S1 9 1. 15 0. 97 0. 82 L171 Y 1. 20 1. 3'o. sa S169 Y 1. 26 1. 10 0. 77 A172 A1. 00 1. 00 1. 00 A170 A1. 00 1. 00 1. 00 A172 C1. 20 0. 86 1. 09 A170 1. 15 1. 06 1. 02 A172 D-0. 15 1. 42 0. 16 1-42 A170 D1. 27 132 0. 88 A172 E-0. 15-0. 44 0. 19 A170 E1. 28 1. 17 0. 99 A172 G 1. 41. 0. 84 1. 07 A170 F 44 1. 17 0. 83 A172 1 1. 70 58 0. 30 A170 G 1. 591 0. 62 0. 96 A172 K0. 95-0. 43 0. 17 A170 I 1. 5S O. M 0. 95 L 1. 20 1. 22 0. 7C A170 K 1. 71 0. 83 0. 9 A172 M0. 84 1. 06 0. 84 A170 L1. 05 0. 85 0. 8', A172 N 0. 37 0, 7 (0. 3a A170 M 1. 03 1. 28 0. 93 A172 P-0. 15 0. 5E 0. 16 A170 N 1. 21 1. 17 0. 96 A172 00. 27 0. 18 034 A170 0. 75 1. 33 0. 80 A172 R0. 44-0. 18 0. 20 A170 01. 15 0. 89 0. 98 A172 S 1. 59 0. 85 0. 96 A170 S 47 0. 47 0. 99 A172 T 1. 1 0. 85 A170 1. 40 0. 72 0. 8e A172 V 1. 40 0. 39 0. 53 A170 V 1. 20 0 74 0. 83 A172 1. 43 0. 45 0. 12 A170 W 1. 04 0. 83 0. 82 A172 Y 0. 87 1. 76 0. 13 A170 Y 0. 80 0. 89 0. 89 S1737 0. 81 2. 72 0. 95 L171 A0. 35 1. 66 0. 79 S173 C 0. 82 3. 07 0. 59 L171 C 0. 56 1. 73 0. 97 S173 E 0. 78 2. 65 0. 90 L171 D-0. 06-0. 13-0. 01 S173 F 0. 96 2. 30 0. 71 L171 F 1. 30 1. 97 0. 87 S173 1. 07 1. 49 0. 95 L171 G 1. 26 133 0. 50 S173 10. 99 2. 22 0. 78 L171 H 1. 67. 1. 07 0. 61 S173 K. 1. 17 3. 01 0. 91 L171 I 1. 53 1. 42 1. 16 S173 L 1. 15 3. 86 0. 77 L171 K 2. 05 1. 53 0. 31 S173 M0. 80 3. 01 0. 84 L171 L 1. 00 1. 00 1. 00 S173 0. 19 2. 66 0. 35 L171 M 0. 53 2. 22 0. 90 S173 1. 09 2. 47 0. 82 L171 N 0. 96 2. 79 0. 40 S173 S 1. 00 1. 00 1. 00 L171 00. 97 1. 93 0. 6, S173 r 1. 06 1. 25 0. 89 Table 1-12 Performance Indic Table 10-12 Perform n' Wild-Type Wild-Type ResJ PAF PAD'Prot. Res./PAF PAD Prot. Pos, Mut. PI PI PI Pos. Mut. PI PI Pi 173 0. 95 2. 54 0. 75 K176 0. 36 128 0. 31 S 1 1. 16 3. 67 0. 67 K176 G1. 01 0. 73 0. 80 173 Y 1. 15 3. 54 0. 81 K176 K 1. 0 (1. 00 1. 00 F174 s 0. 59 2. 09.. 0. 61 K176 L 1. 0. 2 0. 58 F174 C 1. 32 0. 4E 0. 64 K176 M 0. 5# 1. 33 0. 74 F174 F 1. 00 1. 001. 00 C176 ST0. 60 0. 94 0. 85 1 4 1. 60 0 0. 85 1 6 0. 01 0. 78 0. 27 F174 H 0. 93 1. 05 0. 86 K176 0. 59 0. 97 1. 02 F174 K 0. 86 1. 17 0. 76 K176 0. 71 1 1. 06 F174 L1. 05 1. 83 0. 82 K176 S0. 76 0. 72 0. 93 F174 M 0. 91 2. 2C 0. 54 K176 T 1. 04 0 9 ? 0. 70 F174 P 1. 54 1. 4d 0. 1 : K176 V 1. 04 1. 33 0a71 1 4 0. 1 2. 2 0. 1 4 0. 97 0. 701 1 4. 54 146. l 1. 0 4. 46. 8 7 1 1 1 6 0 41 4 O. S2 1 4 F174 1. 16 0. 61. 75 1 8 0. 1 4 96 F174 R0. 70 0. 52 0. 95 K176 Y1. 04 0. 93 0. 60 F174 S1. 16 0. 61 0. 75 P178 A0. 31 4. 39 0. 96 F174 r, 0. 8C 0. 64 0. 62 P178 D 0. 1E 6. 44 0. 93 F174 0. 0 0. 6 0. 82 P178 E Q. 4a 4. 15 1. 05 F174 W0. 96-0. 02 0. 85 P178 G1. 09 2. 95 0. 67 0. 84 1 66. 77 8 134 1. 70 0. 73 1. 70 0 5 1 L 1 8 1 0. M175 E0. 95 1. 43 0. 89 P178 M 0. 53 3. 87 0. 78 M175 G 2. 04 0. 75 0. 67 P178 P0. 06 5. 02 0, 93 M175 L 1. 61 0. 86 1. 19 P178 O 0. 15 3. 64 0. 93 M175 m 1. 00 1, 00 1. 00 P178 s 0. 62-3. 06 0. 95 M175 N 1. 39 1. 02 1. 11 P 178 T 0. 70 2. 28 0. 81 t175 P-0. 2a 0. 08 0. 16 P178 V 0. 67 2. 70 0. 78 M175 01. 56 0. 83 0. 98 P178 w 1. 14 0. 02 0. 64 M175 R 1. 55 0. 86 1. 02 P178 Y 1. 38 6. 91 0. 74 M175 T 2. 21 0. 90 0. 98 F179 A-0. 18-0. 22-0. 02 M175 V 1. 93 0. 81 1. 00 F179 E 0. 02 1. 80 0. 20 M175 w 1. 25 0. 76 1. 14 F179 F1. 00 1. 00 LOO M175 0. 77 0. 72 1. 351 F179 G 0. 03 1. 16 0. 36 K176 A 0. 42 1. 19 0. 84 F179 H 0. 79 0. 93 0. 91 K176 C 0. 5E 1. 01 0. 87 F179 L 1. 15 1. 89 0. 43 K176 D 0. 62 1. 18 0. 74 F179 N 0. 77 0. 95 0. 46 K176 E 0. 67 1. 08 0. 88 F179 P-0. 18-0. 22-0. 02 Ta 1 10-12. Performance Indice Ta 1 1-12 Performanc I li e Wild-Type Wild-Type ResJ PAF PAD Prot. ResJ PAF PAD Prot. Pos. Mut, PI PI PI Pos. Mut. PI PI PI F179 0. 46-0. 87 0 46 D181 1 1. 14 0. 6 0. 54 F179 R-0. 18-0. 2-0. 02 D181 R 1. 23 0. 22. 45 F179 S0. 78 0. 34 0. 62 D181 s 1. 21 0. 5. 6 1 9 0. 70 1. 17 0. 69 D181 1. 2 0. 24 F179 0. 89. 6 0. 62 D181 V OS88-0. 3 0. 21 F179 Y 1. 0, 1. 47 0. 65 D181 W 1. 26-0. 52 0. 28 F180 A 0. 03 2. 70 0. 27 D181 Y1. 29-0. 25 0. 25 F180 C 0. 6s 1. 94 0. 66 A182 A1. 00 1. 00 1. OC F180 E-0. 14-0. 55-0. 02 A182 C 0. 7 0 9 1. 03 F180 F1. 00 1. 00 1. 00 A182 G 0. 92 0. 4 0. 90 1 0 0. 7-5. 0 20-0 14 18-0. 2 1 F180 1 20 2 1. 9 82 0-2 4 F180 S1. 08-6. 98 0. 24 A182 K-0. 14-0. 18-0. 02 1 30 2.. 86 1 4. 2 F180 M 0. 71 4. 3d 0. 86 A182 L-0. 14-0. 18-0. 02 V1 1-014 3. 5. 29 1 2-0. 14 4 FI$O N-014 3. 5 0. 29 A182 N-0. 14 0. 53 0. 1 F180 Q 0. 21-1. 87 0. 36 A182 P-0. 14-1w 0. 12 F180 R 1 0. 64-3. 57 0. 26 A182 00. 03-0. 84 0. 14 tF180 S0. 56-2. 05 0. 29 A182 R0. 25-2. 69 0. 12 F80 T 1. 01-0. 68 0. 3 A182 0 8 0. 85 0. 0 Fizz V 1. 14 3. 24 0. 76 A182 r 1. 14 0. 11 Ot48 F180 1. 11 1. 81 0. 90 A182 W-0. 14-0. 18-0. 02 _o 02 F180 Y 1. 12 2. 99 0. 84 A182 Y-0. 14-0. 1-0. 02 pu81 A 1. 35 1. 23 0. 65 G183 C 0. 56 1. 9 0. 92 D181 _ 1. 09 0. 85 0. 56 G183 D 0. 3a o. ss 0. 62 D181 D1. 00 1. 00 1. 00 G183 F 0. 68 0. 19 0. 75 D181 E l. la 0. 72 0. 78 G183 G1. 00 1. 00 1. 00 D181 F-0. 15-0. 17-0. 01 G183 H 0. 98 0. 95 0. 87 D181 G 1. 09 0. 52 0. 37 G183 L 0. 82 1. 50 0. 47 D181 H-0. 15-0. 17-0. 01 G183 P-0. 18 1. 02 0. 33 D181 1-0. 15-0. 17-0. 01 G183 0. 66-0. 20 0. 97 D181 K 1. 33 0. 47 0. 41 G183 R 0. 92 1. 09 0. 90 D181 L 1. 25 0. 16 0. 16 G183 S 0. 94-0. 08 1. 08 D181 M-0. 15-0. 17-0. 01 G183 V 0. 56-2. 47 0. 57 D181 N-0. 15-0. 17-0. 01 G183 0. 97 1. 45 0. 79 D 181 P 1. 03 0. 66 0. 60 S 184 0. 60 1. 69 131 Table 10-12. Performance In ic Table 10-12. Performances Wild-Type Wild-Type Res./PAF PAD Prot. Resj PAF PAD prot. Pos. Mut. PI PI PI Pos. Mut. PI Pi Pi S184 C 0. 81 239 1. 14 186 1. 01 0. 7 0. 77 S184 D 0. 8d 2. 24 I. 1 1 6 1. 86-5. 42 0, 35 S184 E 0. 94 1. 86-1. 39 18 1 1. 00 1. 00 1. 00 S184 F 1. 0'1. 2, 0. 89 186. 13-03. 1 S184 G 0. 99 0. 82, 1. 1'I186 L 1. 17 1. 14 0. 84 S184 H l. Or 0. 74 1. 07 186 0. 6 1. 3 1. 11 S184 1 0. 92 1. 21 0. 9 186-0. 13-2. 9 0. 25 S184 K 0. 97 1. 61 1. 02 rls6 0. Z-6. 69 0. 25 S184 0. 80 2. 00 0. 9 1 6 S 1. 3 21 0. 65 S184 0. 51. 77 1. 25 1186 T1. 51 0. 23 0. 79 S184 N0. 64 1. 93 1. 03 186 2 4-0. 1 0. 85 0 40-0. 1 S184 00. 89 1. 16 1. 0 1186 Y-0. 13-0. 36-0. 01 1 4 1. 0 1 0. 5 0 S184 s 1. 0 1. 00 S187 A 0. 51 1. 72 0. 86 S 184 r 1. 0i 0. 6 (0 ; 94 S 187 C O. 7C 1. 6, 0. 75 SI 4 0. 0 1. 2 1. 03 187. 9 14 2 184 1. 06 1. 09 0. 84 S187 IF 1. 02 0. 65 0. 73 V185 C 0. 6'0. 8 : 0. 96 S187 G 1. 03 1. 4 0. 88 V185 0. 40-2. 49 0. 21 S187 H 1. 29 1. 51 0. 68 V185 E0. 73 0. 88 0. 76 S187 [138 1. 58 0. 78 V185 F1. 02 1. 20 0. 83 S187 K 1. 45 1. 16 0. 7 V185 G1. 12-3. 67 0. 47 S187 L1. 37 1. 4 0. 75 V185 H 1. 30-0. 58 0. 71 S1 7 0. 49 1. 87 0. 85 V185 1 07 0. 63 1. 03 S187 N, 0. 55 1, 55 0. 90 V1 85 K 1. 3, 0. 79 0. 66 S187 P0. 44-0. 31 0. 78 V185 L 1. 23 0. 93 0. 75 S187 0. 63 0. 35 0. 94 V185 0. 39 1. 46 0. 77 S187 R 1. 04 0. 55 0. 821 V185 0. 77 1. 41 0. 73 S187 S 1. 00 1. 00 1. 00 V185 1. 15 0. 79 0. 57 S187 T 1. 12 0. 23 0. 74 V185 S1. 09 0. 53 0. 75 S187 V 1. 23 0. 58 0. 89 V185 T 1. 11 0. 91 0. 79 S187 W 1. 3 (0. 52 0. 73 V185 V 1. 0t 1, 01 1. 00 S187 Y. 43 0. 80 0. 76 V185 1. 36-0. 44 0. 53 T188 A 0. 97 0. 95 1. 40 V185 Y 1. 37 0. 58 0. 65 T188 C 0. 60 0. 87 2. 04 I186 A1. 46 1. 79 0. 90 T188 3-0. 05-0. 1-0. 02 1186 D-0. 13 4. 29 0. 19 T188 E0. 24 1. 97 0. 44 Table 10-12. Performanc Indi Tabl 10-2 Perf rman In' Wild-Type Wild-Type Res./PAF PAD Prot. Res./PAF PAD Prot. Pos. Mut. PI PI PI Pos. Mut. PI PI PI ri88 F 0. 9e-0. 2C 0. 63 I194 G 0. 10 0. 04 0. 34 188 G 0. 3 0. 132 194 1. 00 1. 00 1. 00 T188 H1. 11-0. 79 0. 74 194 L 0. 80 0. 58 132 188 11. 13 0. 10 1. 85 I194 0. 1. 42. 1 T188 8. OS-0. 14-0. 02 1194 0. 02-0. 40 0. 77 1 L 0. 761 0. 42 1. 76 I194 S 0. 30-0. 15 0. 48 1 8 0. 49 0. 75 1. 60 1194 V037 0. 78 1. 03 188 0. 69 1. 69 1. 24 I194 W0. 04-0. 09 1. 12 18-0. 05-0. 14-0. 02 I194-0. 32-0. 01 1. 01 1 0-0. 05-0. 14-0. 02 F196 A-0. 13-0. 13-0. 02 ri88 R1. 01-0. 47 1. 41 F196 c 1. 74 1. 18 0. 70 T188 s 1. 1 0. 91 1. 52 F196 F 1. 00 1. 0 (1, 00 rlss T l vOC l. OC l. OQ F196 G 1. 59-0. 3 (0. 60 1. 00 1. 0 19 G 1. 9-0 3 60. 1 2 1 53 9 1-0. 1-0-0 14 2 19 I. 1 2. 2 T188 1. 48 0. 09 0. 47 19-0 3-0. 1. 02 D189 A0. 05 1. 18 0. 3 196 L 1. 7 1 7 1 9 D189 c 0. 19. 0. 94 0. 56 Cl196 M 1. 65 0. 71 0. 93 D189 D 0. 03 0. 89 0. 90 F196 N-0. 13-0. 1-0, 0 D189 E 0. 3 0. 7 0. 5 00 0 0. 42 D189 F0. 83 37 0 6'1 F196 Q 1. 0-0. 2 0. 40 D189 G 0. 8 0. 80 0. 83 F196 R-0. 13-0. 13-0. 02 D189 9 1. 25 0. 95 0. 78 F196 S 1. 58-l. S', 0. 29 D189 1 0. 73 1. 27 0. 69 F196 V1. 40 0. 68 0. 51 D189 L130 130 0. 61 F196 1. 01 0. 38 0. 88 D189 M0. 06 0. 88 0. 48 F196 1. 41 0. 97 0. 73 D189 N 0. 22 0. 57 0. 80 D189 P-0. 12 0. 97 0. 67 D189 R 0. 8e 0. 3S 0. 65 D189 S 0. 8E 0. 81 0. 85 D 189 T 1. OC 1. 21 0. 73 D189 V 0. 73 0. 71 0. 72 D189 W1. 09 0. 76 0. 60 1194 0. 29 0. 00 1. 15 I194 _C 0. 27-0. 02 1. 17 I194 0. 07-0. 03 0. 95

GC821-2 EXAMPLE 11 Cloning and Expression of a Sinorhizobium meliloti RSM02162 M. smegmatis Perhydrolase Homologue In this Example, cloning and expression of a S. meliloti perhydrolase homologue are described. The sequences used in cloning and expression are provided. below. The gene RSM02162 (SEQ ID NO : 625) was synthesized by DNA2.0. The gene was given the designation"G00355"and was provided cloned into the commercially available vector, pDRIVE (InvivoGen). The gene was amplified by PCR from this clone using the primer set G00355rbsF/G00355R, Taq DNA polymerase (Roche) as per the manufacturer's directions, with G00355 as the template (10 ng/50 ul reaction) and 10 picomoles (per 50 ul reaction) of each primer. The amplification was carried out in an MJ Research PCR machine using 30 cycles of (1 minute at 95°C ; 1 minute at 55°C ; and 1 minute at 72°C). The amplification of the correct size fragment was confirmed by agarose gel electrophoresis. The fragment was cloned directly into pCR2. 1TOPO (Invitrogen) and transformed into E. coli Top 10 cells (Invitrogen). Transformants were selected on L agar containing carbenicillin (100, ug/ml) at 37°C. The correct construct was confirmed by sequence analysis and designated"pMC355rbs."Figure 20 provides a map of this plasmid.

Primer sequences: G00355rbsF 5'-ggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgc-3' (SEQ ID NO : 626) G00355R 5'-Gcgcgcttagaacagagccgctactttgtcagc-3' (SEQ ID NO : 627) Gene sequence (including stop codon) of RSM02162: 5'-

atggtggaaaaacgttccgttctgtgctttggtgattctctgacttggggctggattccg gtgaaagagagctccccaactctgcgtt acccatacgaacagcgttggaccggtgctatggctgcacgtctgggtgatggttaccaca tcattgaagaaggcctgtccgctcgt actactagcctggacgacccaaacgacgctcgtctgaacggctctacctacctgccgatg gctctggcttctcacctgccactgga tctggtaatcattatgctgggtaceaacgaeaccaaaagctactttcatcgtaccccata cgagattgccaacggcatgggtaaact ggtaggtcaggtcctgacctgtgcaggtggtgttggtacgccttatccagcaccgaaagt cctggtggttgcacctccaccactgg caccaatgccagatccgtggttcgaaggtatgttcggcggtggttacgagaaatctaagg aactgtccggtctgtacaaagcactg gctgatttcatgaaagtggagttcttcgcagcgggtgattgtatctccaccgacggtatc gacggtatccacctgagcgctgaaacc aacatccgcctgggtcatgctattgctgacaaagtagcggctctgttctaa-3' (SEQ ID NO : 625) G00355 Protein sequence : MVEKRSVLCFGDSLTWGWIPVKESSPTLRYPYEQRWTGAMAARLGDGYHIIEEG LSARTTSLDDPNDARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIA NGMGKLVGQVLTCAGGVGTPYPAPKVLWAPPPLAPMPDPWFEGMFGGGYEKS, KELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF (SEQ ID NO : 628) Complete sequence of pDRIVEG00355 : gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggca cgacaggtttcccgactggaaagc gggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggcttt acactttatgcttccggctcgtatgttg tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgcc aagctctaatacgactcactataggg aaagctcggtaccacgcatgctgcagacgcgttacgtatcggatccagaattcgtgattt tagaacagagccgctactttgtcagca atagcatgacccaggcggatgttggtttcagcgctcaggtggataccgtcgataccgtcg gtggagatacaatcacccgctgcga agaactccactttcatgaaatcagccagtgctttgtacagaccggacagttccttagatt tctcgtaaccaccgccgaacataccttc gaaccacggatctggcattggtgccagtggtggaggtgcaaccaccaggactttcggtgc tggataaggcgtaccaacaccacc tgcacaggtcaggacctgacctaccagtttacccatgccgttggcaatctcgtatggggt acgatgaaagtagcttttggtgtcgttg gtacccagcataatgattaccagatccagtggcaggtgagaagccagagccatcggcagg taggtagagccgttcagacgagc gtcgtttgggtcgtccaggctagtagtacgageggacaggccttcttcaatgatgtggta accatcacccagacgtgcagccatag caccggtccaacgctgttcgtatgggtaacgcagagttggggagctctctttcaccggaa tccagccccaagtcagagaatcacc aaagcacagaacggaacgtttttccaccataatctgaattcgtcgacaagcttctcgagc ctaggctagctctagaccacacgtgtg ggggcccgagctcgcggccgctgtattctatagtgtcacctaaatggccgcacaattcac tggccgtcgttttacaacgtcgtgact gggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagct ggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatggaaattgtaagcgttaat attttgttaaaattcgcgttaaatttttgt taaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaa gaatagaccgagatagggttgagtgttg ttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaa aaaccgtctatcagggcgatggccc

actacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaa tcggaaccctaaagggagcccccgat ttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaag gagcgggcgctagggcgctg gcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgcta cagggcgcgtcaggtggcactttt cggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggcattttgccttcctgttttt gctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtg ggttacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttta aagttctgctatgtggcgcggtattatcc cgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttg gttgagtactcaccagtcacagaaaa gcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtga taacactgcggccaacttacttctgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaac tcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggc gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggccctt ceggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatc attgcagcactggggccagatggtaa gccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaa tagacagatcgctgagataggtgcc tcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgat ttaaaacttcatttttaatttaaaaggatctag gtgaagatcctttttgataatctcatgaacaataaaactgtctgcttacataaacagtaa tacaaggggtgttatgagccatattcaac gggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggt ataaatgggctcgcgataatgtcgggc aatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctga aacatggcaaaggtagcgttgccaat gatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgacc atcaagcattttatccgtactcctgatga tgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaata tcctgattcaggtgaaaatattgttgat gcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaac agcgatcgcgtatttcgtctcgctcaggcg caatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggc tggcctgttgaacaagtctggaaagaa atgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcactt gataaccttatttttgacgaggggaaattaat aggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcct atggaactgcctcggtgagttttctcct tcattacagaaacggcritttcaaaaatatggtattgataatcctgatatgaataaattg cagtttcatttgatgctcgatgagtttttctaa gaattaattcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccc cgtagaaaagatcaaaggatcttcttga gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg gtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttc tagtgtagccgtagttaggccaccactt caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttacc gggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggt tcgtgcacacagcccagcttgga gcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgct tcccgaagggagaaaggcggac aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggga aacgcctggtatctttatagtcc tgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcg gagcctatggaaaaacgccagcaacg cggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgt tatcccctgattctgtggataaccgtattaccg cctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtga gcgaggaagcggaaga (SEQ ID NO : 629) Complete sequence pMC355rbs :

Expression of the Homologue from pMC355rbs To express the S. meliloti RSM02162 protein from the plasmid pMC355rbs (See, Figure 20, for a map of this plasmid), a single colony was into a 5 mls of L broth containing 100 pg/ml carbenicillin and grown overnight at 37°C with shaking at 200 rpm. Lysates were prepared by pelleting the cells from 1 ml of the overnight culture by centrifugation and lysed with BugBuster (Novagen). The supernatants were assayed using the pNA activity assay, perhydrolysis assay, and a pNC6 assay (to test its ability to hydrolyze carbon chains longer than C4), as described herein.

Assay Results The following Table (Table 11-1) provides a comparison of the hydrolysis activity of pNA by G00355 as compared to the M. smegmatis perhydrolase Table 11-1. pNA Hydrolysis Activity Rate Compared to Strain pNA Hydrolysis Perhydrolase Rate* E. coli/pMSATNcoI 85 1 E. coli/pMC355rbs 80 0. 94 E. coli/pCR2. 1 34. 6 0. 41

*Rate is absorbance units/min read at 405 nm in a spectrophotometer.

The following Table (Table 11-2) provides a comparison of the perhydrolysis of triacetin by G00355 compared to the M. smegmatis perhydrolase.

Table 11-2. Triacetin Perhydrolysis Activity Perhydrolysis Strain Activity Max Vmax E. coli/pMSATNcoI 1.04 11.88 E. coli/pMC355rbs 1.17 25.05 E. coli/pCR2.1 0.1 2.9 The following Table (Table 11-3) provides a comparison of pNC6 hydrolysis by G00355 compared to the M. smegmatis perhydrolase.

Table 11-3. pNC6 Hydrolysis Activity pNC6 Hydrolysis Rate Compared to Strain Rate* Ms. Perhydrolase E. coli/pMSATNcoI 0. 58 1 E. coli/pMC355rbs 6.57 11.3 E. coli/pCR2. 1 0. 47 0. 8 *Rate is absorbance units/min read at 405 nm in a spectrophotometer.

As these results indicate, the homologue RSM02162 from S. meliloti identified by amino acid sequence homology to the M. smegmatis perhydrolase demonstrated similar, albeit less perhydrolysis activity than the M. smegmatis perhydrolase. However, this enzyme exhibited different substrate specificity, as it was able to hydrolyze pNC6, while the wild-type M. smegmatis perhydrolase cannot.

The results of the pNC6 hydrolysis assay indicated that certain positions/substitutions provided an improvement in the ability of the enzyme to utilize longer chain substrates The positions and substitutions identified in preliminary screens are provided in the following Table. It is not intended that the present invention be limited to these specific positions and substitutions, as it is contemplated that additional positions and/or substitutions will also provide improved activity on longer chain substrates.

Table 11-4. Positions/Substitutions with Improved Activity in PNC6 Assay Wild-Tvse Residue/Positionl Amino Acid Variant 12 G O S54 T 153 P F154 S T V I194 G 196 C G I N P S V EXAMPLE 12 Amplification of Genes Encoding M. smegmatis Perhydrolase Homologues from Environmental Isolates

In this Example, methods used to amplify genes encoding M. smegmatis perhydrolase homologues from environmental isolates are described.

Organisms from soil samples that were positive for the transesterification reaction were purified to single colonies. To amplify the genes by PCR, the degenerate primer sets lAF/5AR and leFlSiR were used in a PCR reaction containing isolated chromosomal DNA from 8 environmental strains exhibiting the transesterification reaction. The PCR reaction was carried out using Taq DNA polymerase (Roche) as per the manufacturer's protocol, with 1 tig of chromosomal DNA added as template and 10 picomoles of each primer in a SO, reaction. The reaction was carried out for 30 cycles of (1 minute at 95°C ; 1 minute at 50°C, and 1 minute at 72°C). Since the partial coding sequence of the perhydrolase gene from Mycobacterium parafortuitum was already isolated, the same strain was used as a positive control. The strains were designated as : 2G, 2D, 9B, 14B, 18D, 19C, 20A. As indicated below, 20A was typed as Mycobacteriumparafortuitum, and 9B is Mycobacterium gilvum. Based on protein homology, it was inferred that 2D is also M. parafortuitum and 14B is Ad gilvum.

Primer Sequences 1AF : 5'-gccaagcgaattctgtgtttcggngaytcnyt-3' (SEQ ID NO : 631) 5AR : 5'-cgattgttcgcctcgtgtgaartgnrtnccrtc-3' (SEQ ID NO : 632) leF: 5'-acggtcctgtgctttggngaytcnyt-3' (SEQ ID NO : 633) 5iR : 5'-ccgctggtcctcatctggrtgntcnccrtc-3' (SEQ ID NO : 634) Amplification with the above primer sets was expected to yield bands of approximately 500 bp. In all cases except 2G, the 1AF/5AR primer set produced a band

of the expected size. In the case of 19C, both primer sets produced bands of the expected size. The-500 bp bands were purified from agarose gels using a gel purification kit (Qiagen) and analyzed by sequencing. While the strains 2G and 19C yielded bands of the expected size with both primer sets they were not the fragments encoding the M. smegmatis perhydrolase homologue.

Partial Sequences of 2D Perhydrolase Homologue and Protein: Gene : 5'-attctgtgtttcggggattccttgacgtggggatggatccctgtcgaagaaggtgtg cccaccgagcggttcccgcgtga cgtccggtggaccggcgtgctggccgacctgctgggcgaccgctacgaggtgatcgagga aggcctgtcggcgcgcacca ccaccgccgacgacccggccgacccccggctcaacggttcgcagtatctgccgtcgtgtc tggccagccatctgccgctg gacctggtgatcctgatgctcggcatcaacgacaccaaggcgaattttggccgcaccccg ttcgacatcgccaccggtat gggagtgcttgccacgcaggtgctcaccagcgccggtggcgtggggaccagctatcccgc gccgcaggtgctgatcgtgg cgccgccgccgctgggcgagctgccccacccctggttcgacctggtgttctccggcggcc gtgagaagaccgccgagttg gcccgcgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggctcg gtgatcagcaccgacggcgt ggacggcacccacttcacacgaggcgaaacaatcga (SEQ ID NO : 635) Protein : ILCFGDSLTWGWIPVEEGVPTERFPRDVRWTGVLADLLGDRYEVIEEGLSARTTT ADDPADPRLNGSQYLPSCLASHLPLDLVILMLGINDTKANFGRTPFDIATGMGVL ATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWFDLVFSGGREKTAELARVYS ALASFMKVPFFDAGSVISTDGVDGTHFTRGETI (SEQ ID NO : 636) Partial Sequences of 9B Perhydrolase Homologue and Protein : Gene : 5'-taccgtcgatgtgtggcctcgtgtgaagtgggtgccgttgccaagcgaattctgtgt ttcggggattcgttgacgtgggg ctggatcccggtcgaggaaggtgtacccacccaacgttttccgaagcgggtgcgctggac cggggtgctggccgacgaac tgggtgctggctatgaggttgtcgaggaggggttgagcgcgcgcaccaccaccgctgacg accctaccgatccccggctg aacggctcggactacctccccgcatgcctggccagceacctgccgctggacctggtgatc ctgatgctcgggaccaacga caccaaggcgaatctgaatcgcacacccgtcgacatcgccagcggaatgggcgtcctggc cacccaggtgctcaccagcg cgggcggggtcggcaccagctacccggccccgcaggtgttgatcgtggcaccgccgccgc tggccgagatgccgcacccg tggttcgagctggtcttcgacggcggccgggagaagaccgcccaactggcccgggtgtac agcgcgctggcgtcgttcat gaaggtgccgttcttcgacgccggatcggtgatcagcaccgacggtgtcgacggcaccca cttcacacgaggcgaaacaa tcgaccgg (SEQ ID NO : 637)

Protein : GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFPKRVRWTGVLADEL GAGYEWEEGLSARTTTADDPTDPRLNGSDYLPACLASHLPLDLVILMLGTNDTK ANLNRTPVDIASGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLAEMPHPWFEL VFDGGREKTAQLARVYSALASFMKVPFFDAGSVISTDGVDGTHFTRGETIDR (SEQ ID NO : 638) Partial Sequences of 14B Perhydrolase Homologue and Protein : Gene : 5'-attctgtgtttcggagattcgttgacgtggggctggatcccggtcgaggaaggtgta cccacccaacgttttccgaagcg. ggtgcgctggaccggggtgctggccgacgaactgggtgctggctatgaggttgtcgagga ggggttgagcgcgcgcacca ccaccgctgacgaccctaccgatccccggctgaacggctcggactacctccccgcatgcc tggccagccacctgccgctg gacctggtgatcctgatgctcgggaccaacgacaccaaggcgaatctgaatcgcacaccc gtcgacatcgccagcggaat gggcgtcctggccacccaggtgctcaccagcgcgggcggggtcggcaccagctacccggc cccgcaggtgttgatcgtgg caccgccgccgctggccgagatgccgcacccgtggttcgagctggtcttcgaeggcggcc gggagaagaccgcccaactg gcccgggtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggatcg gtgatcagcaccgacggtgt cgacggcacccacttcacacgagg (SEQ ID NO : 639) Protein : ILCFGDSLTWGWIPVEEGVPTQRFPKRVRWTGVLADELGAGYEWEEGLSARTT TADDPTDPRLNGSDYLPACLASHLPLDLVILMLGTNDTKANLNRTPVDIASGMGV LATQVLTSAGGVGTSYPAPQVLIVAPPPLAEMPHPWFELVFDGGREKTAQLARV YSALASFMKVPFFDAGSVISTDGVDGTHFTR (SEQ ID NO : 640) Partial Sequences of 20A Perhydrolase Homologue and Protein : Gene : 5'-ttgccaagcggaattctgtgtttcggggattctttgacgtggggatggatccctgtc gaagaaggtgtgcccaccgagcg gttcccgcgtgacgtccggtggaccggcgtgctggccgacctgctgggcgaccgctacga ggtgatcgaggaaggcctgt cggcgcgcaccaccaccgccgacgacccggccgacccccggctcaacggttcgcagtatc tgccgtcgtgtctggccagc catctgccgctggacctggtgatcctgatgctcggcatcaacgacaccaaggcgaatttt ggccgcaccccgttcgacat cgccaccggtatgggagtgcttgccacgcaggtgctcaccagcgccggtggcgtggggac cagctatcccgcgccgcagg tgctgatcgtggcgccgccgccgctgggcgagctgccccacccctggttcgacctggtgt tctccggcggccgtgagaag accgccgagttggcccgcgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttc gacgccggctcggtgatcag caccgacggcgtggacggcacccacttcacacgaggcgaaacaatcga-3' (SEQ ID NO : 641)

Protein : LPSGILCFGDSLTWGWIPVEEGVPTERFPRDVRWTGVLADLLGDRYEVIEEGLSA RTTTADDPADPRLNGSQYLPSCLASHLPLDLVILMLGINDTKANFGRTPFDIATGM GVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWFDLVFSGGREKTAELAR VYSALASFMKVPFFDAGSVISTDGVDGTHFTRGETI (SEQ ID NO : 642) Identification of the Natural Isolates To type the environmental isolates used in this Example, plates of the purified strains were sent to MIDI for 16S rRNA typing. 20A is Mycobacterium parafortuitum, 9B is Mycobacterium gilvum. By protein homology we infer that 2D is also M. parafortuitum and 14B is M. gilvum.

EXAMPLE 13 Sequence and Taxonomic Analyses of Perhydrolase Homologues In this Example, sequence and taxonomic analyses of M. smegmatis perhydrolase homologues are provided Taxonomic Assignment The basic"List of 60"protein sequences accessed from public databases and used for construction of primer sets for screening of metagenomic libraries (BRAIN) was converted into a document illustrating the microbial taxonomic origins of the proteins, as described below. This information was used to produce the following alignment. 1 50 MSAT (------------MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG 14B natural isolate (1)-----------------ILCFGDSLTWGWIPVEEGVPT-QRF2KRVRWTG 20A (1)-------------LPSGILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG 2D natural isolate (1)-----------------ILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG 9B Natural Isolate (1)-GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPT-QRFPKRVRWTG M. parafortuitum CO1 (1)-------------MAKRILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG Sm-RSM05666 (1)--------------MKTVLCYGDSLTWGYDATGSG-----RHALEDRWPS

At-Q8UACO tl)--------------MKTVLAFGDSLTWGADPATG---L--RHPVEHRWPD At-Q8UFG4 I 1)-------------MVKSVLCFGDSLTWGSNAETGG-----RHSHDDLWPS MO91 M4aEl1 (1)--------------MKTILAYGDSLTYGANPIPGGP----RHAYEDRWPT M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGG------RHALEDRWPS P. dejongeii RVM04532 (1)--------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhizobium meliloti (1)---------MEETVARTVLCFGDSNTHGQVPGRGPLDR---YRREQRWGG Q98MY5 Mesorhizobium loti (1)--------------MKTVLCYGDSLTWGYNAEGG------RHALEDRWPS RSM02 162 Sm (1)-----------MVEKRSVLCFGDSLTWGWI PVKESSPT-LRYPYEQRWTG S261 M2aA12 (1)--------------MKNILAFGDSLTWGFVAGQDM-----HPFETRWPN Smal993 Sinorhizobium meliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG Consensus (1) KTILCFGDSLTWGWIPV EG P RHP E RW G 51 100 MSAT (37) VLAQQLGADFEVIE-EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP 14B natural isolate (33) VLADELGAGYEVVE--EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP 20A (37) VLADLLGDRYEVIE--EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 2D natural isolate (33) VLADLLGDRYEVIE--EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 9B Natural Isolate (49) VLADELGAGYEWE--EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP M. parafortuitum CO1 (37) VLADLLGDRYEVIE--EGLSARTTTAEDPADPRL-NGSQYLPSCLASHLP Sm-RSM05666 (32) VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP At-Q8UACO (32) VLEAELAGKAKVHP--EGLGGRTTCYDDHAGPACRNGARALEVALSCHMP At-Q8UFG4 (33) VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M09l-M4aEll (33) ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADRNGARVLPTLLESHSP M1-RML000301 (45) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP P. dejongeii RVM04532 (37) VLAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE-EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP Q98MY5 Mesorhizobium loti (31) VLQASLGGGVQVIA-DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP RSM02162 Sm (39) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP S261 M2aA12 (32) ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP Smal993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP Consensus (51) VLA LGG Y VIE EGLSGRTT DDP D L NGS YLPT LASHLP 101 150 MSAT (84) LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLWQVLUSAGGVGUUYPA 14B natural isolate (80) LDLVILMLGTNDTKANLNRTPVDIA--SGMGVLATQVLTSAGGVGTSYPA 20A (84) LDLVILMLGINDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA 2D natural isolate (80) LDLVILMLGINDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA 9B Natural Isolate (96) LDLVILMLGTNDTKANLNRTPVDIA--SGMGVLATQVLTSAGGVGTSYPA M. parafortuitum C01 (84) LDLVILMLGTNDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA Sm-RSM05666 (80) LDLIVFMLGSNDMKPIIHGTAFGAV--KGIERLVNLVRRHDWPTETE-EG At-Q8UACO (80) LDLVIIMLGTNDIKPVHGGRAEAAV--SGMRRLAQIVETFIYKPREA--V At-Q8UFG4 (83) LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDW-EA M091 M4aEll (81) LDLIVIMLGTNDIKPHHGRTAGEAG--RGMARLVQIIRGHYAGRMQD--E M1-RML000301 (93) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWP--A P. dejongeii RVM04532 (84) LDLVILMLGTNDLKSTFNVPPGEIA--AGAGVLGRMILAGDAGPENR--P Q92XZ1 Sinorhizobium meliloti (87) LDLIIIMLGTNDLKRRFNMPPSEVA--MGIGCLVHDIRELSPGRTGN--D Q98MY5 Mesorhizobium loti (79) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWP--A

RSM02162Sm (86) LDLVIIMLGTNDTKSYFHRTPYEIA-NGMGKLVGQVLTCAGGVGTPYPA S261 M2aA12 (80) LDLVIIMLGTNDIKFAARCRAFDAS--MGMERLIQIVRSANYMKGYK--I Smal993 Sinorhizobium meliloti (97) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA Consensus (101) LDLVIIMLGTNDMKA RTP DIA GMGRLV VLT AGGVG A 151 200 MSAT (132) PKVLWSPPPLAPM-PHPWFQLIF-EGGEQKUUELARVYSALASFMKVPF 14B natural isolate (128) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF 20A (132) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF '2D natural isolate (128) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF 9B Natural Isolate (144) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF M. parafortuitum CO1 (132) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF Sm-RSM05666 (127) PEILIVSPPPLCET--ANSAFAAMFAGGVEQSAMLAPLYRDLADELDCGF At-Q8UACO (126) PKLLIVAPPPCVAG---PGGEPAG-GRDIEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (132) PDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M091 M4aEll (127) PQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF M1-RMLOO (139) PQILIVSPPVVSRT--ENADFREMFAGGDEASKQLAPQYAALADEVGCGF P. dejongeii RVM04532 (130) PQLLLMCPPKVRDLSAMPDLDAKI-PHGAARSAEFPRHYKAQAVALKCEY Q92X21 Sinorhizobium meliloti (133) PEIMIVAPPPMLED--LKEWESIF-SGAQEKSRKLALEFEIMADSLEAHF Q98MY5 Mesorhizobium loti (125) PQILIVSPPWSRT--ENADFREMFAGGDEASKQLRPQYAALADEVGCGF RSM02162 Sm (134) PKVLVWAPPPLAPM-PDPWFEGMF-GGGYEKSKELSGLYKALADFMKVEF S261 M2aA12 (126) PEILIISPPSLVPT--QDEWFNDLWGHAIAESKLFAKHYKRVAEELKVHF Smal993 Sinorhizobium meliloti (145) PKVLWAPPPLAPM-PDPWFEGMF-GGGYEKSKELSGLYKALADFMKVEF Consensus (151) PQVLIVAPPPL EM P FE VF GG EKS LARVY ALAD MKV F 201 241 MSAT (180) FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL----(SEQ ID NO : 643) 14B natural isolate (176) FDAGSVISTDGVDGTHFTR---------------------- (SEQ ID NO : 644) 20A (180) FDAGSVISTDGVDGTHFTRGETI----------- (SEQ ID NO : 645) 2D natural isolate (176) FDAGSVISTDGVDGTHFTRGETI------------------ (SEQ ID NO : 646) 9B Natural Isolate (192) FDAGSVISTDGVDGTHFTRGETIDR---------------- (SEQ ID NO : 647) M. parafortuitum CO1 (180) FDAGSVISTDGVDGIHFTRGEQST----------------- (SEQ ID NO : 648) Sm-RSM05666 (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRMMLGL-- (SEQ ID NO : 649) At-QBUACO (172) FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG--- (SEQ ID NO : 650) At-Q8UFG4 (182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPWRMMLGL-- (SEQ ID NO : 651) M091 M4aEll (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL-- (SEQ ID NO : 652) M1-RML000301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVML---- (SEQ ID NO : 653) P. dejongeii RVM04532 (179) FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG--- (SEQ ID NO : 654) Q92XZ1 Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO : 655) Q98MY5 Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVMLEL--(SEQ ID NO : 656) RSM02162 Sm (182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF----(SEQ ID NO : 657) S261 M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPWKSILAL--(SEQ ID NO : 658) Smal993 Sinorhizobium meliloti (193) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO : 659) Consensus (201) FDAGSVISTD VDGIHLDA T IG AL VR LL (SEQ ID NO : 660)

The alignment tree from the CLUSTALW alignment (which approximates to a phylogenetic tree) suggests 3 or 4 groupings.

From this alignment, a hypothetical protein sequence was constructed from the consensus sequence. Where no consensus existed the site was filled with the Per amino acid; gaps were ignored. This provided a Per-consensus sequence: This consensus sequence was used for a BLASTP search against a non-redundant database. This search identified 55 hits. The majority of the'hits'were GDSL or GDSI type molecules covering a wide range of microbial diversity. However, only the first 14 'hits'had e-values and bit-values in the reliable range. At first sight, this appeared to provide further molecules with a GDSL/N-G/ARTT motif, but this was found to be due to differences in coding (Swiss Prot vs GenBank) The screening of 3 environmental libraries (at BRAIN) resulted in 10 clones with a GDSL motif. A further 2 clones were derived from the BRAIN library. The following Table (Table 13-1) lists the clones and indicates their activity.

Table 13-1. Clones with GDSL Motifs Librar Clone Perhydrolase Activity S248Fa S248 M40cD4No S248Fa S248 M44aA5No S248Fa S248 M18bH12 Not Perhydrolase S248Fa S248M36bC5 NotPerhvdrolase S248Fa S248_M50cD9 Not Perhydrolase S248Fa S248 M2bB11 ? Low S261 S261 M2aA12 Yes S279 S279_M75bA2 Not done S279 S279_M11aC12 Not GDSL S279 S279_M70aE8 ? Low M091 M091 M4aE11 Not tested BRAIN Est114 No BRAIN Estl05Not done

M40cD4 Strongest hit: arylesterase of Brucella melitensis (46% identical). Motifs : GDSL - GAND ; GQTT instead of GRTT. Sequence alignment against the core list of organisms places it close to Caulobacter vibrioides and Brucella melitensis in the alpha- Proteobacteria.

M. sTegmltsKZ ACT (O. S860) _ Ag cbacteriumtumefacEns (0. 2508) Bnjcella maliterEs (0. 2236) Caubbactervitxioides (0. 2960) M40cD4 (0. 2796)

M44aA5 Strongest hit: Acyl-CoA thioesterase of Pseudomonas aeruginosa (43% identical). Motifs: GDSL-GGND; no GRTT or equivalent. Sequence alignment against the core list of organisms places it close to Pseudomonas sp in the gamma- Proteobacteria. M. smegmatis ACT (0. 4490) Neisseria rneningitidis (0. 3799) M44aA5 (0. 3369) Pseudomonas aeruginosa thioesterase (0. 1468) 'Pseudomonas syringae (0. 1418) Vibrio cholerae (0. 2079) Yersinia pestis (0. 2124)

GC821-2 M2bBll Strongest hit: arylesterase of Brucella melitensis. Motifs: GDSL-GAND; no GRTT or equivalent. Sequence alignment against the core list of organisms shows no strong association placing it between the alpha- and gamma-Proteobacteria. M. smegmafis ACT (0. 5146) Escherichia coli (0. 0531) Salmonella enterica subsp. enterica (0. 0547) Yersinia pestis (0. 1846) --Vbdo cholera (0. 1522) - Vibrio mimicus (0. 1628) Pseudomonas aeruginosa thioesterase (0. 1480) Pseudomonas syringe (0. 1406) Brucella melitensis (0. 3147) '. Caulobacter vibrioides (0. 3043) M2bB11 (0. 3201)

M2aA12 Strongest hit: arylesterase of Agrobacterium tumefaciens (42% identical) Motifs: GDSL-GRTT-GTND. Sequence alignment against the core list of organisms places it close to Agrobacterium tumefaciens in the alpha-Proteobacteria. M. smegmatisACr (0. 1730) Agrobactedum rhizogenes Q9KWA6_hypprot (0. 0928) - Sinorhizobium melikwnt (0. 0815) _ iWcterdejongeii RVM04532 (0. 3004) | Agrobacterium rhizDgenes Q9KWB1 (0. 1338) Sinorhizobium rreliloti HYPPROT (0. 1368) Agrobactertumefaciensaryiesterase (0. 2428) Agrobacterium tumefaciens aryylest (0. 0084) _, tAgsbacteriumtumefaciensQ8UFG4 (0. 0103) | I AgrobacteriumtumefacensO9Z109 (0. 0280) Sinorhizobium n-elilot ARYLEST (0. 1363) Mezorhiapbium loti ARYLEST (0. 1978) S261_M2aA12 (0. 2553) Ralstoria pickettii (0. 2291) Ralstonia solanaceranum (O. 2143)

M75bA2 Strongest hit: incomplete. BLAST search revealed nothing significant. Motifs: GDSL-GTND; no GRTT or equivalent. Sequence alignment against the core list of organisms shows no convincing associations. The closest neighbors appear to be the Vibrio-Aeromonas groups of the gamma-Proteobacteria. M. smegmatis ACT (0. 4020) M75bA2prelim (0. 4499) _ Vibrio cholerae_lecithinase (0. 1. 816) <Vibrio hatveyi (0. 0751) ~ Vibrio parahaemolyticus (0. 0780) , Vbrio vulnificus (0. 1362) Legionella pneumophila (0. 3459) Aeromonas hydrophila (0. 0330) Aeromonas salmonicida (0. 0017) lAeromonas salmonicida_GPLCACr (0. 0013) Salmonella typhimurium SseJ (0. 3861) M70aE8 Strongest hit: acyl-CoA thioesterase from E. coli (30% identical), and aryl esterase hydrolase from Vibrio mimicus (27% identical). Based on incomplete sequence GDSL-type esterase (BRAIN) from Neisseria meningitidis (50% identical). Motifs: GDSL-GGND; no GRTT-replaced with GRTV. Sequence alignment against the core list of organisms shows the closest association to Neisseria meningitidis, a member of the beta-Proteobacteria. M. srnegmatisACT (0. 4419) M70aE8 (0. 2787) Neisseda meningitidis (0. 3023) Pseudomonas aeruginosa thioesterase (0. 1473) Pseudomonas syringe (0. 1412) Salmonella enterica subsp. enterica (0. 1527) Yersinia pestis (0. 1562) Vibdo cholera (0. 1509) brio mimicus (0. 1641)

M4aEll Strongest hit: arylesterase from Agrobacterium tumefaciens (59% identity) Motifs : GDSL-GRTT-GTND. Sequence alignment against the core list of organisms shows the closest association to members of the alpha-Proteobacteria such as Agrobacterium. M. sn egmatis ACT (0. 1697) Agrobacterium rhizogenes Q9KWA6 hypprot (0. 0937) - Sinorhizobiummelifoti-hypProt (0. 0806) Pmsthecobacterdejongeii RVAA04532 (0. 2938) Agrobacterium rhizogenes Q9KWB1 (0. 1312) SinorhizDbiummeliloti HYPPROT (0. 1394) Agrobacter tumefaciens arylesterase (0. 2473) Agrobacterium tumefaciens arylest (0. 0084) r, CAgrobactenumtumefaciens Q8UFG4 (0. 0103) Agrobactedum tumefaciens Q9ZI09 (0. 0278) Sinorhizobium melilot ARYLEST (0. 1324) Meorhhizobium loti ARYLEST (0. 1872) M091_M4aE11 (0. 2509) Ralstonia pickettii (0. 2262) Raistonia solanacerarum (0. 2198)

Est114 Strongest hit: phosphatidylcholine sterol acyltransferase from Aeromonas hydrophila (gamma-Proteobacteria) (30% identical). Motifs : GDSL-GPND ; no GRTT but GATT may be an equivalent. Sequence alignment against the core list of organisms shows the closest association to Acidophilium sp. and Aeromonas/Vibrio within the gamma-Proteobacteria.

M. smegmatis ACT (0. 4350) Salmonella typhimurium SseJ (0. 3837) Acidophilium sp. (0. 3439) Est114 (0. 3317) _ Vibrio cholerae_iecithinase (0. 1843) Vbrio harveyi (0. 0752) 'brio parahaerralyticus (0. 0786) Vibrio vulnificus (0. 1340) Aeromonas hydrophila (0. 0318) LAeromonas salmonicida (0. 0017) IAeromonassalmonicida_GPLCACT (0. 0013) Legionella pneumophila (0. 3367)

Est105 Strongest hit: Pseudomonas aeruginosa outer membrane esterase, and hypothetical protein Pseudomonas putida (27% identical). Motifs : GDSL-GAND, no GRTT or equivalent. Sequence alignment against the core list of organisms shows the closest association to members of the gamma-Proteobacteria. M. smegmatis ACT (0. 5011) l m Est105 (0. 3462) Moraxella bovis (0. 2785) Moraxella catarrhalis (0. 2835) Photorhabdus luminescens (0. 2935) Salmonella typhimurium (0. 2467) Serratia liquefaciens (0. 2440) Pseudomonas aeruginosa (0. 2136) Pseudomonas putida (0. 2128)

An overall alignment of these clones/sequences (here shown underlined) indicates that they are scattered throughout the alignment tree of strains indicating that the metagenomic screening has provided a variety of sequences and not a limited diversity. Mamums ACT (0. 1786) Agrobactedum rhizogenes Q9KWAB_hypprot (0. 0856) Sinorhizobfum melllati-hypProt (0. 088 'Prosthecobacter deJongeli RVM04532 (02883) Agrobactedum rhlmgenes Q9içNB1 (0. 1362) Sinorhizoblum meliloff HYPPROT (0. 1344) Agrobader tumefadens arylesterase (02454) S261 M2aAI2 (0. 2474) M091 K44aEl 1 (0. 2663) Arobactedum tumefadens aryiest (0. 0059) n Agrobadedum tumefadens Q8UFG4 (0. 0081) Agrobacterium tumefadens Q9ZI09 (0. 0283) SinohIzoblum melllot ARYLEST (0. 1445) Mezahizoblum loff ARYLEST (0. 1917) Ralatonle pidceti (0. 2256) Rnlstonia sdanacerarum (0. 2247) Agrobadedum tumefadens (0. 2359) Brucella melitensis (0. 2292) Caulobacter Abdoides (0. 2913) MQcD4 (. 2897) Chromobacterium vidaceum (0. 2778) Pseudomonas aeruginosa thloesterase (0. 1441) L Pseudomonas sydngae (0. 1444) Eschedchia coil (0. 0545) SalrtronNla entedca subsp. entaica (0. 0534) Yerslnla pesBa (0. 1548) Vibdo chderaa (0. 1574) Vibrio mimicus (0. 1576) M2bRl 1 (0. 3019) M44aA5 (0. 3104) M70aEg (0. 2760) Netssecia mentngIBdis (0. 2938) Gostridium acatobutylicum (0. 3920) Badllus cerous (0. 3422) Enterococas feecalis (0. 3133) Ladococcus lacfis subsp. Iactis (0. 3256) Bacillus subUlis_hypProt (0. 4082) Acidophiilum sp. (0. 3852) w (0. 3606) Aeromonas hydrophile (0. 0321) Aeramonas Imonidda (0. 0015) Aeromonas salmonicida_GPLCACT (0. 0015) Legionella pneumophila (0. 3704) _-Vibdo chderae iedthisse (0. 1862) bdo harveyi (0. 0770) 'Vfbrio perahaemolydcus (0. 0765) I Vibriovulnlflcus (0. 1485) Salmonella typhimurium SseJ (0. 4293) Arabidopsis thaliana (0. 3980) Homo sapiens (0. 3911) Maraxella bovis (0. 2868) Moraxeua catarrhalls (0. 2882) Est105 (0. 3982) Photorhabdus luminescens (0. 3038) Salmonella typhimurium (0. 2500) Serratia Iiquefadens (0. 2469) Pseudomonas aeruginosa (0. 2114) Pseudomonas putida (0. 2182) Aspergillus aculeatus (0. 4168) _ I Badllus subiDis_yesT (0. 3105) Bacillus subtilis_yesY (0. 2952) Erwinia chrysanthemi (0. 4168) M75bAZ (0. 4432) bacillus subtilis bfmBB (0. 4620) Bacillus subUlls-fabD (0. 4598) ßaållus subUlis YxiM (OA516) Badllus subtilis YusK (0. 4480) Bacillus subtilis-YhdO (0. 4364) Homo sapiens PAF (0. 4039) Streptococcus pneumoniae (0. 4082) Listeria monocytogenes (0. 4028) Pseudomonas aeruginosa HypProt (0. 3119) Sreptomyces avemmiülis (0. 3212) Streptomyces diastatochromogenes (0. 4150) Streptomyces scabiei (0. 4230)

Gene Mining for GRTT-Type Esterases (clones with perhydrolase activity) Sinorhizobium meliloti Smal993-hypothetical protein Sme Motifs: GDSL-ARTT-GTND Sinorhizobium melilotiQ92XZl-hypothetical protein Sme Motifs: GDSN-GRTT-GTND Mesorhizobium loti Q98MY5-arylesterase_Mlo Motifs: GDSL-GRTT-GAND Moraxella bovis AAK53448 (lipase) Motifs : GDSL-GSND, no GRTT or equivalent in this sequence order.

(perhydrolase activity low, questionable sequence) Agrobacterium tumefaciens Q8UACO Motifs: GDSL-GRTT-GTND Agrobacterium tumefaciens Q8UFG4 Motifs: GDSL-GRTT-GTND Mesorhizobium loti RML000301 Motifs: GDSL-GRTT-GAND Sinorhizobium meliloti RSM05666 Motifs: GDSL-GRTT-GSND (this clone was inactive for perhydrolase activity; and probably represents a false negative) Sinorhizobium meliloti RSM02162 Motifs: GDSL-ARTT-GTND Prosthecobacter dejongeii RVM05432 Motifs: GDSN-GRTT-GTND

A GDSxi-x2RTT-Gx3ND motif characterizes the active clones/sequences, where: X1 = L or N X2 =AorG X3 = T or A or S The Moraxella bovis AAK53448 sequence does not fit this pattern and is excluded from the alignment analysis provided below: Multiple Sequence Alignment of Active Clones/Sequences 1 50 ACT MSMEG (------------MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG Q98MY5 Mesorhizobium loti (1)-----------,--MKTVLCYGDSLTWGYNAEGGR------HALEDRWPS Smal993 Sinorhizobium meliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG Q92XZ1 Sinorhizobium meliloti (1)---------MEETVARTVLCFGDSNTHGQVPGRGPLDR---YRREQRWGG P. dejongeii RVM04532 il)--------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG RSM05666 Sm (1)--------------MKTVLCYGDSLTWGYDATGSG-----RHALEDRWPS RSM02162Sm (1)-----------MVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG At-Q8UACO (1)--------------MKTVLAFGDSLTWGADPATGLR-----HPVEHRWPD At-Q8UFG4 (1)-------------MVKSVLCFGDSLTWGSNAETGG-----RHSHDDLWPS M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGGR------HALEDRWPS S261 M2aA12 (1)--------------NKNILAFGDSLTWGFVAGQDA-----RHPFETRWPN MO91 M4aE11 (1)--------------MKTILAYGDSLTYGANPIPGG-PR---HAYEDRWPT Consensus (1) MKTVLCFGDSLTWGY P G RHA E RWP 51 100 ACT MSMEG (37) VLAQQLGADFEVIE--EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP Q98MY5 Mesorhizobium loti (31) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP Smal993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE--EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP P. dejongeii RVM04532 (37) VLAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP RSM05666 Sm (32) VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP RSM02162Sm (39) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP At-Q8UACO (32) VLEAELAGKAKVHP--EGLGGRTTCYDDHAGPACRNGARALEVALSCHMP At-Q8UFG4 (33) VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M1-RML000301 (45) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP S261 M2aA12 (32) ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP M091 M4aE11 (33) ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADRNGARVLPTLLESHSP Consensus (51) VL A LGG VIE EGL GRTTAHDD A RNGAR LPT L SHAP 101 150 ACT MSMEG (84) LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLWQVLUSAGGVGUUYPA

Q98MY5 Mesorhizobium loti (79) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWPAP- Smal993 Sinorhizobium meliloti (97) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA Q92XZ1 Sinorhizobium meliloti (87) LDLIIIMLGTNDLKRRFNMPPSEVA--MGIGCLVHDIRELSPGRTGN--- P. dejongeii RVM04532 (84) LDLVILMLGTNDLKSTFNVPPGEIA--AGAGVLGRMILAGDAGPENR-PP RSM05666 Sm (80) LDLIVFMLGSNDMKPIIHGTAFGAV--KGIERLVNLVRRHDWPTETEEG- RSM02162 Sm (86) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA At-Q8UACO (80) LDLVIIMLGTNDIKPVHGGRAEAAVS--GMRRLAQIVETFIYKPREAVP- At-Q8UFG4 (83) LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDWEAP M1-RML000301 (93) IDLIVIMLGANDMKPWIHGNPVAAX--QGIQRLIDIVRGNDYPFDWPAP- S261 M2aA12 (80) LDLVIIMLGTNDIKFAARCRAFDAS--MGMERLIQIVRSANYMKGYKIP- M091 M4aEll (81) LDLIVIMLGTNDIKPHHGRTAGEAG--RGMARLVQIIRGHYAGRMQDEP- Consensus (101) LDLVIIMLGTNDMKP H P EAA GM RLV IVR YG P 151 200 ACT MSMEG (132) PKVLWSPPPLAPMPHPWFQLIFE--GGEQKUUELARVYSALASFMKVPF Q98MY5 Mesorhizobium loti (126)-QILIVSPPWSRTENADFREMFAG--GDEASKQLAPQYAALADEVGCGF Smal993 Sinorhizobium meliloti (145) PKVLWAPPPLAPMPDPWFEGMFG--GGYEKSKELSGLYKALADFMKVEF Q92XZ1 Sinorhizobium meliloti (132) DPEIMIVAPPPMLEDLKEWESIFS--GAQEKSRKLALEFEIMADSLEAHF P. dejongeii RVM04532 (131) QLLLMCPPKVRDLSAMPDLDAKIP--HGAARSAEFPRHYKAQAVALKCEY RSM05666 Sm (127) PEILIVSPPPLCETANSAFAAMFAG--GVEQSAMLAPLYRDLADELDCGF RSM02162 Sm (134) PKVLWAPPPLAPMPDPWFEGMFG--GGYEKSKELSGLYKALADFMKVEF At-Q8UACO (127)-KLLIVAPPPCVAGPGGEPAGGRD----IEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (133)-DVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M1-RML000301 (140)-QILIVSPPWSRTENADFREMFAG--GDEASKQLAPQYAALADEVGCGF S261 M2aA12 (127)-EILIISPPSLVPTQDEWFNDLWG--HAIAESKLFAKHYKRVAEELKVHF M091 M4aEll (128)-QIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF Consensus (151) ILIVSPPPL T DF AMFG G E SK LA YKALADELK F 201 241 ACT MSMEG (180) FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL---- (SEQ ID NO : 662) Q98MY5 Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVMLEL-- (SEQ ID NO : 663) Smal993 Sinorhizobium meliloti (193) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO : 664) Q92XZ1 Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO : 665) P. dejongeii RVM04532 (179) FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG--- (SEQ ID NO : 666) RSM05666 Sm (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRRMMLGL-- (SEQ ID NO : 667) RSM02162 Sm (182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO : 668) At-Q8UACO (172) FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG--- (SEQ ID NO : 669) At-Q8UFG4 (182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL-- (SEQ ID NO : 670) M1-RML000301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML---- (SEQ ID NO : 671) S261 M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL-- (SEQ ID NO : 672) M091 M4aEll (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL-- (SEQ ID NO : 673) Consensus (201) FDAGTVA TSPVDGIHLDAENTR IG ALA WR LLG (SEQ ID NO : 674)

A guide tree (i.e., an approximation of a phylogenetic tree) of the CLUSTALW alignment of active clones/sequences is provided below. ACT MSMEG Sma1993 Sinorhizobium meliloti _ RSM02162_Sm P. dejongeii RVM04532 Q92XZ1 Sinorhizobiummetitoti Q98MY5 Mesorhizobium loti IMI-RMLOO0301 RSM05666 Sm At-Ci8UFG4 At » Q8UACO S261_M2aA12 I M091_M4aE11

Table 13-2. Similarity and Identity of Clones/Sequences Compared to M. smegmatis Perhydrolase Clone/Sequence % % Identity Similarity Sinorhizobium meliloti Smal993 55. 5 71. 6 Sinorhizobium meliloti Q92XZ1 38.7 54. 7 Mesorhizobium loti 098MY5 38. 8 53. 4 Moraxella bovis AAK53448 5. 0 9. 7 Agrobacterium tumefaciens 08UACO 36. 7 47. 7 Agrobacterium tumefaciens Q8UFG4 37. 1 50. 4 Mesorhizobium loti RML000301 34. 8 50. 9 Sinorhizobium meliloti RSM05666 37. 4 52. 5 Sinorhizobium meliloti RSM02162 58. 3 75. 2 Prosthecobacter dejongeii RVM05432 41. 6 55. 7 S261_M2aA12 39. 3 54. 3 M091 M4aEll 34. 7 50.2

Based on the results, the active clones were found to have an overall identity to M. smegmatis perhydrolase of 38. 7-58. 3%. Moraxella bovis AAK53448 was found to be an exception and the (translated) amino acid sequence is questionable.

Redundancy From the analyses above, it was evident that some redundancy exists in the alignment provided at the beginning of this Example that will have added undue weighting to the consensus sequence. Also, further GDSL-GRTT sequences were added.

Thus, in the revised alignment below, the following changes were made: Removed: Natural isolate 14B Natural isolate 2D RSM02162Sm Q98MY5 Mesorhizobium loti Added: BAB16197 (Arh II) BAB16192 (Arh I) NP 00197751 (Mlo II) NP 00216984 (Bce) NP 522806 (Rso) Non-redundant alignment: 1 50 20A (1)-------------LPSGILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG 9B Natural Isolate (1)-GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFP-KRVRWTG M. parafortuitum CO1 (1)-------------MAKRILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG MSAT (1)-------------MAKRILCFGDSLTWGWV2VEDGAPTERFA-2DVRWTG

Sm-RSM05666 (1)--------------MKTVLCYGDSLTWGYDATG-----SGRHALEDRWPS At-Q8UACO (1)--------------MKTVLAFGDSLTWGADPAT-----GLRHPVEHRWPD At-Q8UFG4 (1)-------------MVKSVLCFGDSLTWGSNAET-----GGRHSHDDLWeS M091 M4aEl1 il)--------------MKTILAYGDSLTYGANPIP----GGPRHAYEDRWPT M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAE------GGRHALEDRWPS P. dejongeii RVM04532 (1)--------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhlzobium meLiloti (1)-----MEETVARTVLCFGDSNTHGQVPG-RGPLDRYR-REQRNGG S261 M2aA12 (1)--------------MKNILAFGDSLTWGFVAG-----QDARHPFETRWPN Smal993 Sinorhizobium meliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPTLRYP-YEQRWTG ZP 00197751 (1)--------------MKTILCYGDSLTWGYDAVG-----PSRHAYEDRWPS ZP 00216984 (1)----------MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFA-REERWTG BAB16192 (1)-----MICHKGGEEMRSVLCYGDSNTHGQIPG--GSPLDRYG-PNERWPG BAB16197 (1)-----------MAESRSILCFGDSLTWGWIPVPESSPTLRYP-FEQRWTG NP 522806 (l)--------------MQQILLYSDSLSWGIIPG-----TRRRLPFAARWAG Consensus (1) MKTILCFGDSLTWGWIPV P RR E RW G 51 100 20A (37) VLADLLGDRYEVIE---EGLSARTTTADDPADPRLN-GSQYLPSCLASHL 9B Natural Isolate. (49) VLADELGAGYEVVE---EGLSARTTTADDPTDPRLN-GSDYLPACLASHL M. parafortuitum CO1 (37) VLADLLGDRYEVIE---EGLSARTTTAEDPADPRLN-GSQYLPSCLASHL MSAT (37) VLAQQLGADFEVIE---EGLSARTTNIDDPTDPRLN-GASYLPSCLATHL Sm-RSM05666 (32) VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHA At-Q8UACO (32) VLEAELAGKAKVHP---EGLGGRTTCYDDHAGPACRNGARALEVALSCHM At-Q8UFG4 (33) VLQKALGSDVHVIFT-HEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHA M091 M4aE11 (33) ALEQGLGGKARVIA---EGLGGRTTVHDDWFANADRNGARVLPTLLESHS M1-RML000301 (45) VLQASLGGGVQVIA---DGLNGRTTAFDDHLAGADRNGARLLPTALTTHA P. dejongeii RVM04532 (37) VLAKALGAGFRVIE---EGQNGRTTVHEDPLNICRK-GKDYLPACLESHK Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE---EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHA S261 M2aA12 (32) ALAAGLGGKARVIE---EGQNGRTTVFDDAATFESRNGSVALPLLLISHQ Smal993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE---EGLSARTTSLDDPNDARLN-GSTYLPMALASHL ZP 00197751 (32) VLQGRLGSSARVIA---EGLCGRTTAFDDWVAGADRNGARILPTLLATHS ZP 00216984 (40) VLAQTLGASWRVIE---EGLPARTTVHDDPIEGRHKNGLSYLRACVESHL BAB16192 (43) VLRRELGSQWYVIE---EGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHA BAB16197 (39) AMAAALGDGYSIIE---EGLSARTTSVEDPNDPRLN-GSAYLPMALASHL NP 522806 (32) VMEHALQAQGHAVRIVEDCLNGRTTVLDDPARPGRN-GLQGLAQRIEAHA Consensus (51) VLA LGA Y VIE EGL GRTT DDP D RNGA YLP L SH 101 150 20A (83) PLDLVILMLGINDTKANFGRTPFD--IATGMGVLATQVLTSAGG-VGTSY 9B Natural Isolate (95) PLDLVILMLGTNDTKANLNRTPVD--IASGMGVLATQVLTSAGG-VGTSY M. parafortuitum CO1 (83) PLDLVILMLGTNDTKANFGRTPFD--IATGMGVLATQVLTSAGG-VGTSY MSAT (83) PLDLVIIMLGTNDTKAYFRRTPLD--IALGMSVLVTQVLTSAGG-VGTTY Sm-RSM05666 (79) PLDLIVFMLGSNDMKPIIHGTAFG--AVKGIERLVNLVRRHDWPT--ETE At-Q8UACO (79) PLDLVIIMLGTNDIKPVHGGRAEA--AVSGMRRLAQIVETFIYK---PRE At-Q8UFG4 (82) PLDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQV--SDW MO91 M4aE11 (80) PLDLIVIMLGTNDIKPHHGRTAGE--AGRGMARLVQIIRGHYAG---RMQ M1-RML000301 (92) PIDLIVIMLGANDMKPWIHGNPVA--AKQGIQRLIDIVRGHDYP---FDW

P. dejongeii RVM04532 (83) PLDLVILMLGTNDLKSTFNVPPGE--IAAGAGVLGRMILAGDA---GPEN Q92XZ1 Sinorhizobium meliloti (86) PLDLIIIMLGTNDLKRRFNMPPSE--VAMGIGCLVHDIRELSP---GRTG S261 M2aA12 (79) PLDLVIIMLGTNDIKFAARCRAFD--ASMGMERLIQIVRSANYM---KGY Smal993 Sinorhizobium meliloti (96) PLDLVIIMLGTNDTKSYFHRTPYE--IANGMGKLVGQVLTCAGG-VGTPY ZP00197751 (79) PLDLVIVMLGTNDMKSFVCGRAIG-AKQGMERIVQIIRGQPYS--FNY ZP 00216984 (87) PVDVWLMLGTNDLKTRFSVTPAD--IATSVGVLLAKIAACGA---GPSG BAB16192 (90) ILDLVIIMLGTNDLKARFGQPPSE--VAMGIGCLVYDIRELAP---GPGG BAB16197 (85) PLDLVIILLGTNDTKSYFRRTPYE--IANGMGKLAGQVLTSAGG-IGTPY NP522806 (81) PLALVILMLGTNDFQAIFRHTAQD--AAQGVAQLVRAIRQAPIEP---GM Consensus (101) PLDLVIIMLGTNDLKA F TP D IA GMGRLV VR G G Y 151 200 20A (130) PAPQVLIVAPPPLGELPHPWFDL--VFSGGREKTAELARVYSALASFMKV 9B Natural Isolate (142) PAPQVLIVAPPPLAEMPHPWFEL--VFDGGREKTAQLARVYSALASFMKV M. parafortuitum CO1 (130) PAPQVLIVAPPPLGELPHPWFDL--VFSGGREKTAELARVYSALASFMKV MSAT (130) PAPKVLWSSPPPLAPMPHPWFQL--IFEGGEQKTTELARVYSALASFMKV Sm-RSM05666 (125) EGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAP--LYRDLADELDC At-Q8UACO (124) AVPKLLIVAPPPCVAGP--GGEPAGGRDIEQSMRLAP--LYRKLAAELGH At-Q8UFG4 (130) EAPDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDC M091 M4aEll (125) DEPQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKV M1-RML000301 (137) PAPQILIVSPPWSRTENADFREMFAGGDEASKQLAP--QYAALADEVGC P. dejongeii RVM04532 (128) RPPQLLLMCPPKVRDLSAMPDLDAKIPHGAAR-SAEFPRHYKAQAVALKC Q92XZ1 Sinorhizobium meliloti (131) NDPEIMIVAPPPMLEDLKEWES---IFSGAQEKSRKLALEFEIMADSLEA S261 M2aA12 (124) KIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAK--HYKRVAEELKV Smal993 Sinorhizobium meliloti (143) PAPKVLWAPPPLAPMPDPWFEG--MFGGGYEKSKELSGLYKALADFMKV ZP 00197751 (124) KVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAP--LYAALAQQTGC Z (132) ASPKLVLMAPAPIVEVGFLGEI---FAGGAAK-SRQLAKRYEQVASDAGA BAB16192 (135) KPPEIMWAPPPMLDDIKEWEP--IFSGAQEKSRRLALEFEIIADSLEV BAB16197 (132) PAPKLLIVSPPPLAPMPDPWFEG--MEGGGYEKSLELAKQYKALANFLKV NP 522806 (126) PVPPVLIWPPAITAPAGAMADK---FADAQPKCAGLAQAYRATAQTLGC Consensus (151) AP ILIVAPPPL E WF IFGGA KS LA YKALA LKV 201 248 20A (178) PFFDAGSVISTDGVDGTHFTRGETI----------------------- (SEQ ID NO : 675) 9B Natural Isolate (190) PFFDAGSVISTDGVDGTHFTRGETIDR--------------------- (SEQ ID NO : 676) M. parafortuitum CO1 (178) PFFDAGSVISTDGVDGIHFTRGEQST---------------------- (SEQ ID NO : 677) MSAT (178) PFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL--------- (SEQ ID NO : 678) Sm-RSM05666 (173) GFFDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRRMMLGL------- (SEQ ID NO : 679) At-Q8UACO (170) HFFDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG-------- (SEQ ID NO : 680) At-QBUFG4 (180) GFFDAGSVARTTPVDGVHLDAENTRAIGRGLEPWRMMLGL------- (SEQ ID NO : 681) M091 M4aEll (175) HFFDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL------- (SEQ ID NO : 682) M1-RML000301 (185) GFFDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVML--------- (SEQ ID NO : 683) P. dejongeii RVM04532 (177) EYFNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG-------- (SEQ ID NO : 684) Q92XZ1 Sinorhizobium meliloti (178) HFFDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA----- (SEQ ID No : 685) S261_M2aA12 (172) HFFDAGTVAVADKTDGGHLDAVNTKAIGVALVPWKSILAL------- (SEQ ID NO : 686) Smal993 Sinorhizobium meliloti (191) EFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF--------- (SEQ ID NO : 687 ZP 00197751 (172) AFFDAGTVARTTPLDGIHLDAENTRAIGAGLEPWRQALGL------- (SEQ ID NO : 688)

ZP 00216984 (178) HFLDAGAIVEVSPVDGVHFAADQHRVLGQRVAALLQQIA--------- (SEQ ID NO : 689) BAB16192 (182) HFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGWR------- (SEQ ID N0 : 690) BAB16197 (180) DFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAAA (SEQ ID NO : 691) NP 522806 (173) HVFDANSVTPASRVDGIHLDADQHAQLGRAMAQWGTLLAQ------- (SEQ ID NO : 692) Consensus (201) FFDAGSV TSPVDGIHLDAENTR LG ALA VR IL (SEQ ID NO : 693) The guide tree to the CLUSTALW alignment (which approximates to a phylogenetic tree) clearly indicates 3 groupings: 1) GDSL-ARTT group including Act 2) GDSL-GRTT group composed of members of the Rhizobiales and the metagenome; and 3) Intermediate group of mixed motifs.

It is also contemplated that the results suggest some form of gene duplication and mutation events in the Rhizobiales and lateral gene transfer to Mycobacterium. 20A Mvcobacterium Darafortuitum M. Darafortuitum C01 9B Natural Isolate Mvcobacterium silvu GDSL-ARTT KCA-r -GDSL-ARTT MSAT M. smeematts Sma1993 Sinorhizobium meliloti _ BAB161 97 Arobacterium tumefaciens- Prothecobacter. deionseii RVM04532 Q92XZ1 Sinorhizobium meliloti BAB16192 A. tumefaciens ZP 00216984 Burkholderia cenacia NP 522806 Ralstonia solanacearum- At-Q8UACO A. tumefaciens Sm-RSM05666 Sinorhizobium melilot At-Q8UFG4 A. tumefaciens MI-RML000301 Mesorhizobium loti GDSL-GRTT -ZP 00197751 Mesorhizobium loti M091 M4a S261 M2a Metagenome

Using the non-redundant alignment a new Act consensus was constructed called "Act chimera".

An alignment of Act-chimera with Ms Act (Chimera align) indicates 91.6% similarity and 86.0% identity, as indicated below.

1 50 MSAT (1) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIE Act-Chimera (1)--KTILCFGDSLTWGWIPVEDGAPTERRAPEVRWTGVLAQQLGADYEVIE Consensus (1) K ILCFGDSLTWGWIPVEDGAPTER APDVRWTGVLAQQLGADFEVIE 51 100 MSAT (51) EGLSARTTNIDDPTDPRLN-GASYLPSCLATHLPLDLVIIMLGTNDTKAY Act-Chimera (49) EGLSGRTTNIDDPTDPRLRNGASYLPSCLASHLPLDLVIIMLGTNDLKAY Consensus (51) EGLSARTTNIDDPTDPRL GASYLPSCLASHLPLDLVIIMLGTND KAY 101 150 MSAT (100) FRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLWSPPPLAPMPHPW Act-Chimera (99) FRRTPLDIALGMGRLVTQVRTSAGGVGTTYPAPKILIVAPPPLAEMPHPW Consensus (101) FRRTPLDIALGM LVTQV TSAGGVGTTYPAPKILIVAPPPLA MPHPW 151 200 MSAT (150) FQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEA Act-Chimera (149) FQLIFGGAEQKSTELARVYKALASFLKVPFFDAGSVISTSPVDGIHLDAE Consensus (151) FQLIF GAEQKSTELARVY ALASFLKVPFFDAGSVIST VDGIH 201 217 MSAT (200) NNRDLGVALAEQVRSLL (SEQ ID NO : 695) Act-Chimera (199) NTRDLGVALAEQVRSIL (SEQ ID NO : 694) Consensus (201) N RDLGVALAEQVRSIL (SEQ ID NO : 696) A BLASTP search with Act-chimera did not reveal any further sequences.

The Act-chimera is"forced"on the Per sequence at the positions where no consensus exists. However, a basic'unforced'consensus sequence did not provide any more information from a blastp search or from alignment analysis. Thus, comparison with the most distant homologues in the blastp'hit'list was considered more useful in defining the important residues/positions in Act sequence space. This was a useful exercise, as these sequences were not used in the non-redundant alignment.

For example, Rhodopirellula baltica (NP_865748 ; Psp; a Planctomycetes and quite different from either Mycobacterium or Rhizobiales), was compared as shown below.

1 50 MSAT (1) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLA---QQLGADFE NP 865746 (1)-MHSILIYGDSLSWGIIPGTR----RRFAFHQRWPGVMEIELRQTGIDAR Consensus (1) IL FGDSLSWG IP RFA RW GVL Q G D 51 100 MSAT (48) VIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTK NP 865746 (46) VIEDCLNGRRTVLEDPIKPGRNGLDGLQQRIEINSPLSLWLFLGTNDFQ Consensus (51) VIED L AR T IDDP P NG L I PL LVII LGTND 101 150 MSAT (98) AYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLWSPPPLAPMPH NP 865746 (96) SVHEFHAEQSAQGLALLV--DAIRRSPFEPGMPTPKILLVAPPTVHH-PK Consensus (101) A A GLALLV P PKILLVAPP L P 151 200 MSAT (148) PWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFT NP 865746 (143) LDMAAKFQNAETKSTGLADAIRKVSTEHSCEFFDAATVTTTSWDGVHLD Consensus (151) F. AE KST LA LAS FFDAASV ST VDGIH 201 222 MSAT (198) EANNRDLGVALAEQVRSLL--- (SEQ ID NO : 695) NP 865746 (193) QEQHQALGTALASTIAEILADC (SEQ ID NO : 697) Consensus (201) N LG ALA I IL (SEQ ID NO : 698) The following is an alignment with Ralstonia eutropha (Reu): 1 50 MSAT (1)---------MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLA-- ZP_00166901 (1) MPLTAPSEVDPLQILVYADSLSWGIVPGTR----RRLPFPVRWPGRLELG Consensus (1) IL FADSLSWG VP R VRW G L 51 100 MSAT (40)--QQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLV ZP00166901 (47) LNADGGAPVRIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALV Consensus (51) GA IIED L AR T DDP P NG L I H PL LV 101 150 MSAT (88) IIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLW ZP00166901 (97) VLMLGNNDFQSMHPHNAWHAAQGVGALV--HAIRTAPIEPGMPVPPILVV Consensus (101) IIMLG ND A A GM LV A I P P ILVV

151 200 MSAT (138) SPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVIS ZP00166901 (145) VPPPIRT-PCGPLAPKFAGGEHKWAGLPEALRELCATVDCSLFDAGTVIQ Consensus (151) PPPI P F GGE K L L A M FDAGSVI 201 237 MSAT (188) TDGVDGIHFTEANNRDLGVALAEQVRSLL-------- (SEQ ID NO : 695) ZP00166901 (194) SSAVDGVHLDADAHVALGDALQPVVRALLAESSGHPS (SEQ ID NO : 699) Consensus (201) S AVDGIH LG AL VRALL (SEQ ID NO : 700) Based on these results, the following conclusions were made. A BLASTS nr- database search with a perhydrolase consensus sequence revealed GDSL or GDSI lipases/esterases from a wide diversity of organisms. However, only 12 or 14 of these were reliable homologues of Per. Nearly all of these were derived from 1 small group of bacteria, namely the Rhizobiales (i. e., Gram-negative soil bacteria belonging the alpha- Proteobacteria). A few members of the beta-Proteobacteria were found, but no Mycobacterium sp. This provides an indication that the perhydrolase (Per) gene/protein is not widely distributed in nature.

The Mycobacterium protein is characterized by the GDSL-ARTT motif, whereas most of the Rhizobiales are characterized by a GDSL-GRTT motif. There are also some mixed or intermediate motifs (e. g., GDSN-GRTT, GDSN-ARTT and SDSL-GRTT).

This may indicate gene duplication and mutation event and lateral gene transfer. The consensus residues identified in these experiments were L6, W14, R27, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, and G205.

Using the non-redundant alignment and comparison with distant homologues the follow sequence space can be defined starting at position 5 of the M. smegmatis perhydrolase and ending at position 195, with perhydrolase shown in residues in bold.

In sum, it is clear from the analyses above that the active clones/sequences with a GDSxi-x2RTT-GX3ND motif have all been found among the alpha-Proteobacteria- Gram-negative bacteria associated with the soil rhizosphere. This is in sharp contrast to the prototype perhydrolase from M. smegmatis-a high GC content Gram-positive bacterium assigned to the class Actinobacteria. This division is illustrated in Figure 2, which provides a phylogenetic tree, showing the major branches of the bacteria and the origin of the active clones/sequences compared to M. smegmatis.

EXAMPLE 14 Native Molecular Weight Estimation of Homologues of the Perhydrolase In this Example, experiments conducted to estimate the native molecular weights of M. smegmatis perhydrolase homologues are described.

Preparation of Samples for Purification (Size Determination) A single colony of the desired strains was inoculated in 50ml Terrific Broth and incubated overnight at 37°C with shaking at 200 rpm. The cells were pelleted by centrifugation for 10 minutes at 7000 rpm in a Sorvall SuperSpeed Centrifuge. The pellets were then resuspended in 10 ml 25mM Bis-Tris (pH 6.5) and lysed by passage through a French pressure cell twice. The lysates were then centrifuged at 15000 rpm in a Sorvall SuperSpeed Centrifuge. The soluble fraction was heat treated at 55°C for 1 hour to precipitate cellular proteins. The samples were then centrifuged at 10000 rpm in a Sorvall SuperSpeed Centrifuge and the soluble fractions used for further purification or assay.

Sizing Columns The supernatants (prepared as described above) were run on a Sephadex 200 sizing column in 20 mM phosphate (pH 8. 0), with a flow rate of 0.5 ml/min. The column was calibrated prior to running the samples with MW standards (listed below) and purified M. smegmatis perhydrolase protein. The crude sample elution volumes were determined by collecting 0.5 ml fractions, and assaying the fractions for pNB activity.

Molecular weights and elution volumes of the standards: Thyroglobulin MW 669 kDa: elution volume 16ml Aldolase MW 158 kDa: elution volume 24 ml Ovalbumin MW 43 kDa: elution volume 26 ml Ribonuclease MW 14 kDa : elution volume 32 ml Perhydrolase elution volume 24 ml Results The following Table (Table 14-1) provides the elution volume of some of the M. smegmatis perhydrolase homologues identified herein.

Table 14-1. Elution Volume (Estimated Molecular Weight) of M. smegmatis se ! ase Homologues Homologue Sample Elution Volume (. ml ! LO_SmeI 24 pET26 Smell24 ET26_MlO 24 pET26b Stm24 ET26b Mbo 24 M70aEB pET26 32 pET26 m2aA12 24 ET26b S2487am 32 S. meliloti RSM02162 G00355 24 PET_M2aA12 (5261 ! 24 M. smegmatis Perhydrolase 24

The data in the above Table and the assay results obtained for these homologues indicated that these enzymes have an amino acid sequence similar to the M. smegmatis perhydrolase. As with the M. smegmatis perhydrolase, these homologues exhibit perhydrolysis activity as multimers. As described herein, the perhydrolase is an octamer, while the homologues, although they elute in a similar volume, are contemplated to be dimers, trimers, tetramers, hexamers, and/or octamers.

EXAMPLE 15 Crystal Structure of Perhydrolase In this Example, the crystallographic analysis of the perhydrolase is described.

Perhydrolase crystals were obtained under two conditions: 2.0 M [NH4] 2SO4, 2% PEG400,0. 1 M Tris pH 7.1 (giving triclinic, PI crystals) and 1.0 M ammonium dihydrogen phosphate, and O. 1M sodium citrate pH 5.6 (giving tetragonal, P4 crystals) Both crystal forms gave suitable diffraction beyond 2. OA resolution. Derivative protein for a MAD phase determination using selenium replacing sulfur containing methionine resulting in a protein molecule having four selenomethionines the N-terminal methionine is cleaved proteolytically. Of the two forms, triclininc P 1 a= 83. 77A b=90. 07A c= 112. 115A a=73. 32° = 77. 30° y=88. 07° and P4a=b=98. 18A c=230. 12A, the P4 crystal gave data that was possible to use for structure determination. Three wavelength MAD datasets were collected at wavelengths corresponding to the Se absorption edge, near the inflection point and a third, away from the absorption edge.

GC821-2 Three hundred and thirty-three frames (0.3 degree oscillations per frame) for each wavelength with 1 sec exposure time were collected from a single tetragonal space group P4 crystal. The structure could be solved with either SOLVE or SHELX computer programs giving similar solutions for the 32 possible Se positions. The map was fitted using the program"O". It was possible to trace electron density for residues 3-216 in each of the eight independent molecules. The final structure of these eight molecules was refined using CNS. The current crystallographic R-factor is 21%. The coordinates are provided below. CRYST1 98. 184 98. 184 230. 119 90. 00 90. 00 90. 00 SCALE1 0. 010185 0. 000000 0. 000000 0. 000000 SCALE2 0. 000000 0. 010185 0. 000000 0. 000000 SCALE3 0. 000000 0. 000000 0. 004346 0. 000000 ATOM 1 CB LYS 3-8. 167-61. 964 18. 588 1. 000 40. 95 ATOM 2 CG LYS 3-8. 685-63. 192 19. 323 1. 000 22. 95 ATOM 3 CD LYS 3-8. 635-64. 400 18. 399 1. 000 14. 97 ATOM 4 CE LYS 3-7. 963-65. 575 19. 090 1. 000 19. 83 ATOM 5 NZ LYS 3-7. 359-66. 511 18. 099 1. 000 44. 28 ATOM 6 C LYS 3-9. 684-60. 377 17. 426 1. 000 13. 89 ATOM 7 0 LYS 3-9. 087-59. 356 17. 767 1. 000 12. 50 ATOM 8 N LYS 3-8. 000-61. 626 16. 153 1. 000 15. 57 ATOM 9 CA LYS 3-8. 919-61. 686 17. 284 1. 000 20. 71 ATOM 10 N ARG 4-10. 987-60. 381 17. 166 1. 000 24. 56 ATOM 11 CA ARG 4-11. 695-59. 097 17. 204 1. 000 22. 65 ATOM 12 CB ARG 4-12. 299-58. 822 15. 822 1. 000 21. 44 ATOM 13 CG ARG 4-11. 232-58. 465 14. 792 1. 000 21. 56 ATOM 14 CD ARG 4-11. 845-58. 181 13. 431 1. 000 29. 29 ATOM 15 NE ARG 4-11. 660-56. 790 13. 020 1. 000 32. 87 ATOM 16 CZ ARG 4-12. 643-56. 013 12. 585 1. 000 30. 24 ATOM 17 NH1 ARG 4-13. 879-56. 487 12. 494 1. 000 17. 82 ATOM 18 NH2 ARG 4-12. 399-54. 760 12. 229 1. 000 44. 53 ATOM 19 C ARG 4-12. 735-59. 054 18. 308 1. 000 14. 59 ATOM 20 0 ARG 4-13. 604-59. 909 18. 456 1. 000 18. 72 ATOM 21 N ILE 5-12. 639-58. 012 19. 131 1. 000 13. 45 ATOM 22 CA ILE 5-13. 549-57. 882 20. 263 1. 000 12. 08 ATOM 23 CB ILE 5-12. 747-57. 835 21. 578 1. 000 15. 40 ATOM 24 CG2 ILE 5-13. 678-57. 677 22. 765 1. 000 5. 80 ATOM 25 CG1 ILE 5-11. 811-59. 034 21. 741 1. 000 11. 66 ATOM 26 CD1 ILE 5-10. 437-58. 632 22. 232 1. 000 19. 35 ATOM 27 C ILE 5-14. 420-56. 640 20. 142 1. 000 8. 96

ATOM 28 0 ILE 5-13. 905-55. 529 20. 021 1. 000 13. 31 ATOM 29 N LEU 6-15. 736-56. 833 20. 169 1. 000 13. 04 ATOM 30 CA LEU 6-16. 675-55. 728 20. 059 1. 000 8. 54 ATOM 31 CB LEU 6-17. 879-56. 087 19. 178 1. 000 7. 42 ATOM 32 CG LEU 6-18. 959-54. 996 19. 120 1. 000 14. 12 ATOM 33 CD1 LEU 6-18. 446-53. 783 18. 359 1. 000 12. 19 ATOM 34 CD2 LEU 6-20. 245-55. 512 18. 494 1. 000 27. 94 ATOM 35 C LEU 6-17. 170-55. 293 21. 436 1. 000 2. 72 ATOM 36 0 LEU 6-17. 719-56. 101 22. 179 1. 000 13. 36 ATOM 37 N CYS 7-16. 978-54. 020 21. 756 1. 000 1. 38 ATOM 38 CA CYS 7-17. 472-53. 469 23. 011 1. 000 3. 17 ATOM 39 CB CYS 7-16. 411-52. 582 23. 667 1. 000 7. 01 ATOM 40 SG CYS 7-14. 867-53. 471 23. 992 1. 000 11. 21 ATOM 41 C CYS 7-18. 755-52. 685 22. 776 1. 000 0. 65 ATOM 42 0 CYS 7-18. 756-51. 627 22. 145 1. 000 4. 76 ATOM 43 N PHE 8-19. 859-53. 228 23. 281 1. 000 0. 00 ATOM 44 CA PHE 8-21. 147-52. 568 23. 053 1. 000 1. 14 ATOM 45 CB PHE 8-22. 115-53. 578 22. 443 1. 000 5. 54 ATOM 46 CG PHE 8-23. 421-53. 000 21. 937 1. 000 3. 36 ATOM 47 CD1 PHE 8-23. 456-52. 212 20. 800 1. 000 0. 89 ATOM 48 CD2 PHE 8-24. 602-53. 262 22. 614 1. 000 1. 39 ATOM 49 CE1 PHE 8-24. 644-51. 683 20. 333 1. 000 0. 00 ATOM 50 CE2 PHE 8-25. 793-52. 733 22. 148 1. 000 4. 42 ATOM 51 CZ PHE 8-25. 818-51. 944 21. 012 1. 000 2. 71 ATOM 52 C PHE 8-21. 677-51. 978 24. 346 1. 000 4. 46 ATOM 53 0 PHE 8-21. 873-52. 672 25. 348 1. 000 6. 98 ATOM 54 N GLY 9-21. 923-50. 666 24. 384 1. 000 5. 61 ATOM 55 CA GLY 9-22. 396-50. 109 25. 646 1. 000 5. 44 ATOM 56 C GLY 9-22. 860-48. 673 25. 522 1. 000 5. 66 ATOM 57 0 GLY 9-23. 229-48. 222 24. 440 1. 000 14. 54 ATOM 58 N ASP 10-22. 837-47. 964 26. 641 1. 000 3. 89 ATOM 59 CA ASP 10-23. 322-46. 596 26. 734 1. 000 5. 17 ATOM 60 CB ASP 10-24. 331-46. 467 27. 880 1. 000 2. 99 ATOM 61 CG ASP 10-23. 807-47. 052 29. 175 1. 000 7. 05 ATOM 62 OD1 ASP 10-22. 617-46. 829 29. 494 1. 000 17. 93 ATOM 63 OD2 ASP 10-24. 564-47. 738 29. 895 1. 000 10. 98 ATOM 64 C ASP 10-22. 154-45. 642 26. 939 1. 000 5. 15 ATOM 65 0 ASP 10-21. 022-45. 940 26. 556 1. 000 5. 62 ATOM 66 N SER 11-22. 423-44. 497 27. 554 1. 000 9. 02 ATOM 67 CA SER 11-21. 394-43. 493 27. 802 1. 000 3. 43 ATOM 68 CB SER 11-22. 014-42. 331 28. 585 1. 000 7. 25 ATOM 69 OG SER 11-22. 640-42. 813 29. 763 1. 000 18. 93 ATOM 70 C SER 11-20. 199-44. 046 28. 561 1. 000 7. 58 ATOM 71 0 SER 11-19. 089-43. 508 28. 501 1. 000 16. 71 ATOM 72 N LEU 12-20. 393-45. 133 29. 308 1. 000 6. 56 ATOM 73 CA LEU 12-19. 264-45. 696 30. 046 1. 000 16. 41 ATOM 74 CB LEU 12-19. 711-46. 759 31. 042 1. 000 17. 05

ATOM 75 CG LEU 12-20. 598-46. 336 32. 210 1. 000 18. 22 ATOM 76 CD1 LEU 12-20. 866-47. 527 33. 123 1. 000 7. 48 ATOM 77 CD2 LEU 12-19. 973-45. 184 32. 988 1. 000 10. 83 ATOM 78 C LEU 12-18. 269-46. 285 29. 048 1. 000 14. 99 ATOM 79 0 LEU 12-17. 065-46. 307 29. 267 1. 000 6. 10 ATOM 80 N THR 13-18. 828-46. 764 27. 940 1. 000 14. 77 ATOM 81 CA THR 13-18. 014-47. 347 26. 876 1. 000 8. 83 ATOM 82 CB THR 13-18. 828-48. 381 26. 080 1. 000 6. 87 ATOM 83 OG1 THR 13-19. 109-49. 487 26. 949 1. 000 10. 08 ATOM 84 CG2 THR 13-18. 033-48. 940 24. 914 1. 000 16. 85 ATOM 85 C THR 13-17. 490-46. 245 25. 970 1. 000 4. 56 ATOM 86 0 THR 13--16. 315-46. 220 25. 616 1. 000 11. 71 ATOM 87 N TRP 14-18. 376-45. 317 25. 612 1. 000 5. 57 ATOM 88 CA TRP 14-17. 992-44. 210 24. 742 1. 000 7. 21 ATOM 89 CB TRP 14-19. 208-43. 329 24. 453 1. 000 6. 90 ATOM 90 CG TRP 14-18. 917-42. 183 23. 537 1. 000 11. 88 ATOM 91 CD2 TRP 14-18. 731-40. 813 23. 924 1. 000 13. 72 ATOM 92 CE2 TRP 14-18. 483-40. 081 22. 745 1. 000 11. 95 ATOM 93 CE3 TRP 14-18. 752-40. 147 25. 152 1. 000 10. 63 ATOM 94 CD1 TRP 14-18. 779-42. 222 22. 181 1. 000 8. 28 ATOM 95 NE1 TRP 14-18. 517-40. 963 21. 694 1. 000 7. 16 ATOM 96 CZ2 TRP 14-18. 255-38. 705 22. 763 1. 000 5. 39 ATOM 97 CZ3 TRP 14-18. 526-38. 783 25. 168 1. 000 12. 55 ATOM 98 CH2 TRP 14-18. 282-38. 084 23. 981 1. 000 12. 81 ATOM 99 C TRP 14-16. 880-43. 353 25. 327 1. 000 5. 41 ATOM 100 0 TRP 14-16. 107-42. 745 24. 582 1. 000 4. 90 ATOM 101 N GLY 15-16. 794-43. 283 26. 652 1. 000 8. 94 ATOM 102 CA GLY 15-15. 794-42. 475 27. 318 1. 000 4. 51 ATOM 103 C GLY 15-16. 249-41. 098 27. 755 1. 000 10. 98 ATOM 104 0 GLY 15-15. 480-40. 136 27. 646 1. 000 15. 11 ATOM 105 N TRP 16-17. 471-40. 952 28. 255 1. 000 23. 34 ATOM 106 CA TRP 16-17. 988-39. 691 28. 792 1. 000 15. 10 ATOM 107 CB TRP 16-19. 408-39. 890 29. 327 1. 000 6. 11 ATOM 108 CG TRP 16-20. 139-38. 694 29. 846 1. 000 1. 78 ATOM 109 CD2 TRP 16-21. 229-38. 008 29. 213 1. 000 8. 98 ATOM 110 CE2 TRP 16-21. 613-36. 942 30. 051 1. 000 7. 76 ATOM 111 CE3 TRP 16-21. 923-38. 186 28. 009 1. 000 15. 66 ATOM 112 CD1 TRP 16-19. 927-38. 021 31. 016 1. 000 0. 35 ATOM 113 NE1 TRP 16-20. 798-36. 973 31. 154 1. 000 8. 35 ATOM 114 CZ2 TRP 16-22. 649-36. 063 29. 734 1. 000 5. 16 ATOM 115 CZ3 TRP 16-22. 952-37. 317 27. 692 1. 000 5. 34 ATOM 116 CH2 TRP 16-23. 306-36. 269 28. 551 1. 000 4. 72 ATOM 117 C TRP 16-17. 059-39. 154 29. 881 1. 000 7. 85 ATOM 118 0 TRP 16-16. 846-39. 815 30. 899 1. 000 3. 97 ATOM 119 N VAL 17-16. 533-37. 952 29. 685 1. 000 5. 45 ATOM 120 CA VAL 17-15. 750-37. 256 30. 695 1. 000 12. 08 ATOM 121 CB VAL 17-14. 822-36. 191 30. 082 1. 000 17. 55

ATOM 122 CG1 VAL 17-14. 084-35. 443 31. 185 1. 000 11. 59 ATOM 123 CG2 VAL 17-13. 841-36. 807 29. 099 1. 000 7. 77 ATOM 124 C VAL 17-16. 673-36. 565 31. 696 1. 000 13. 86 ATOM 125 0 VAL 17-17. 390-35. 618 31. 351 1. 000 1. 02 ATOM 126 N PRO 18-16. 660-37. 034 32. 936 1. 000 8. 38 ATOM 127 CD PRO 18-15. 770-38. 071 33. 476 1. 000 8. 64 ATOM 128 CA PRO 18-17. 572-36. 501 33. 948 1. 000 9. 99 ATOM 129 CB PRO 18-17. 201-37. 294 35. 208 1. 000 12. 31 ATOM 130 CG PRO 18-15. 817-37. 789 34. 954 1. 000 7. 46 ATOM 131 C PRO 18-17. 327-35. 017 34. 191 1. 000 13. 05 ATOM 132 0 PRO 18-16. 163-34. 619 34. 306 1. 000 18. 63 ATOM 133 N VAL 19-18. 381-34. 211 34. 266 1. 000 6. 92 ATOM 134 CA VAL 19-18. 214-32. 793 34. 585 1. 000 9. 29 ATOM 135 CB VAL 19-18. 482-31. 856 33. 388 1. 000 5. 33 ATOM 136 CG1 VAL 19-17. 377-31. 995 32. 354 1. 000 6. 78 ATOM 137 CG2 VAL 19-19. 850-32. 150 32. 796 1. 000 3. 72 ATOM 138 C VAL 19-19. 151-32. 380 35. 710 1. 000 12. 02 ATOM 139 0 VAL 19-20. 217-32. 962 35. 913 1. 000 14. 52 ATOM 140 N GLU 20-18. 771-31. 351 36. 467 1. 000 17. 17 ATOM 141 CA GLU 20-19. 662-30. 994 37. 575 1. 000 13. 30 ATOM 142 CB GLU 20-18. 918-30. 130 38. 595 1. 000 25. 34 ATOM 143 CG GLU 20-18. 276-30. 968 39. 702 1. 000 31. 46 ATOM 144 CD GLU 20-16. 871-30. 487 40. 017 1. 000 35. 91 ATOM 145 OE1 GLU 20-16. 143-30. 157 39. 055 1. 000 40. 11 ATOM 146 OE2 GLU 20-16. 507-30. 431 41. 210 1. 000 45. 47 ATOM 147 C GLU 20-20. 913-30. 294 37. 080 1. 000 7. 56 ATOM 148 0 GLU 20-21. 964-30. 361 37. 723 1. 000 11. 30 ATOM 149 N ASP 21-20. 852-29. 610 35. 936 1. 000 19. 38 ATOM 150 CA ASP 21-22. 099-28. 983 35. 471 1. 000 23. 47 ATOM 151 CB ASP 21-21. 815-27. 740 34. 640 1. 000 17. 53 ATOM 152 CG ASP 21-21. 114-27. 991 33. 326 1. 000 14. 93 ATOM 153 OD1 ASP 21-20. 984-29. 159 32. 908 1. 000 26. 78 ATOM 154 OD2 ASP 21-20. 685-26. 996 32. 694 1. 000 8. 74 ATOM 155 C ASP 21-22. 959-29. 988 34. 707 1. 000 19. 54 ATOM 156 0 ASP 21-23. 988-29. 627 34. 131 1. 000 22. 49 ATOM 157 N GLY 22-22. 550-31. 250 34. 697 1. 000 13. 19 ATOM 158 CA GLY 22-23. 279-32. 377 34. 166 1. 000 15. 71 ATOM 159 C GLY 22-23. 507-32. 377 32. 659 1. 000 20. 02 ATOM 160 0 GLY 22-23. 370-33. 431 32. 036 1. 000 23. 32 ATOM 161 N ALA 23-23. 846-31. 235 32. 138 1. 000 26. 40 ATOM 162 CA ALA 23-24. 265-30. 672 30. 873 1. 000 28. 79 ATOM 163 CB ALA 23-24. 483-29. 192 31. 152 1. 000 32. 86 ATOM 164 C ALA 23-23. 309-30. 988 29. 745 1. 000 22. 68 ATOM 165 0 ALA 23-22. 922-32. 189 29. 753 1. 000 40. 02 ATOM 166 N PRO 24-22. 847-30. 255 28. 748 1. 000 12. 97 ATOM 167 CD PRO 24-22. 892-28. 855 28. 309 1. 000 15. 92 ATOM 168 CA PRO 24-22. 051-31. 028 27. 767 1. 000 5. 31

ATOM 169 CB PRO 24-22. 024-30. 134 26. 520 1. 000 4. 03 ATOM 170 CG PRO 24-22. 002-28. 762 27. 105 1. 000 6. 80 ATOM 171 C PRO 24-20. 622-31. 273 28. 222 1. 000 14. 45 ATOM 172 0 PRO 24-20. 034-30. 591 29. 056 1. 000 19. 65 ATOM 173 N THR 25-20. 062-32. 310 27. 600 1. 000 13. 21 ATOM 174 CA THR 25-18. 685-32. 690 27. 894 1. 000 11. 82 ATOM 175 CB THR 25-18. 691-33. 772 28. 987 1. 000 12. 19 ATOM 176 OG1 THR 25-17. 348-34. 104 29. 355 1. 000 19. 38 ATOM 177 CG2 THR 25-19. 372-35. 027 28. 454 1. 000 0. 00 ATOM 178 C THR 25-18. 009-33. 160 26. 620 1. 000 14. 10 ATOM 179 0 THR 25-18. 555-33. 019 25. 518 1. 000 16. 46 ATOM 180 N GLU 26-16. 818-33. 724 26. 762 1. 000 12. 30 ATOM 181 CA GLU 26-16. 157-34. 314 25. 598 1. 000 13. 24 ATOM 182 CB GLU 26-14. 909-33. 518 25. 225 1. 000 15. 75 ATOM 183 CG GLU 26-15. 211-32. 066 24. 873 1. 000 25. 45 ATOM 184 CD GLU 26-15. 451-31. 152 26. 056 1. 000 27. 41 ATOM 185 OE1 GLU 26-14. 687-31. 210 27. 048 1. 000 22. 86 ATOM 186 OE2 GLU 26-16. 416-30. 347 26. 012 1. 000 17. 32 ATOM 187 C GLU 26-15. 850-35. 775 25. 891 1. 000 8. 80 ATOM 188 0 GLU 26-16. 279-36. 316 26. 909 1. 000 2. 55 ATOM 189 N ARG 27-15. 121-36. 421 25. 001 1. 000 13. 28 ATOM 190 CA ARG 27-14. 783-37. 838 25. 124 1. 000 12. 71 ATOM 191 CB ARG 27-14. 857-38. 447 23. 726 1. 000 6. 07 ATOM 192 CG ARG 27-14. 491-39. 908 23. 585 1. 000 4. 38 ATOM 193 CD ARG 27-14. 879-40. 387 22. 186 1. 000 11. 29 ATOM 194 NE ARG 27-14. 974-41. 840 22. 110 1. 000 13. 10 ATOM 195 CZ ARG 27-15. 191-42. 517 20. 992 1. 000 9. 74 ATOM 196 NH1 ARG 27-15. 337-41. 868 19. 842 1. 000 11. 38 ATOM 197 NH2 ARG 27-15. 262-43. 839 21. 029 1. 000 0. 00 ATOM 198 C ARG 27-13. 413-38. 031 25. 746 1. 000 8. 79 ATOM 199 0 ARG 27-12. 534-37. 181 25. 579 1. 000 17. 59 ATOM 200 N PHE 28-13. 183-39. 133 26. 461 1. 000 12. 29 ATOM 201 CA PHE 28-11. 826-39. 379 26. 955 1. 000 9. 91 ATOM 202 CB PHE 28-11. 783-40. 575 27. 900 1. 000 10. 13 ATOM 203 CG PHE 28-12. 084-40. 263 29. 355 1. 000 11. 54 ATOM 204 CD1 PHE 28-11. 250-39. 431 30. 084 1. 000 8. 88 ATOM 205 CD2 PHE 28-13. 194-40. 802 29. 979 1. 000 11. 27 ATOM 206 CE1 PHE 28-11. 535-39. 156 31. 408 1. 000 8. 90 ATOM 207 CE2 PHE 28-13. 486-40. 533 31. 305 1. 000 5. 41 ATOM 208 CZ PHE 28-12. 647-39. 703 32. 020 1. 000 0. 61 ATOM 209 C PHE 28-10. 901-39. 635 25. 770 1. 000 11. 56 ATOM 210 0 PHE 28-11. 370-40. 112 24. 736 1. 000 13. 14 ATOM 211 N ALA 29-9. 612-39. 349 25. 896 1. 000 13. 02 ATOM 212 CA ALA 29-8. 674-39. 656 24. 818 1. 000 13. 91 ATOM 213 CB ALA 29-7. 275-39. 163 25. 151 1. 000 6. 49 ATOM 214 C ALA 29-8. 662-41. 157 24. 545 1. 000 15. 68 ATOM 215 0 ALA 29-8. 937-41. 954 25. 446 1. 000 31. 74

ATOM 216 N PRO 30-8. 345-41. 537 23. 314 1. 000 11. 44 ATOM 217 CD PRO 30-7. 982-40. 660 22. 192 1. 000 12. 10 ATOM 218 CA PRO 30-8. 326-42. 955 22. 936 1. 000 18. 85 ATOM 219 CB PRO 30-7. 822-42. 956 21. 494 1. 000 16. 38 ATOM 220 CG PRO 30-7. 283-41. 593 21. 244 1. 000 14. 74 ATOM 221 C PRO 30-7. 386-43. 767 23. 826 1. 000 13. 40 ATOM 222 0 PRO 30-7. 570-44. 969 23. 979 1. 000 8. 18 ATOM 223 N ASP 31-6. 396-43. 115 24. 412 1. 000 22. 50 ATOM 224 CA ASP 31-5. 426-43. 715 25. 312 1. 000 26. 63 ATOM 225 CB ASP 31-4. 170-42. 841 25. 398 1. 000 30. 41 ATOM 226 CG ASP 31-3. 792-42. 143 24. 108 1. 000 39. 21 ATOM 227 OD1 ASP 31-2. 577-42. 086 23. 802 1. 000 39. 00 ATOM 228 OD2 ASP 31-4. 673-41. 634 23. 375 1. 000 37. 50 ATOM 229 C ASP 31-5. 985-43. 926 26. 721 1. 000 17. 49 ATOM 230 0 ASP 31-5. 482-44. 784 27. 450 1. 000 25. 27 ATOM 231 N VAL 32-6. 989-43. 150 27. 092 1. 000 14. 45 ATOM 232 CA VAL 32-7. 592-43. 125 28. 421 1. 000 12. 64 ATOM 233 CB VAL 32-7. 966-41. 683 28. 814 1. 000 10. 68 ATOM 234 CG1 VAL 32-8. 580-41. 609 30. 199 1. 000 13. 66 ATOM 235 CG2 VAL 32.-6. 742-40. 774 28. 752 1. 000 20. 51 ATOM 236 C VAL 32-8. 808-44. 042 28. 507 1. 000 9. 73 ATOM 237 0 VAL 32-8. 890-44. 834 29. 452 1. 000 2. 23 ATOM 238 N ARG 33-9. 722-43. 964 27. 553 1. 000 10. 63 ATOM 239 CA ARG 33-10. 888-44. 824 27. 410 1. 000 6. 85 ATOM 240 CB ARG 33-11. 369-44. 833 25. 961 1. 000 16. 41 ATOM 241 CG ARG 33-12. 281-43. 727 25. 488 1. 000 21. 19 ATOM 242 CD ARG 33-12. 464-43. 806 23. 974 1. 000 26. 66 ATOM 243 NE ARG 33-11. 862-42. 659 23. 309 1. 000 30. 35 ATOM 244 CZ ARG 33-11. 493-42. 567 22. 044 1. 000 31. 60 ATOM 245 NH1 ARG 33-11. 658-43. 585 21. 214 1. 000 34. 85 ATOM 246 NH2 ARG 33-10. 952-41. 433 21. 610 1. 000 52. 70 ATOM 247 C ARG 33-10. 600-46. 279 27. 775 1. 000 9. 71 ATOM 248 0 ARG 33-9. 603-46. 830 27. 300 1. 000 16. 85 ATOM 249 N TRP 34-11. 450-46. 924 28. 577 1. 000 10. 64 ATOM 250 CA TRP 34-11. 166-48. 311 28. 952 1. 000 6. 46 ATOM 251 CB TRP 34-12. 149-48. 855 29. 979 1. 000 12. 45 ATOM 252 CG TRP 34-13. 561-49. 106 29. 583 1. 000 6. 95 ATOM 253 CD2 TRP 34-14. 104-50. 199 28. 835 1. 000 9. 27 ATOM 254 CE2 TRP 34-15. 493-49. 986 28. 723 1. 000 5. 43 ATOM 255 CE3 TRP 34-13. 571-51. 345 28. 240 1. 000 14. 72 ATOM 256 CD1 TRP 34-14. 622-48. 298 29. 888 1. 000 4. 49 ATOM 257 NE1 TRP 34-15. 786-48. 820 29. 374 1. 000 4. 03 ATOM 258 CZ2 TRP 34-16. 337-50. 864 28. 050 1. 000 8. 19 ATOM 259 CZ3 TRP 34-14. 405-52. 216 27. 572 1. 000 12. 73 ATOM 260 CH2 TRP 34-15. 778-51. 976 27. 479 1. 000 8. 32 ATOM 261 C TRP 34-11. 111-49. 214 27. 723 1. 000 7. 27 ATOM 262 0 TRP 34-10. 393-50. 222 27. 767 1. 000 11. 53

ATOM 263 N THR 35-11. 839-48. 887 26. 659 1. 000 1. 15 ATOM 264 CA THR 35-11. 730-49. 673 25. 431 1. 000 5. 29 ATOM 265 CB THR 35-12. 708-49. 239 24. 331 1. 000 3. 10 ATOM 266 OG1 THR 35-12. 629-47. 820 24. 163 1. 000 15. 85 ATOM 267 CG2 THR 35-14. 146-49. 549 24. 726 1. 000 5. 16 ATOM 268 C THR 35-10. 307-49. 555 24. 882 1. 000 14. 32 ATOM 269 0 THR 35-9. 738-50. 494 24. 333 1. 000 12. 77 ATOM 270 N GLY 36-9. 756-48. 361 25. 060 1. 000 15. 72 ATOM 271 CA GLY 36-8. 392-48. 056 24. 689 1. 000 15. 87 ATOM 272 C GLY 36-7. 407-48. 785 25. 583 1. 000 14. 86 ATOM 273 0 GLY 36-6. 374-49. 252 25. 101 1. 000 22. 97 ATOM 274 N VAL 37-7. 686-48. 905 26. 884 1. 000 12. 48 ATOM 275 CA VAL 37-6. 696-49. 577 27. 728 1. 000 11. 76 ATOM 276 CB VAL 37-6. 921-49. 365 29. 229 1. 000 10. 95 ATOM 277 CG1 VAL 37-6. 092-50. 382 30. 009 1. 000 0. 00 ATOM 278 CG2 VAL 37-6. 577-47. 940 29. 630 1. 000 10. 31 ATOM 279 C VAL 37-6. 707-51. 081 27. 471 1. 000 16. 75 ATOM 280 0 VAL 37-5. 669-51. 735 27. 494 1. 000 14. 29 ATOM 281 N LEU 38-7. 911-51. 586 27. 238 1. 000 14. 60 ATOM 282 CA LEU 38-8. 094-52. 999 26. 917 1. 000 11. 25 ATOM 283 CB LEU 38-9. 573-53. 266 26. 660 1. 000 12. 92 ATOM 284 CG LEU 38-9. 975-54. 663 26. 198 1. 000 15. 77 ATOM 285 CD1 LEU 38-9. 747-55. 691 27. 293 1. 000 0. 00 ATOM 286 CD2 LEU 38-11. 425-54. 677 25. 733 1. 000 24. 28 ATOM 287 C LEU 38-7. 224-53. 347 25. 720 1. 000 7. 67 ATOM 288 0 LEU 38-6. 408-54. 262 25. 740 1. 000 13. 04 ATOM 289 N ALA 39,-7. 404-52. 568 24. 659 1. 000 9. 64 ATOM 290 CA ALA 39-6. 603-52. 667 23. 451 1. 000 3. 53 ATOM 291 CB ALA 39-6. 894-51. 487 22. 530 1. 000 6. 32 ATOM 292 C ALA 39-5. 112-52. 704 23. 761 1. 000 9. 32 ATOM 293 0 ALA 39-4. 411-53. 632 23. 367 1. 000 28. 59 ATOM 294 N GLN 40-4. 653-51. 665 24. 456 1. 000 21. 51 ATOM 295 CA GLN 40-3. 251-51. 553 24. 833 1. 000 18. 93 ATOM 296 CB GLN 40-2. 974-50. 365 25. 744 1. 000 28. 00 ATOM 297 CG GLN 40-3. 597-49. 034 25. 378 1. 000 37. 51 ATOM 298 CD GLN 40-3. 070-47. 877 26. 214 1. 000 40. 85 ATOM 299 OE1 GLN 40-1. 998-47. 335 25. 933 1. 000 61. 34 ATOM 300 NE2 GLN 40-3. 809-47. 475 27. 248 1. 000 9. 83 ATOM 301 C GLN 40-2. 822-52. 851 25. 525 1. 000 10. 96 ATOM 302 0 GLN 40-1. 856-53. 475 25. 106 1. 000 18. 66 ATOM 303 N GLN 41-3. 563-53. 239 26. 552 1. 000 15. 02 ATOM 304 CA GLN 41-3. 253-54. 423 27. 337 1. 000 22. 27 ATOM 305 CB GLN 41-4. 258-54. 582 28. 484 1. 000 16. 69 ATOM 306 CG GLN 41-4. 064-53. 605 29. 624 1. 000 14. 55 ATOM 307 CD GLN 41-2. 788-53. 852 30. 406 1. 000 16. 86 ATOM 308 OE1 GLN 41-2. 759-54. 650 31. 344 1. 000 13. 75 ATOM 309 NE2 GLN 41-1. 731-53. 158 30. 008 1. 000 21. 79

ATOM 310 C GLN 41-3. 261-55. 694 26. 493 1. 000 28. 40 ATOM 311 0 GLN 41-2. 442-56. 589 26. 703 1. 000 26. 71 ATOM 312 N LEU 42-4. 190-55. 776 25. 546 1. 000 28. 62 ATOM 313 CA LEU 42-4. 373-57. 007 24. 780 1. 000 26. 50 ATOM 314 CB LEU 42-5. 707-56. 920 24. 012 1. 000 19. 31 ATOM 315 CG LEU 42-6. 934-57. 122 24. 914 1. 000 16. 32 ATOM 316 CD1 LEU 42-8. 226-57. 077 24. 119 1. 000 10. 94 ATOM 317 CD2 LEU 42-6. 810-58. 438 25. 673 1. 000 15. 03 ATOM 318 C LEU 42-3. 217-57. 312 23. 846 1. 000 23. 29 ATOM 319 0 LEU 42-2. 770-58. 457 23. 728 1. 000 20. 82 ATOM 320 N GLY 43-2. 693-56. 312 23. 141 1. 000 22. 18 ATOM 321 CA GLY 43-1. 605-56. 590 22. 215 1. 000 18. 95 ATOM 322 C GLY 43-2. 086-56. 793 20. 791 1. 000 23. 97 ATOM 323 0 GLY 43-3. 284-56. 838 20. 514 1. 000 27. 50 ATOM 324 N ALA 44-1. 136-56. 927 19. 879 1. 000 22. 72 ATOM 325 CA ALA 44-1. 317-57. 012 18. 448 1. 000 24. 25 ATOM 326 CB ALA 44 0. 048-56. 939 17. 755 1. 000 13. 44 ATOM 327 C ALA 44-2. 034-58. 272 17. 990 1. 000 23. 83 ATOM 328 0 ALA 44-2. 146-58. 520 16. 787 1. 000 17. 77 ATOM 329 N ASP 45-2. 524-59. 086 18. 917 1. 000 21. 59 ATOM 330 CA ASP 45-3. 230-60. 298 18. 495 1. 000 17. 80 ATOM 331 CB ASP 45-2. 705-61. 491 19. 296 1. 000 18. 22 ATOM 332 CG ASP 45-1. 201-61. 625 19. 113 1. 000 24. 69 ATOM 333 OD1 ASP 45-0. 710-61. 174 18. 053 1. 000 34. 10 ATOM 334 OD2 ASP 45-0. 517-62. 159 20. 007 1. 000 33. 14 ATOM 335 C ASP 45-4. 732-60. 107 18. 647 1. 000 11. 82 ATOM 336 0 ASP 45-5. 535-60. 992 18. 364 1. 000 23. 89 ATOM 337 N PHE 46-5. 097-58. 914 19. 097 1. 000 9. 27 ATOM 338 CA PHE 46-6. 485-58. 519 19. 253 1. 000 12. 25 ATOM 339 CB PHE 46-6. 909-58. 479 20. 722 1. 000 14. 52 ATOM 340 CG PHE 46-6. 474-59. 693 21. 529 1. 000 11. 99 ATOM 341 CD1 PHE 46-5. 160-59. 814 21. 956 1. 000 12. 17 ATOM 342 CD2 PHE 46-7. 383-60. 690 21. 846 1. 000 8. 34 ATOM 343 CE1 PHE 46-4. 760-60. 917 22. 683 1. 000 13. 46 ATOM 344 CE2 PHE 46-6. 990-61. 794 22. 575 1. 000 6. 30 ATOM 345 CZ PHE 46-5. 680-61. 904 22. 998 1. 000 8. 44 ATOM 346 C PHE 46-6. 725-57. 149 18. 615 1. 000 13. 30 ATOM 347 0 PHE 46-5. 816-56. 366 18. 366 1. 000 27. 22 ATOM 348 N GLU 47-7. 992-56. 883 18. 349 1. 000 12. 78 ATOM 349 CA GLU 47-8. 469-55. 616 17. 833 1. 000 9. 15 ATOM 350 CB GLU 47-8. 667-55. 644 16. 325 1. 000 11. 20 ATOM 351 CG GLU 47-8. 791-54. 276 15. 670 1. 000 21. 84 ATOM 352 CD GLU 47-9. 726-54. 293 14. 474 1. 000 25. 88 ATOM 353 OE1 GLU 47-9. 575-55. 205 13. 632 1. 000 30. 74 ATOM 354 OE2 GLU 47-10. 602-53. 408 14. 388 1. 000 7. 59 ATOM 355 C GLU 47-9. 781-55. 280 18. 550 1. 000 11. 37 ATOM 356 0 GLU 47-10. 722-56. 071 18. 545 1. 000 11. 73

ATOM 357 N VAL 48-9. 775-54. 103 19. 160 1. 000 10. 53 ATOM 358 CA VAL 48-10. 954-53. 604 19. 843 1. 000 8. 11 ATOM 359 CB VAL 48-10. 595-52. 826 21. 115 1. 000 9. 71 ATOM 360 CG1 VAL 48-11. 842-52. 251 21. 773 1. 000 15. 31 ATOM 361 CG2 VAL 48-9. 849-53. 732 22. 085 1. 000 7. 41 ATOM 362 C VAL 48-11. 745-52. 714 18. 882 1. 000 12. 72 ATOM 363 0 VAL 48-11. 147-51. 879 18. 203 1. 000 10. 16 ATOM 364 N ILE 49-13. 046-52. 943 18. 862 1. 000 13. 04 ATOM 365 CA ILE 49-14. 031-52. 170 18. 122 1. 000 14. 10 ATOM 366 CB ILE 49-14. 879-53. 068 17. 203 1. 000 16. 77 ATOM 367 CG2 ILE 49-15. 735-52. 214 16. 285 1. 000 1. 57 ATOM 368 CG1 ILE 49-14. 049-54. 081 16. 415 1. 000 18. 10' ATOM 369 CD1 ILE 49-14. 687-54. 559 15. 133 1. 000 14. 33 ATOM 370 C ILE 49-14. 930-51. 406 19. 091 1. 000 9. 02 ATOM 371 0 ILE 49-15. 531-52. 013 19. 983 1. 000 15. 82 ATOM 372 N GLU 50-15. 000-50. 085 18. 932 1. 000 5. 34 ATOM 373 CA GLU 50-15. 730-49. 277 19. 911 1. 000 12. 03 ATOM 374 CB GLU 50-14. 967-47. 984 20. 222 1. 000 10. 36 ATOM 375 CG GLU 50-13. 623-48. 203 20. 889 1. 000 7. 32 ATOM 376 CD GLU 50-12. 768-46. 966 21. 056 1. 000 7. 06 ATOM 377 OE1 GLU 50-12. 744-46. 077 20. 177 1. 000 5. 78 ATOM 378 OE2 GLU 50-12. 079-46. 870 22. 101 1. 000 25. 19 ATOM 379 C GLU 50-17. 145-48. 962 19. 446 1. 000 6. 79 ATOM 380 0 GLU 50-17. 358-48. 318 18. 423 1. 000 8. 80 ATOM 381 N GLU 51-18. 118-49. 429 20. 225 1. 000 9. 34 ATOM 382 CA GLU 51-19. 524-49. 179 19. 924 1. 000 16. 23 ATOM 383 CB GLU 51-20. 173-50. 400 19. 270 1. 000 15. 22 ATOM 384 CG GLU 51-19. 757-50. 596 17. 820 1. 000 18. 39 ATOM 385 CD GLU 51-20. 348-49. 531 16. 917 1. 000 17. 99 ATOM 386 OE1 GLU 51-21. 352-48. 912 17. 332 1. 000 26. 29 ATOM 387 OE2 GLU 51-19. 820-49. 309 15. 809 1. 000 15. 93 ATOM 388 C GLU 51-20. 295-48. 788 21. 184 1. 000 10. 51 ATOM 389 0 GLU 51-21. 202-49. 495 21. 623 1. 000 7. 29 ATOM 390 N GLY 52-19. 906-47. 655 21. 751 1. 000 5. 90 ATOM 391 CA GLY 52-20. 533-47. 140 22. 961 1. 000 3. 93 ATOM 392 C GLY 52-21. 329-45. 887 22. 635 1. 000 6. 21 ATOM 393 0 GLY 52-20. 785-44. 950 22. 057 1. 000 16. 40 ATOM 394 N LEU 53-22. 607-45. 890 22. 989 1. 000 11. 68 ATOM 395 CA LEU 53-23. 498-44. 764 22. 710 1. 000 7. 60 ATOM 396 CB LEU 53-24. 627-45. 195 21. 792 1. 000 4. 45 ATOM 397 CG LEU 53-25. 576-44. 164 21. 185 1. 000 3. 84 ATOM 398 CD1 LEU 53-26. 721-43. 872 22. 141 1. 000 15. 09 ATOM 399 CD2 LEU 53-24. 856-42. 874 20. 817 1. 000 3. 41 ATOM 400 C LEU 53-24. 035-44. 204 24. 023 1. 000 5. 05 ATOM 401 0 LEU 53-24. 664-44. 920 24. 801 1. 000 5. 74 ATOM 402 N SER 54-23. 771-42. 918 24. 251 1. 000 9. 85 ATOM 403 CA SER 54-24. 192-42. 296 25. 502 1. 000 10. 24

ATOM 404 CB SER 54-23. 797-40. 819 25. 524 1. 000 7. 63 ATOM 405 OG SER 54-22. 395-40. 683 25. 640 1. 000 4. 65 ATOM 406 C SER 54-25. 695-42. 448 25. 691 1. 000 7. 74 ATOM 407 0 SER 54-26. 438-42. 326 24. 717 1. 000 10. 39 ATOM 408 N ALA 55-26. 127-42. 713 26. 920 1. 000 0. 00 ATOM 409 CA ALA 55-27. 554-42. 749 27. 218 1. 000 0. 00 ATOM 410 CB ALA 155-28. 209-41. 474 26. 713 1. 000 0. 00 ATOM 411 C ALA 55-28. 235-43. 982 26. 640 1. 000 6. 11 ATOM 412 0 ALA 55-29. 442-44. 179 26. 816 1. 000 2. 57 ATOM 413 N ARG 56-27. 474-44. 843 25. 971 1. 000 8. 50 ATOM 414 CA ARG 56-27. 997-46. 084 25. 433 1. 000 5. 94 ATOM 415 CB ARG 56-26. 919-46. 868 24. 672 1. 000 0. 00 ATOM 416 CG. ARG 56-27. 420-48. 244 24. 247 1. 000 2. 73 ATOM 417 CD ARG 56-26. 467-48. 951 23. 307 1. 000 0. 00 ATOM 418 NE ARG 56-26. 552-48. 440 21. 935 1. 000 6. 44 ATOM 419 CZ ARG 56-25. 465-48. 325 21. 170 1. 000 11. 18 ATOM 420 NH1 ARG 56-24. 283-48. 678 21. 666 1. 000 0. 00 ATOM 421 NH2 ARG 56-25. 549-47. 861 19. 928 1. 000 1. 13 ATOM 422 C ARG 56-28. 539-47. 009 26. 526 1. 000 12. 43 ATOM 423 0 ARG 56-27. 886-47. 179 27. 556 1. 000 10. 16 ATOM 424 N, THR 57-29. 697-47. 592 26. 262 1. 000 9. 24 ATOM 425 CA THR 57-30. 376-48. 548 27. 120 1. 000 9. 36 ATOM 426 CB THR 57-31. 855-48. 161 27. 315 1. 000 4. 78 ATOM 427 OG1 THR 57-32. 608-48. 509 26. 146 1. 000 3. 70 ATOM 428 CG2 THR 57-31. 992-46. 656 27. 484 1. 000 0. 00 ATOM 429 C THR 57-30. 284-49. 953 26. 532 1. 000 10. 18 ATOM 430 0 THR 57-29. 873-50. 099 25. 378 1. 000 12. 60 ATOM 431 N THR 58-30. 648-50. 987 27. 286 1. 000 5. 87 ATOM 432 CA THR 58-30. 574-52. 349 26. 769 1. 000 1. 65 ATOM 433 CB THR 58-30. 850-53. 410 27. 853 1. 000 5. 35 ATOM 434 OG1 THR 58-32. 151-53. 196 28. 413 1. 000 12. 48 ATOM 435 CG2 THR 58-29. 859-53. 311 29. 002 1. 000 11. 47 ATOM 436 C THR 58-31. 556-52. 569 25. 624 1. 000 1. 31 ATOM 437 0 THR 58-31. 162-52. 902 24. 506 1. 000 7. 78 ATOM 438 N ASN 59-32. 856-52. 404 25. 867 1. 000 4. 91 ATOM 439 CA ASN 59-33. 810-52. 604 24. 772 1. 000 11. 25 ATOM 440 CB ASN 59-34. 150-54. 090 24. 624 1. 000 9. 19 ATOM 441 CG ASN 59-35. 186-54. 548 25. 629 1. 000 9. 50 ATOM 442 OD1 ASN 59-35. 293-54. 000 26. 725 1. 000 13. 36 ATOM 443 ND2 ASN 59-35. 965-55. 556 25. 263 1. 000 4. 31 ATOM 444 C ASN 59-35. 070-51. 775 24. 960 1. 000 8. 67 ATOM 445 0 ASN 59-36. 172-52. 160 24. 574 1. 000 12. 75 ATOM 446 N ILE 60-34. 938-50. 587 25. 548 1. 000 10. 46 ATOM 447 CA ILE 60-36. 128-49. 752 25. 722 1. 000 10. 70 ATOM 448 CB ILE 60-36. 572-49. 721 27. 198 1. 000 11. 36 ATOM 449 CG2 ILE 60-35. 465-49. 223 28. 112 1. 000 0. 00 ATOM 450 CG1 ILE 60-37. 872-48. 940 27. 417 1. 000 8. 05

ATOM 451 CD1 ILE 60-38. 291-48. 800 28. 860 1. 000 27. 90 ATOM 452 C ILE 60-35. 879-48. 350 25. 177 1. 000 16. 37 ATOM 453 0 ILE 60-34. 813-47. 773 25. 374 1. 000 28. 53 ATOM 454 N ASP 61-36. 861-47. 811 24. 470 1. 000 18.. 37 ATOM 455 CA ASP 61-36. 838-46. 520 23. 821 1. 000 12. 62 ATOM 456 CB ASP 61-38. 110-46. 353 22. 977 1. 000 12. 58 ATOM 457 CG ASP 61-38. 111-47. 199 21. 725 1. 000 12. 09 ATOM 458 OD1 ASP 61-37. 044-47. 723 21. 349 1. 000 16. 37 ATOM 459 OD2 ASP 61-39. 197-47. 332 21. 122 1. 000 23. 20 ATOM 460 C ASP 61-36. 796-45. 350 24. 794 1. 000 11. 54 ATOM 461 0 ASP 61-37. 626-45. 279 25. 702 1. 000 8. 66 ATOM 462 N ASP 62-35. 860-44. 428 24. 603 1. 000 8. 03 ATOM 463 CA ASP 62-35. 844-43. 228 25. 431 1. 000 14. 39 ATOM 464 CB ASP 62-34. 430-42. 656 25. 565 1. 000 13. 94 ATOM 465 CG ASP 62-34. 384-41. 598 26. 656 1. 000 18. 06 ATOM 466 OD1 ASP 62-33. 609-41. 768 27. 622 1. 000 13. 05 ATOM 467 OD2 ASP 62-35. 129-40. 604 26. 536 1. 000 20. 19 ATOM 468 C ASP 62-36. 759-42. 162 24. 844 1. 000 13. 14 ATOM 469 0 ASP 62-36. 506-41. 698 23. 731 1. 000 14. 36 ATOM 470 N PRO 63-37. 800-41. 751 25. 553 1. 000 8. 49 ATOM 471 CD PRO 63-38. 102-42. 088 26. 951 1. 000 4. 73 ATOM 472 CA PRO 63-38. 805-40. 853 24. 972 1. 000 16. 60 ATOM 473 CB PRO 63-39. 802-40. 646 26. 123 1. 000 11. 61 ATOM 474 CG PRO 63-39. 020-40. 960 27. 352 1. 000 8. 04 ATOM 475 C PRO 63-38. 251-39. 504 24. 531 1. 000 19. 70 ATOM 476 0 PRO 63-38. 924-38. 738 23. 835 1. 000 10. 26 ATOM 477 N THR 64-37. 024-39. 180 24. 922 1. 000 22. 29 ATOM 478 CA THR 64-36. 429-37. 908 24. 534 1. 000 19. 30 ATOM 479 CB THR 64-35. 852-37. 191 25. 769 1. 000 20. 62 ATOM 480 OG1 THR 64-34. 550-37. 713 26. 045 1. 000 30. 42 ATOM 481 CG2 THR 64-36. 718-37. 467 26. 992 1. 000 7. 89 ATOM 482 C THR 64-35. 329-38. 087 23. 497 1. 000 19. 22 ATOM 483 0 THR 64-34. 609-37. 132 23. 183 1. 000 11. 15 ATOM 484 N ASP 65-35. 189-39. 301 22. 965 1. 000 15. 61 ATOM 485 CA ASP 65-34. 139-39. 542 21. 967 1. 000 18. 78 ATOM 486 CB ASP 65-32. 777-39. 286 22. 605 1. 000 20. 50 ATOM 487 CG ASP 65-31. 613-39. 348 21. 638 1. 000 17. 33 ATOM 488 OD1 ASP 65-31. 767-39. 935 20. 550 1. 000 19. 33 ATOM 489 OD2 ASP 65-30. 538-38. 810 21. 983 1. 000 15. 26 ATOM 490 C ASP 65-34. 241-40. 945 21. 382 1. 000 14. 84 ATOM 491 0 ASP 65-33. 982-41. 936 22. 060 1. 000 8. 38 ATOM 492 N PRO 66-34. 638-41. 026 20. 115 1. 000 15. 75 ATOM 493 CD PRO 66-34. 896-39. 870 19. 235 1. 000 23. 61 ATOM 494 CA PRO 66-34. 882-42. 301 19. 441 1. 000 9. 14 ATOM 495 CB PRO 66-35. 693-41. 871 18. 206 1. 000 14. 38 ATOM 496 CG PRO 66-35. 210-40. 494 17. 902 1. 000 16. 45 ATOM 497 C PRO 66-33. 621-43. 029 18. 995 1. 000 8. 15

ATOM 498 0 PRO 66-33. 695-44. 041 18. 283 1. 000 12. 38 ATOM 499 N ARG 67-32. 446-42. 557 19. 404 1. 000 11. 98 ATOM 500 CA ARG 67-31. 209-43. 225 19. 020 1. 000 7. 77 ATOM 501 CB ARG 67-30. 081-42. 211 18. 831 1. 000 8. 16 ATOM 502 CG ARG 67-30. 162-41. 308 17. 614 1. 000 7. 27 ATOM 503 CD ARG 67-29. 078-40. 228 17. 713 1. 000 11. 05 ATOM 504 NE ARG 67-29. 378-39. 266 18. 769 1. 000 11. 17 ATOM 505 CZ ARG 67-28. 768-38. 115 19. 001 1. 000 13. 35 ATOM 506 NH1 ARG 67-27. 756-37. 708 18. 245 1. 000 3. 80 ATOM 507 NH2 ARG 67-29. 168-37. 347 20. 010 1. 000 9. 93 ATOM 508 C ARG 67-30. 728-44. 239 20. 048 1. 000 8. 92 ATOM 509 0 ARG 67-29. 714-44. 887 19. 774 1. 000 13. 65 ATOM 510 N LEU 68-31. 389-44. 365 21. 191 1. 000 9. 14 ATOM 511 CA LEU 68-30. 805-45. 057 22. 335 1. 000 13. 92 ATOM 512 CB LEU 68-31. 052-44. 223 23. 608 1. 000 7. 80 ATOM 513 CG LEU 68-30. 899-42. 707 23. 481 1. 000 8. 78 ATOM 514 CD1 LEU 68-31. 285-41. 987 24. 770 1. 000 13. 12 ATOM 515 CD2 LEU 68-29. 477-42. 333 23. 090 1. 000 3. 77 ATOM 516 C LEU 68-31. 299-46. 478 22. 571 1. 000 16. 19 ATOM 517 0 LEU 68-30. 895-47. 092 23. 574 1. 000 5. 21 ATOM 518 N ASN 69-32. 139-47. 056 21. 716 1. 000 7. 75 ATOM 519 CA ASN 69-32. 520-48. 457 21. 927 1. 000 6. 53 ATOM 520 CB ASN 69-33. 807-48. 842 21. 198 1. 000 6. 25 ATOM 521 CG ASN 69-34. 377-50. 172 21. 658 1. 000 11. 70 ATOM 522 OD1 ASN 69-33. 732-51. 219 21. 664 1. 000 2. 64 ATOM 523 ND2 ASN 69-35. 646-50. 164 22. 057 1. 000 10. 84 ATOM 524 C ASN 69-31. 406-49. 404 21. 480 1. 000 8. 62 ATOM 525 0 ASN 69-31. 204-49. 617 20. 287 1. 000 14. 61 ATOM 526 N GLY 70-30. 697-49. 972 22. 452 1. 000 8. 79 ATOM 527 CA GLY 70-29. 582-50. 854 22. 212 1. 000 1. 64 ATOM 528 C GLY 70-29. 911-52. 031 21. 316 1. 000 6. 17 ATOM 529 0 GLY 70-29. 189-52. 293 20. 355 1. 000 12. 06 ATOM 530 N ALA 71-30. 982-52. 744 21. 622 1. 000 1. 39 ATOM 531 CA ALA 71-31. 442-53. 885 20. 843 1. 000 5. 92 ATOM 532 CB ALA 71-32. 688-54. 457 21. 529 1. 000 3. 81 ATOM 533 C ALA 71-31. 766-53. 565 19. 392 1. 000 4. 67 ATOM 534 0 ALA 71-31. 565-54. 391 18. 490 1. 000 0. 00 ATOM 535 N SER 72-32. 295-52. 371 19. 121 1. 000 3. 88 ATOM 536 CA SER 72-32. 687-52. 033 17. 752 1. 000 6. 33 ATOM 537 CB SER 72-33. 678-50. 870 17. 759 1. 000 4. 05 ATOM 538 OG SER 72-33. 023-49. 637 18. 004 1. 000 25. 62 ATOM 539 C SER 72-31. 468-51. 730 16. 884 1. 000 7. 90 ATOM 540 0 SER 72-31. 568-51. 720 15. 658 1. 000 12. 06 ATOM 541 N TYR 73-30. 315-51. 505 17. 498 1. 000 8. 51 ATOM 542 CA TYR 73-29. 070-51. 210 16. 789 1. 000 8. 77 ATOM 543 CB TYR 73-28. 394-50. 029 17. 478 1. 000 10. 31 ATOM 544 CG TYR 73-27. 124-49. 453 16. 913 1. 000 11. 92

ATOM 545 CD1 TYR 73-27. 113-48. 329 16. 090 1. 000 8. 49 ATOM 546 CE1 TYR 73-25. 931-47. 812 15. 586 1. 000 1. 47 ATOM 547 CD2 TYR 73-25. 888-50. 018 17. 201 1. 000 10. 36 ATOM 548 CE2 TYR 73-24. 704-49. 512 16. 703 1. 000 9. 07 ATOM 549 CZ TYR 73-24. 727-48. 398 15. 890 1. 000 5. 36 ATOM 550 OH TYR 73-23. 544-47. 902 15. 391 1. 000 10. 80 ATOM 551 C TYR 73-28. 148-52. 419 16. 730 1. 000 13. 31 ATOM 552 0 TYR 73-27. 404-52. 630 15. 764 1. 000 10. 40 ATOM 553 N LEU 74-28. 172-53. 261 17. 759 1. 000 8. 99 ATOM 554 CA LEU 74-27. 204-54. 342 17. 901 1. 000 7. 76 ATOM 555 CB LEU 74-27. 554-55. 155 19. 155 1. 000 9. 47 ATOM 556 CG LEU 74-26. 402-55. 532 20. 080 1. 000 10. 36 ATOM 557 CD1 LEU 74-26. 786-56. 729 20. 939 1. 000 25. 33 ATOM 558 CD2 LEU 74-25. 137-55. 819 19. 288 1. 000 13. 92 ATOM 559 C LEU 74-27. 088-55. 253 16. 687 1. 000 5. 72 ATOM 560 0 LEU 74-25. 980-55. 383 16. 141 1. 000 7. 01 ATOM 561 N PRO 75-28. 141-55. 907 16. 219 1. 000 6. 99 ATOM 562 CD PRO 75-29. 553-55. 794 16. 615 1. 000 1. 55 ATOM 563 CA PRO 75-27. 965-56. 896 15. 140 1. 000 7. 57 ATOM 564 CB PRO 75-29. 384-57. 401 14. 855 1. 000 5. 01 ATOM 565 CG PRO 75-30. 158-57. 063 16. 086 1. 000 6. 27 ATOM 566 C PRO 75-27. 364-56. 285 13. 882 1. 000 4. 16 ATOM 567 0 PRO 75-26. 651-56. 971 13. 158 1. 000 4. 35 ATOM 568 N SER 76-27. 640-55. 014 13. 615 1. 000 6. 22 ATOM 569 CA SER 76-27. 050-54. 322 12. 473 1. 000 0. 00 ATOM 570 CB SER 76-27. 758-52. 978 12. 261 1. 000 0. 00 ATOM 571 OG SER 76-29. 120-53. 249 11. 920 1. 000 0. 00 ATOM 572 C SER 76-25. 554-54. 127 12. 674 1. 000 0. 69 ATOM 573 0 SER 76-24. 767-54. 280 11. 740 1. 000 4. 06 ATOM 574 N CYS 77-25. 202-53. 802 13. 911 1. 000 2. 82 ATOM 575 CA CYS 77-23. 851-53. 599 14. 384 1. 000 2. 99 ATOM 576 CB CYS 77-23. 878-53. 202 15. 868 1. 000 0. 00 ATOM 577 SG CYS 77-22. 325-52. 508 16. 451 1. 000 8. 78 ATOM 578 C CYS 77-22. 962-54. 831 14. 225 1. 000 13. 77 ATOM 579 0 CYS 77-21. 828-54. 700 13. 755 1. 000 12. 12 ATOM 580 N LEU 78-23. 455-55. 996 14. 621 1. 000 15. 71 ATOM 581 CA LEU 78-22. 751-57. 268 14. 538 1. 000 10. 13 ATOM 582 CB LEU 78-23. 617-58. 387 15. 129 1. 000 2. 73 ATOM 583 CG LEU 78-23. 777-58. 354 16. 651 1. 000 7. 98 ATOM 584 CD1 LEU 78-24. 866-59. 319 17. 085 1. 000 3. 36 ATOM 585 CD2 LEU 78-22. 451-58. 676 17. 330 1. 000 8. 53 ATOM 586 C LEU 78-22. 385-57. 650 13. 106 1. 000 9. 88 ATOM 587 0 LEU 78-21. 222-57. 855 12. 761 1. 000 12. 55 ATOM 588 N ALA 79-23. 407-57. 748 12. 271 1. 000 11. 93 ATOM 589 CA ALA 79-23. 297-58. 022 10. 848 1. 000 2. 98 ATOM 590 CB ALA 79-24. 699-58. 042 10. 255 1. 000 0. 32 ATOM 591 C ALA 79-22. 393-57. 026 10. 127 1. 000 7. 73

ATOM 592 0 ALA 79-21. 724-57. 408 9. 163 1. 000 13. 15 ATOM 593 N THR 80-22. 337-55. 774 10. 560 1. 000 10. 93 ATOM 594 CA THR 80-21. 427-54. 757 10. 044 1. 000 6. 56 ATOM 595 CB THR 80-21. 703-53. 373 10. 669 1. 000 9. 10 ATOM 596 OG1 THR 80-23. 013-52. 897 10. 320 1. 000 4. 47 ATOM 597 CG2 THR 80-20. 722-52. 328 10. 148 1. 000 8. 02 ATOM 598 C THR 80-19. 970-55. 117 10. 317 1. 000 10. 87 ATOM 599 0 THR 80-19. 103-55. 052 9. 450 1. 000 12. 66 ATOM 600 N HIS 81-19. 659-55. 512 11. 548 1. 000 13. 90 ATOM 601 CA HIS 81-18. 282-55. 720 11. 978 1. 000 13. 04 ATOM 602 CB HIS 81-18. 119-55. 195 13. 418 1. 000 15. 15 ATOM 603 CG HIS 81-18. 279-53. 704 13. 502 1. 000 10. 10 ATOM 604 CD2 HIS 81-19. 202-52. 927 14. 111 1. 000 6. 25 ATOM 605 ND1 HIS 81-17. 404-52. 833 12. 889 1. 000 7. 20 ATOM 606 CE1 HIS 81-17. 775-51. 589 13. 117 1. 000 7. 73 ATOM 607 NE2 HIS 81-18. 867-51. 616 13. 863 1. 000 6. 24 ATOM 608 C HIS 81-17. 827-57. 166 11. 896 1. 000 9. 61 ATOM 609 0 HIS 81-16. 674-57. 460 12. 216 1. 000 10. 35 ATOM 610 N LEU 82-18. 689-58. 081 11. 470 1. 000 4. 74. ATOM 611 CA LEU 82-18. 257-59. 461 11. 247 1. 000 6. 06 ATOM 612 CB LEU 82-19. 399-60. 263 10. 631 1. 000 6. 90 ATOM 613 CG LEU 82-20. 535-60. 716 11. 541 1. 000 6. 83 ATOM 614 CD1 LEU 82-21. 388-61. 774 10. 851 1. 000 11. 79 ATOM 615 CD2 LEU 82-19. 987-61. 246 12. 856 1. 000 23. 45 ATOM 616 C LEU 82-17. 042-59. 500 10. 337 1. 000 6. 51 ATOM 617 0 LEU 82-16. 972-58. 722 9. 375 1. 000 1. 45 ATOM 618 N PRO 83-16. 056-60. 360 10. 556 1. 000 7. 15 ATOM 619 CD PRO 83-14. 823-60. 374 9. 731 1. 000 0. 00 ATOM 620 CA PRO 83-16. 043-61. 394 11. 583 1. 000 5. 44 ATOM 621 CB PRO 83-14. 941-62. 341 11. 067 1. 000 9. 33 ATOM 622 CG PRO 83-13. 968-61. 405 10. 415 1. 000 7. 09 ATOM 623 C PRO 83-15. 638-60. 922 12. 973 1. 000 10. 31 ATOM 624 0 PRO 83-14. 716-60. 125 13. 110 1. 000 16. 21 ATOM 625 N LEU 84-16. 319-61. 434 13. 994 1. 000 14. 34 ATOM 626 CA LEU 84-16. 009-61. 132 15. 382 1. 000 10. 66 ATOM 627 CB LEU 84-17. 165-60. 373 16. 049 1. 000 7. 23 ATOM 628 CG LEU 84-17. 485-59. 010 15. 434 1. 000 2. 01 ATOM 629 CD1 LEU 84-18. 843-58. 518 15. 902 1. 000 8. 19 ATOM 630 CD2 LEU 84-16. 382-58. 019 15. 766 1. 000 5. 93 ATOM 631 C LEU 84-15. 734-62. 386 16. 203 1. 000 7. 34 ATOM 632 0 LEU 84-16. 299-63. 447 15. 945 1. 000 8. 40 ATOM 633 N ASP 85-14. 879-62. 247 17. 208 1. 000 8. 68 ATOM 634 CA ASP 85-14. 607-63. 332 18. 146 1. 000 10. 21 ATOM 635 CB ASP 85-13. 093-63. 433 18. 382 1. 000 15. 96 ATOM 636 CG ASP 85-12. 338-63. 789 17. 117 1. 000 11. 01 ATOM 637 OD1 ASP 85-12. 343-64. 975 16. 727 1. 000 9. 49 ATOM 638 OD2 ASP 85-11. 739-62. 878 16. 518 1. 000 28. 18

ATOM 639 C ASP 85-15. 313-63. 142 19. 477 1. 000 0. 00 ATOM 640 0 ASP 85-15. 778-64. 067 20. 137 1. 000 5. 48 ATOM 641 N LEU 86-15. 414-61. 907 19. 958 1. 000 7. 62 ATOM 642 CA LEU 86-16. 080-61. 695 21. 243 1. 000 8. 84 ATOM 643 CB LEU 86-15. 085-61. 690 22. 403 1. 000 12. 15 ATOM 644 CG LEU 86-15. 655-61. 580 23. 822 1. 000 13. 98 ATOM 645 CD1 LEU 86-16. 562-62. 757 24. 151 1. 000 7. 12 ATOM 646 CD2 LEU 86-14. 535-61. 477 24. 850 1. 000 10. 28 ATOM 647 C LEU 86-16. 841-60. 374 21. 221 1. 000 6. 69 ATOM 648 0 LEU 86-16. 327-59. 409 20. 649 1. 000 8. 05 ATOM 649 N VAL 87-18. 013-60. 361 21. 842 1. 000 4. 26 ATOM 650 CA VAL 87-18. 752-59. 127 22. 049 1. 000 2. 21 ATOM 651 CB VAL 87-20. 150-59. 126 21. 413 1. 000 8. 44 ATOM 652 CG1 VAL 87-20. 848-57. 808 21. 722 1. 000 2. 51 ATOM 653 CG2 VAL 87-20. 104-59. 352 19. 911 1. 000 0. 00 ATOM 654 C VAL 87-18. 893-58. 869 23. 551 1. 000 7. 05 ATOM 655 0 VAL 87-19. 472-59. 660 24. 289 1. 000 5. 76 ATOM 656 N ILE 88-18. 351-57. 746 24. 010 1. 000 7. 24 ATOM 657 CA ILE 88-18. 499-57. 336 25. 400 1. 000 6. 18 ATOM 658 CB ILE 88-17. 233-56. 652 25. 938 1. 000 6. 54 ATOM 659 CG2 ILE 88-17. 458-56. 098 27. 333 1. 000 11. 40 ATOM 660 CG1 ILE 88-16. 001-57. 559 25. 902 1. 000 6. 21 ATOM 661 CD1 ILE 88-14. 734-56. 856 26. 339 1. 000 7. 20 ATOM 662 C ILE 88-19. 693-56. 394 25. 506 1. 000 4. 68 ATOM 663 0 ILE 88-19. 817-55. 458 24. 716 1. 000 10. 14 ATOM 664 N ILE 89-20. 574-56. 672 26. 457 1. 000 7. 74 ATOM 665 CA ILE 89-21. 765-55. 857 26. 645 1. 000 12. 20 ATOM 666 CB ILE 89-23. 052-56. 635 26. 306 1. 000 12. 51 ATOM 667 CG2 ILE 89-24. 253-55. 703 26. 339 1. 000 11. 52 ATOM 668 CG1 ILE 89-22. 981-57. 390 24. 979 1. 000 6. 47 . ATOM 669 CD1 ILE 89-24. 250-58. 111 24. 597 1. 000 8. 71 ATOM 670 C ILE 89-21. 861-55. 340 28. 078 1. 000 11. 05 ATOM 671 0 ILE 89-22. 169-56. 106 28. 989 1. 000 3. 02 ATOM 672 N MET 90-21. 590-54. 049 28. 236 1. 000 7. 01 ATOM 673 CA MET 90-21. 808-53. 359 29. 492 1. 000 11. 48 ATOM 674 CB MET 90-20. 535-52. 721 30. 043 1. 000 9. 27 ATOM 675 CG MET 90-20. 756-52. 097 31. 415 1. 000 10. 33 ATOM 676 XD MET 90-19. 202-51. 706 32. 246 1. 000 17. 92 ATOM 677 CE MET 90-18. 544-50. 475 31. 124 1. 000 12. 70 ATOM 678 C MET 90-22. 872-52. 262 29. 325 1. 000 12. 90 ATOM 679 0 MET 90-22. 524-51. 143 28. 954 1. 000 0. 00 ATOM 680 N LEU 91-24. 108-52. 639 29. 604 1. 000 8. 70 ATOM 681 CA LEU 91-25. 292-51. 802 29. 511 1. 000 10. 58 ATOM 682 CB LEU 91-26. 114-52. 105 28. 254 1. 000 9. 42 ATOM 683 CG LEU 91-25. 573-51. 564 26. 932 1. 000 4. 10 ATOM 684 CD1 LEU 91-26. 427-52. 046 25. 772 1. 000 0. 00 ATOM 685 CD2 LEU 91-25. 506-50. 044 26. 961 1. 000 2. 02

ATOM 686 C LEU 91-26. 169-52. 031 30. 734 1. 000 2. 21 ATOM 687 0 LEU 91-25. 989-53. 066 31. 388 1. 000 10. 59 ATOM 688 N GLY 92-27. 087-51. 117 31. 025 1. 000 4. 69 ATOM 689 CA GLY 92-27. 963-51. 321 32. 172 1. 000 7. 16 ATOM 690 C GLY 92-28. 189-50. 092 33. 027 1. 000 0. 00 ATOM 691 0 GLY 92-29. 266-49. 924 33. 603 1. 000 8. 09 ATOM 692 N THR 93-27. 204-49. 219 33. 133 1. 000 0. 16 ATOM 693 CA THR 93-27. 241-48. 005 33. 929 1. 000 9. 42 ATOM 694 CB THR 93-25. 927-47. 205 33. 768 1. 000 17. 05 ATOM 695 OG1 THR 93-24. 811-48. 063 34. 024 1. 000 26. 81 ATOM 696 CG2 THR 93-25. 847-46. 068 34. 778 1. 000 0. 34 ATOM 697 C THR 93-28. 386-47. 075 33. 551 1. 000 9. 26 ATOM 698 0 THR 93-29. 037-46. 491 34. 419 1. 000 14. 18 ATOM 699 N ASN 94-28. 614-46. 927 32. 250 1. 000 0. 69 ATOM 700 CA ASN 94-29. 609-45. 981 31. 755 1. 000 5. 12 ATOM 701 CB ASN 94-29. 333-45. 677 30. 274 1. 000 9. 42 ATOM 702 CG ASN 94-27. 990-44. 983 30. 120 1. 000 10. 74 ATOM 703 OD1 ASN 94-27. 679-44. 062 30. 873 1. 000 21. 66 ATOM 704 ND2 ASN 94-27. 175-45. 417 29. 174 1. 000 18. 23 ATOM 705 C ASN 94-31. 029-46. 481 31. 986 1. 000 5. 80 ATOM 706 0 ASN 94-31. 889-45. 654 32. 317 1. 000 4. 04 ATOM 707 N ASP 95-31. 282-47. 777 31. 863 1. 000 4. 02 ATOM 708 CA ASP 95-32. 568-48. 411 32. 137 1. 000 7. 86 ATOM 709 CB ASP 95-32. 522-49. 913 31. 880 1. 000 5. 49 ATOM 710 CG ASP 95-32. 090-50. 392 30. 521 1. 000 10. 09 ATOM 711 OD1 ASP 95-30. 998-50. 021 30. 040 1. 000 16. 22 ATOM 712 OD2 ASP 95-32. 843-51. 184 29. 907 1. 000 15. 98 ATOM 713 C ASP 95-33. 020-48. 208 33. 591 1. 000 9. 17 ATOM 714 0 ASP 95-34. 188-48. 361 33. 958 1. 000 0. 43 ATOM 715 N THR 96-32. 051-47. 882 34. 421 1. 000 11. 45 ATOM 716 CA THR 96-32. 122-47. 529 35. 823 1. 000 16. 75 ATOM 717 CB THR 96-30. 697-47. 638 36. 412 1. 000 24. 78 ATOM 718 OG1 THR 96-30. 607-48. 784 37. 274 1. 000 17. 62 ATOM 719 CG2 THR 96-30. 350-46. 409 37. 229 1. 000 12. 12 ATOM 720 C THR 96-32. 697-46. 132 35. 997 1. 000 12. 12 ATOM 721 0 THR 96-33. 047-45. 678 37. 088 1. 000 10. 94 ATOM 722 N LYS 97-32. 820-45. 406 34. 883 1. 000 12. 18 ATOM 723 CA LYS 97-33. 387-44. 060 34. 954 1. 000 14. 27 ATOM 724 CB LYS 97-33. 247-43. 336 33. 620 1. 000 13. 25 ATOM 725 CG LYS 97-31. 996-42. 477 33. 500 1. 000 11. 50 ATOM 726 CD LYS 97-31. 819-41. 935 32. 086 1. 000 3. 08 ATOM 727 CE LYS 97-30. 344-41. 856 31. 717 1. 000 0. 00 ATOM 728 NZ LYS 97-30. 131-41. 152 30. 416 1. 000 0. 00 ATOM 729 C LYS 97-34. 848-44. 112 35. 403 1. 000 12. 44 ATOM 730 0 LYS 97-35. 636-44. 914 34. 911 1. 000 8. 04 ATOM 731 N ALA 98-35. 179-43. 246 36. 355 1. 000 11. 97 ATOM 732 CA ALA 98-36. 454-43. 218 37. 047 1. 000 4. 97

ATOM 733 CB ALA 98-36. 522-41. 982 37. 943 1. 000 3. 36 ATOM 734 C ALA 98-37. 641-43. 246 36. 100 1. 000 12. 00 ATOM 735 0 ALA 98-38. 651-43. 905 36. 355 1. 000 22. 61 ATOM 736 N TYR 99-37. 535-42. 518 34. 988 1. 000 12. 39 ATOM 737 CA TYR 99-38. 695-42. 403 34. 107 1. 000 7. 25 ATOM 738 CB TYR 99-38. 521-41. 297 33. 087 1. 000 9. 11 ATOM 739 CG TYR 99-37. 300-41. 251 32. 217 1. 000 15. 58 ATOM 740 CD1 TYR 99-37. 261-41. 912 30. 995 1. 000 13. 09 ATOM 741 CE1 TYR 99-36. 144-41. 874 30. 186 1. 000 9. 06 ATOM 742 CD2 TYR 99-36. 173-40. 533 32. 598 1. 000 14. 48 ATOM 743 CE2 TYR 99-35. 051-40. 482 31. 796 1. 000 15. 13 ATOM 744 CZ TYR 99-35. 044-41. 154 30. 591 1. 000 11. 74 ATOM 745 OH TYR 99-33. 925-41. 102 29. 794 1. 000 6. 20 ATOM 746 C TYR 99-38. 990-43. 726 33. 413 1. 000. 11. 25 ATOM 747 0 TYR 99-40. 121-43. 927 32. 963 1. 000 12. 89 ATOM 748 N PHE 100-37. 993-44. 606 33. 351 1. 000 4. 63 ATOM 749 CA PHE 100-38. 237-45. 908 32. 731 1. 000 1. 01 ATOM 750 CB PHE 100-36. 903-46. 556 32. 348 1. 000 3. 41 ATOM 751 CG PHE 100-36. 316-45. 980 31. 070 1. 000 11. 77 ATOM 752 CD1 PHE 100-35. 018-45. 506 31. 032 1. 000 7. 50 ATOM 753 CD2 PHE 100-37. 080-45. 919 29. 917 1. 000 16. 94 ATOM 754 CE1 PHE 100-34. 489-44. 981 29. 868 1. 000 7. 31 ATOM 755 CE2 PHE 100-36. 557-45. 398 28. 748 1. 000 12. 92 ATOM 756 CZ PHE 100-35. 260-44. 925 28. 722 1. 000 7. 58 ATOM 757 C PHE 100-39. 051-46. 829 33. 628 1. 000 6. 94 ATOM 758 0 PHE 100-39. 711-47. 750 33. 131 1. 000 9. 31 ATOM 759 N ARG 101-39. 032-46. 629 34. 943 1. 000 12. 10 ATOM 760 CA ARG 101-39. 783-47. 468 35. 869 1. 000 12. 96 ATOM 761 CB ARG 101-41. 294-47. 296 35. 695 1. 000 16. 21 ATOM 762 CG ARG 101-41. 890-45. 959 36. 087 1. 000 19. 51 ATOM 763 CD ARG 101-43. 376-45. 918 35. 740 1. 000 25. 82 ATOM 764 NE ARG 101-43. 818-44. 553 35. 466 1. 000 31. 88 ATOM 765 CZ ARG 101-43. 797-43. 583 36. 373 1. 000 33. 97 ATOM 766 NH1 ARG 101-43. 355-43. 839 37. 599 1. 000 43. 49 ATOM 767 NH2 ARG 101-44. 206-42. 361 36. 067 1. 000 44. 85 ATOM 768 C ARG 101-39. 472-48. 955 35. 704 1. 000 12. 20 ATOM 769 0 ARG 101-40. 376-49. 782 35. 878 1. 000 12. 48 ATOM 770 N ARG 102-38. 238-49. 319 35. 378 1. 000 8. 86 ATOM 771 CA ARG 102-37. 887-50. 733 35. 264 1. 000 11. 00 ATOM 772 CB ARG 102-36. 899-50. 962 34. 115 1. 000 6. 96 ATOM 773 CG ARG 102-37. 497-50. 805 32. 720 1. 000 9. 64 ATOM 774 CD ARG 102-36. 518-51. 198 31. 624 1. 000 8. 07 ATOM 775 NE ARG 102-37. 140-51. 842 30. 474 1. 000 4. 64 ATOM 776 CZ ARG 102-36. 540-52. 606 29. 571 1. 000 7. 34 ATOM 777 NH1 ARG 102-35. 240-52. 877 29. 628 1. 000 1. 45 ATOM 778 NH2 ARG 102-37. 232-53. 131 28. 567 1. 000 6. 11 ATOM 779 C ARG 102-37. 320-51. 275 36. 577 1. 000 11. 09

ATOM 780 0 ARG 102-36. 734-50. 567 37. 394 1. 000 10. 02 ATOM 781 N THR 103-37. 497-52. 573 36. 785 1. 000 11. 01 ATOM 782 CA THR 103-36. 898-53. 307 37. 893 1. 000 12. 65 ATOM 783 CB THR 103-37. 844-54. 376 38. 462 1. 000 7. 64 ATOM 784 OG1 THR 103-38. 083-55. 384 37. 468 1. 000 11. 29 ATOM 785 CG2 THR 103-39. 199-53. 771 38. 790 1. 000 15. 33 ATOM 786 C THR 103-35. 618-53. 966 37. 390 1. 000 10. 55 ATOM 787 0 THR 103-35. 409-53. 986 36. 173 1. 000 9. 17 ATOM 788 N PRO 104-34. 765-54. 474 38. 264 1. 000 10. 17 ATOM 789 CD PRO 104-34. 799-54. 363 39. 731 1. 000 14. 03 ATOM 790 CA PRO 104-33. 598-55. 230 37. 803 1. 000 6. 81 ATOM 791 CB PRO 104-32. 968-55. 748 39. 094 1. 000 5. 25 ATOM 792 CG PRO 104-33. 402-54. 759 40. 129 1. 000 8. 07 ATOM 793 C PRO 104-34. 010-56. 400 36. 911 1. 000 5. 89 ATOM 794 0 PRO 104-33. 251-56. 728 35. 998 1. 000 5. 49 ATOM 795 N LEU 105-35. 164-56. 994 37. 173 1. 000 2. 55 ATOM 796 CA LEU 105-35. 690-58. 071 36. 341 1. 000 10. 27 ATOM 797 CB LEU 105-36. 989-58. 642 36. 890 1. 000 11. 51 ATOM 798 CG LEU 105-37. 304-60. 122 36. 695 1. 000 16. 39 ATOM 799 CD1 LEU 105-38. 804-60. 319 36. 480 1. 000 4. 05 ATOM 800 CD2 LEU 105.-36. 533-60. 744 35. 542 1. 000 15. 49 ATOM 801 C LEU 105-35. 923-57. 566 34. 915 1. 000 14. 30 ATOM 802 0 LEU 105-35. 415-58. 168 33. 969 1. 000 14. 22 ATOM 803 N ASP 106-36. 686-56. 484 34. 791 1. 000 11. 11 ATOM 804 CA ASP 106-36. 922-55. 878 33. 482 1. 000 8. 08 ATOM 805 CB ASP 106-37. 636-54. 538 33. 621 1. 000 14. 02 ATOM 806 CG'ASP 106-39. 046-54. 638 34. 152 1. 000 13. 88 ATOM 807 OD1 ASP 106-39. 726-55. 653 33. 875 1. 000 19. 94 ATOM 808 OD2 ASP 106-39. 479-53. 686 34. 843 1. 000 4. 29 ATOM 809 C ASP 106-35. 607-55. 668 32. 734 1. 000 7. 79 ATOM 810 0 ASP 106-35. 504-55. 987 31. 554 1. 000 10. 52 ATOM 811 N ILE 107-34. 614-55. 131 33. 438 1. 000 5. 00 ATOM 812 CA ILE 107-33. 321-54. 814 32. 845 1. 000 6. 63 ATOM 813 CB ILE 107-32. 444-54. 016 33. 828 1. 000 14. 49 ATOM 814 CG2 ILE 107-31. 125-53. 622 33. 184 1. 000 7. 24 ATOM 815 CG1 ILE 107-33. 146-52. 790 34. 415 1. 000 16. 93 ATOM 816 CD1 ILE 107-32. 174-51. 779 34. 992 1. 000 19. 38 ATOM 817 C ILE 107-32. 564-56. 059 32. 405 1. 000 5. 12 ATOM 818 0 ILE 107-31. 877-56. 024 31. 381 1. 000 4. 80 ATOM 819 N ALA 108-32. 691-57. 148 33. 157 1. 000 5. 34 ATOM 820 CA ALA 108-32. 021-58. 398 32. 812 1. 000 4. 25 ATOM 821 CB ALA 108-32. 089-59. 399 33. 956 1. 000 2. 49 ATOM 822 C ALA 108-32. 637-59. 018 31. 568 1. 000 2. 89 ATOM 823 0 ALA 108-31. 952-59. 619 30. 738 1. 000 11. 68 ATOM 824 N LEU 109-33. 956-58. 864 31. 449 1. 000 0. 00 ATOM 825 CA LEU 109-34. 609-59. 401 30. 251 1. 000 6. 18 ATOM 826 CB LEU 109-36. 125-59. 391 30. 435 1. 000 12. 37

ATOM 827 CG LEU 109-36. 674-60. 463 31. 386 1. 000 15. 66 ATOM 828 CD1 LEU 109-37. 985-60. 004 32. 001 1. 000 27. 44 ATOM 829 CD2 LEU 109-36. 854-61. 794 30. 672 1. 000 3. 14 ATOM 830 C LEU 109-34. 171-58. 620 29. 022 1. 000 10. 30 ATOM 831 0 LEU 109-34. 035-59. 139 27. 915 1. 000 18. 00 ATOM 832 N GLY 110-33. 918-57. 323 29. 193 1. 000 11. 78 ATOM 833 CA GLY 110-33. 426-56. 535 28. 069 1. 000 8. 26 ATOM 834 C GLY 110-32. 028-56. 976 27. 666 1. 000 7. 06 ATOM 835 0 GLY 110-31. 757-57. 155 26. 482 1. 000 18. 68 ATOM 836 N MET 111-31. 149-57. 149 28. 651 1. 000 5. 04 ATOM 837 CA MET 111-29. 812-57. 661 28. 414 1. 000 4. 52 ATOM 838 CB MET 111-28. 962-57. 717 29. 683 1. 000 1. 61 ATOM 839 CG MET 111-27. 663-58. 503 29. 542 1. 000 0. 00 ATOM 840 XD MET 111-26. 456-57. 694 28. 453 1. 000 16. 83 ATOM 841 CE MET 111-25. 895-56. 355 29. 497 1. 000 5. 08 ATOM 842 C MET 111-29. 915-59. 066 27. 821 1. 000 6. 40 ATOM 843 0 MET 111-29. 098-59. 476 27. 005 1. 000 8. 66 ATOM 844 N SER 112-30. 937-59. 795 28. 270 1. 000 9. 55 ATOM 845 CA SER 112-31. 140-61. 133 27. 731 1. 000 8. 05 ATOM 846 CB SER 112-32. 322-61. 821 28. 405 1. 000 10. 37 ATOM 847 OG SER 112-33. 488-61. 744 27. 609 1. 000 8. 11 ATOM 848 C SER 112-31. 341-61. 034 26. 217 1. 000 6. 07 ATOM 849 0 SER 112-30. 761-61. 823 25. 471 1. 000 9. 26 ATOM 850 N VAL 113-32. 142-60. 065 25. 803 1. 000 4. 80 ATOM 851 CA VAL 113-32. 424-59. 788. 24. 401 1. 000 9. 22 ATOM 852 CB VAL 113-33. 414-58. 615 24. 266 1. 000 9. 35 ATOM 853 CG1 VAL 113-33. 350-57. 979 22. 886 1. 000 0. 53 ATOM 854 CG2 VAL 113-34. 830-59. 090 24. 567 1. 000 15. 43 ATOM 855 C'VAL 113-31. 149-59. 490 23. 616 1. 000 18. 19 ATOM 856 0 VAL 113-31. 027-59. 900 22. 456 1. 000 17. 08 ATOM 857 N LEU 114-30. 199-58. 791 24. 235 1. 000 16. 22 ATOM 858 CA LEU 114-28. 948-58. 431 23. 570 1. 000 9. 05 ATOM 859 CB LEU 114-28. 220-57. 329 24. 341 1. 000 4. 93 ATOM 860 CG LEU 114-28. 938-55. 983 24. 427 1. 000 6. 23 ATOM 861 CD1 LEU 114-28. 122-54. 973 25. 221 1. 000 8. 47 ATOM 862 CD2 LEU 114-29. 228-55. 450 23. 032 1. 000 0. 00 ATOM 863 C LEU 114-28. 018-59. 628 23. 407 1. 000 5. 15 ATOM 864 0 LEU 114-27. 310-59. 762 22. 410 1. 000 8. 05 ATOM 865 N VAL 115-28. 028-60. 503 24. 403 1. 000 5. 78 ATOM 866 CA VAL 115-27. 223-61. 717 24. 373 1. 000 8. 93 ATOM 867 CB VAL 115-27. 202-62. 383 25. 762 1. 000 8. 05 ATOM 868 CG1 VAL 115-26. 501-63. 729 25. 720 1. 000 0. 00 ATOM 869 CG2 VAL 115-26. 543-61. 439 26. 759 1. 000 0. 00 ATOM 870 C VAL 115-27. 763-62. 685 23. 330 1. 000 9. 50 ATOM 871 0 VAL 115-27. 007-63. 390 22. 662 1. 000 9. 58 ATOM 872 N THR 116-29. 087-62. 715 23. 179 1. 000 8. 15 ATOM 873 CA THR 116-29. 688-63. 617 22. 199 1. 000 8. 38

ATOM 874 CB THR 116-31. 222-63. 622 22. 327 1. 000 12. 50 ATOM 875 OG1 THR 116-31. 575-64. 207 23. 585 1. 000 13. 40 ATOM 876 CG2 THR 116-31. 848-64. 479 21. 233 1. 000 10. 82 ATOM 877 C THR 116-29. 316-63. 241 20. 771 1. 000 5. 56 ATOM 878 0 THR 116-29. 011-64. 127 19. 966 1. 000 5. 27 ATOM 879 N GLN 117-29. 345-61. 945 20. 473 1. 000 8. 17 ATOM 880 CA GLN 117-28. 956-61. 430 19. 160 1. 000'9. 93 ATOM 881 CB GLN 117-29. 166-59. 920 19. 080 1. 000 3. 66 ATOM 882 CG GLN 117-30. 592-59. 440 19. 279 1. 000 6. 21 ATOM 883 CD GLN 117-30. 699-57. 933 19. 390 1. 000 7. 09 ATOM 884 OE1 GLN 117-29. 801-57. 260 19. 896 1. 000 12. 85 ATOM 885 NE2 GLN 117-31. 811-57. 376 18. 914 1. 000 7. 39 ATOM 886 C GLN 117-27. 499-61. 761 18. 847 1. 000 11. 60 ATOM 887 0 GLN 117-27. 105-62. 023 17. 706 1. 000 9. 03 ATOM 888 N VAL 118-26. 652-61. 751 19. 879 1. 000 11. 77 ATOM 889 CA VAL 118-25. 258-62. 146 19. 659 1. 000 8. 34 ATOM 890 CB VAL 118-24. 340-61. 768 20. 831 1. 000 0. 49 ATOM 891 CG1 VAL 118-22. 892-62. 118 20. 499 1. 000 21. 94 ATOM 892 CG2 VAL 118-24. 452-60. 291 21. 169 1. 000 3. 31 ATOM 893 C VAL 118-25. 166-63. 652 19. 417 1. 000 10. 48 ATOM 894 0 VAL 118-24. 354-64. 107 18. 607 1. 000 10. 54 ATOM 895 N LEU 119-25. 993-64. 431 20. 112 1. 000 7. 97 ATOM 896 CA LEU 119-25. 916-65. 885 19. 993 1. 000 8. 73 ATOM 897 CB LEU 119-26. 679-66. 572 21. 135 1. 000 8. 06 ATOM 898 CG LEU 119-25. 981-66. 556 22. 498 1. 000 21. 06 ATOM 899 CD1 LEU 119-26. 800-67. 296 23. 548 1. 000 5. 53 ATOM 900 CD2 LEU 119-24. 580-67. 150 22. 403 1. 000 21. 96 ATOM 901 C LEU 119-26. 446-66. 362 18. 649 1. 000 5. 78 ATOM 902 0 LEU 119-26. 022-67. 409 18. 153 1. 000 14. 06 ATOM 903 N THR 120-27. 364-65. 608 18. 053 1. 000 8. 82 ATOM 904 CA THR 120-27. 964-65. 985 16. 780 1. 000 0. 00 ATOM 905 CB THR 120-29. 497-65. 798 16. 815 1. 000 6. 15 ATOM 906 OG1 THR 120-29. 805-64. 405 16. 969 1. 000 10. 14 ATOM 907 CG2 THR 120-30. 121-66. 535 17. 994 1. 000 0. 76 ATOM 908 C THR 120-27. 419-65. 198 15. 594 1. 000 10. 30 ATOM 909 0 THR'120-28. 061-65. 190 14. 537 1. 000 13. 46 ATOM 910 N SER 121-26. 272-64. 533 15. 700 1. 000 11. 26 ATOM 911 CA SER 121-25. 774-63. 675 14. 636 1. 000 7. 70 ATOM 912 CB SER 121-25. 000-62. 487 15. 240 1. 000 5. 36 ATOM 913 OG SER 121-23. 826-62. 954 15. 886 1. 000 3. 70 ATOM 914 C SER 121-24. 852-64. 353 13. 629 1. 000 7. 89 ATOM 915 0 SER 121-24. 360-63. 660 12. 730 1. 000 13. 24 ATOM 916 N ALA 122-24. 603-65. 645 13. 755 1. 000 11. 50 ATOM 917 CA ALA 122-23. 748-66. 370 12. 820 1. 000 12. 48 ATOM 918 CB ALA 122-23. 820-67. 868 13. 098 1. 000 3. 73 ATOM 919 C ALA 122-24. 124-66. 083 11. 370 1. 000 7. 92 ATOM 920 0 ALA 122-25. 311-66. 050 11. 042 1. 000 8. 42

ATOM 921 N GLY 123-23. 125-65. 859 10. 529 1. 000 7. 14 ATOM 922 CA GLY 123-23. 316-65. 625 9. 115 1. 000 3. 98 ATOM 923 C GLY 123-23. 643-64. 196 8. 735 1. 000 12. 34 ATOM 924 0 GLY 123-23. 445-63. 822 7. 571 1. 000 1. 55 ATOM 925 N GLY 124-24. 132-63. 404 9. 683 1. 000 19. 09 ATOM 926 CA GLY 124-24. 506-62. 016 9. 471 1. 000 13. 26 ATOM 927 C GLY 124-25. 277-61. 809 8. 186 1. 000 10. 25 ATOM 928 0 GLY 124-26. 403-62. 278 8. 018 1. 000 10. 97 ATOM 929 N VAL 125-24. 684-61. 110 7. 217 1. 000 12. 50 ATOM 930 CA VAL 125-25. 365-60. 956 5. 930 1. 000 9. 40 ATOM 931 CB VAL 125-25. 557-59. 477 5. 559 1. 000 14. 11 ATOM 932 CG1 VAL 125-26. 156-59. 326 4. 168 1. 000 13. 51 ATOM 933 CG2 VAL 125-26. 455-58. 786 6. 578 1. 000 22. 31 ATOM 934 C VAL 125-24. 588-61. 675 4. 833 1. 000 6. 71 ATOM 935 0 VAL 125-23. 580-61. 151 4. 368 1. 000 4. 54 ATOM 936 N GLY 126-25. 047-62. 850 4. 427 1. 000 14. 20 ATOM 937 CA GLY 126-24. 466-63. 654 3. 377 1. 000 9. 15 ATOM 938 C GLY 126-23. 012-64. 018 3. 580 1. 000 10. 06 ATOM 939 0 GLY 126-22. 225-64. 068 2. 629 1. 000 4. 29 ATOM 940 N THR 127-22. 595-64. 295 4. 811 1. 000 6. 29 ATOM 941 CA THR 127-21. 214-64. 701 5. 050 1. 000 3. 83 ATOM 942 CB THR 127-20. 470-63. 707 5. 957 1. 000 8. 35 ATOM 943 OG1 THR 127-20. 719-64. 001 7. 339 1. 000 16. 55 ATOM 944 CG2 THR 127-20. 987-62. 295 5. 716 1. 000 11. 34 ATOM 945 C THR 127-21. 143-66. 099 5. 663 1. 000 1. 10 ATOM 946 0 THR 127-22. 159-66. 699 6. 001 1. 000 4. 52 ATOM 947 N THR 128-19. 921-66. 590 5. 790 1. 000 9. 21 ATOM 948 CA THR 128-19. 546-67. 893 6. 299 1. 000 8. 72 ATOM 949 CB THR 128-18. 451-68. 505 5. 397 1. 000 10. 99 ATOM 950 OG1 THR 128-17. 447-67. 497 5. 236 1. 000 7. 85 ATOM 951 CG2 THR 128-18. 976-68. 853 4. 015 1. 000 3. 45 ATOM 952 C THR 128-18. 995-67. 821 7. 718 1. 000 13. 03 ATOM 953 0 THR 128-18. 450-68. 788 8. 255 1. 000 8. 50 ATOM 954 N TYR 129-19. 127-66. 646 8. 315 1. 000 10. 20 ATOM 955 CA TYR 129-18. 542-66. 357 9. 615 1. 000 7. 58 ATOM 956 CB TYR 129-18. 323-64. 853 9. 722 1. 000 8. 22 ATOM 957 CG TYR 129-17. 246-64. 280 8. 835 1. 000 11. 97 ATOM 958 CD1 TYR 129-17. 514-63. 176 8. 031 1. 000 8. 62 ATOM 959 CE1 TYR 129-16. 547-62. 636 7. 211 1. 000 7. 23 ATOM 960 CD2 TYR 129-15. 970-64. 827 8. 799 1. 000 12. 10 ATOM 961 CE2 TYR 129-14. 991-64. 290 7. 982 1. 000 16. 92 ATOM 962 CZ TYR 129-15. 288-63. 196 7. 193 1. 000 16. 10 ATOM 963 OH TYR 129-14. 315-62. 655 6. 383 1. 000 11. 56 ATOM 964 C TYR 129-19. 416-66. 840 10. 765 1. 000 9. 63 ATOM 965 0 TYR 129-20. 644-66. 723 10. 714 1. 000 13. 75 ATOM 966 N PRO 130-18. 789-67. 380 11. 804 1. 000 8. 51 ATOM 967 CD PRO 130-17. 336-67. 523 12. 004 1. 000 10. 11

ATOM 968 CA PRO 130-19. 549-67. 914 12. 938 1. 000 5. 53 ATOM 969 CB PRO 130-18. 522-68. 804 13. 647 1. 000 8. 51 ATOM 970 CG PRO 130-17. 227-68. 097 13. 397 1. 000 11. 17 ATOM 971 C PRO 130-19. 983-66. 791 13. 872 1. 000 7. 77 ATOM 972 0 PRO 130-19. 500-65. 667 13. 730 1. 000 2. 72 ATOM 973 N ALA 131-20. 873-67. 117 14. 799 1. 000 7. 61 ATOM 974 CA ALA 131-21. 305-66. 205 15. 844 1. 000 2. 73 ATOM 975 CB ALA 131-22. 537-66. 747 16. 554 1. 000 0. 00 ATOM 976 C ALA 131-20. 174-65. 984 16. 842 1. 000 8. 30 ATOM 977 0 ALA 131-19. 502-66. 942 17. 223 1. 000 12. 18 ATOM 978 N PRO 132-19. 937-64. 752 17. 273 1. 000 14. 28 ATOM 979 CD PRO 132-20. 610-63. 516 16. 842 1. 000 11. 04 ATOM 980 CA PRO 132-18. 901-64. 505 18. 284 1. 000 12. 37 ATOM 981 CB PRO 132-18. 696-62. 992 18. 181 1. 000 14. 35 ATOM 982 CG PRO 132-20. 032-62. 472 17. 753 1. 000 12. 70 ATOM 983 C PRO 132-19. 395-64. 884 19. 675 1. 000 12. 80 ATOM 984 0 PRO 132-20. 608-65. 027 19. 856 1. 000 21. 24 ATOM 985 N LYS 133-18. 497-65. 051 20. 641 1. 000 14. 17 ATOM 986 CA LYS 133-18. 903-65. 337 22. 017 1. 000 14. 31 ATOM 987 CB LYS 133-17. 760-65. 881 22. 869 1. 000 14. 22 ATOM 988 CG LYS 133-17. 050-67. 101 22. 317 1. 000 13. 51 ATOM 989 CD LYS 133-15. 746-67. 358 23. 057 1. 000 18. 76 ATOM 990 CE LYS 133-15. 463-68. 849 23. 174 1. 000 21. 23 ATOM 991 NZ LYS 133-15. 154-69. 237 24. 580 1. 000 37. 08 ATOM 992 C LYS 133-19. 441-64. 066 22. 667 1. 000 10. 23 ATOM 993 0 LYS 133-19. 319-62. 982 22. 091 1. 000 4. 45 ATOM 994 N VAL 134-20. 032-64. 194 23. 853 1. 000 4. 74 ATOM 995 CA VAL 134-20. 562-63. 000 24. 507 1. 000 10. 55 ATOM 996 CB VAL 134-22. 106-62. 964 24. 490 1. 000 11. 86 ATOM 997 CG1 VAL 134-22. 586-61. 523 24. 423 1. 000 0. 00 ATOM 998 CG2 VAL 134-22. 659-63. 778 23. 334 1. 000 29. 88 ATOM 999 C VAL 134-20. 129-62. 885 25. 963 1. 000 12. 01 ATOM 1000 0 VAL 134-20. 215-63. 837 26. 736 1. 000 27. 94 ATOM 1001 N LEU 135-19. 676-61. 703 26. 357 1. 000 12. 21 ATOM 1002. CA LEU 135-19. 364-61. 443 27. 757 1. 000 14. 41 ATOM 1003 CB LEU 135-17. 975-60. 835 27. 898 1. 000 17. 37 ATOM 1004 CG LEU 135-17. 123-61. 223 29. 105 1. 000 18. 57 ATOM 1005 CD1 LEU 135-15. 993-60. 213 29. 264 1. 000 4. 42 ATOM 1006 CD2 LEU 135-17. 932-61. 341 30. 387 1. 000 6. 01 ATOM 1007 C LEU 135-20. 397-60. 497 28. 360 1. 000 17. 03 ATOM 1008 0 LEU 135-20. 485-59. 326 27. 984 1. 000 14. 19 ATOM 1009 N VAL 136-21. 196-60. 988 29. 303 1. 000 19. 10 ATOM 1010 CA VAL 136-22. 167-60. 110 29. 954 1. 000 14. 45 ATOM 1011 CB VAL 136-23. 344-60. 925 30. 511 1. 000 13. 65 ATOM 1012 CG1 VAL 136-24. 272-60. 045 31. 335 1. 000 8. 06 ATOM 1013 CG2 VAL 136-24. 080-61. 596 29. 362 1. 000 0. 00 ATOM 1014 C VAL 136-21. 498-59. 327 31. 073 1. 000 10. 63

ATOM 1015 0 VAL 136-20. 929-59. 948 31. 971 1. 000 7. 12 ATOM 1016 N VAL 137-21. 556-57. 997 31. 027 1. 000 7. 93 ATOM 1017 CA VAL 137-20. 882-57. 215 32. 056 1. 000 6. 63 ATOM 1018 CB VAL 137-19. 699-56. 397 31. 497 1. 000 6. 08 ATOM 1019 CG1 VAL 137.-19. 115-55. 512 32. 595 1. 000 6. 59 ATOM 1020 CG2 VAL 137-18. 609-57. 291 30. 936 1. 000 10. 34 ATOM 1021 C VAL 137-21. 828-56. 255 32. 775 1. 000 6. 02 ATOM 1022 0 VAL 137-22. 319-55. 273 32. 219 1. 000 11. 10 ATOM 1023 N SER 138-22. 061-56. 558 34. 040 1. 000 6. 05 ATOM 1024 CA SER 138-22. 800-55. 715 34. 972 1. 000 9. 77 ATOM 1025 CB SER 138-23. 139-56. 523 36. 223 1. 000 16. 98 ATOM 1026 OG SER 138-23. 850-55. 804 37. 202 1. 000 19. 18 ATOM 1027 C SER 138-21. 944-54. 496 35. 276 1. 000 8. 41 ATOM 1028 0 SER 138-20. 779-54. 646 35. 652 1. 000 13. 52 ATOM 1029 N PRO 139-22. 459-53. 287 35. 096 1. 000 12. 22 ATOM 1030 CD PRO 139-23. 803-52. 952 34. 599 1. 000 11. 54 ATOM 1031 CA PRO 139-21. 657-52. 087 35. 389 1. 000 6. 14 ATOM 1032 CB PRO 139-22. 422-51. 015 34. 608 1. 000 7. 78 ATOM 1033 CG PRO 139-23. 848-51. 455 34. 731 1. 000 3. 74 ATOM 1034 C PRO 139-21. 620-51. 775 36. 875 1. 000 3. 92 ATOM 1035 0 PRO 139-22. 460-52. 217 37. 664 1. 000 1Q. 47 ATOM 1036 N PRO 140-20. 636-51. 014 37. 347 1. 000 8. 52 ATOM 1037 CD PRO 140-19. 524-50. 412 36. 611 1. 000 3. 33 ATOM 1038 CA PRO 140-20. 591-50. 724 38. 788 1. 000 13. 50 ATOM 1039 CB PRO 140-19. 251-50. 012 38. 971 1. 000 12. 27 ATOM 1040 CG PRO 140-18. 843-49. 543 37. 623 1. 000 6. 73 ATOM 1041 C PRO 140-21. 748-49. 832 39. 228 1. 000 15. 77 ATOM 1042 0 PRO 140-22. 321-49. 073 38. 445 1. 000 21. 96 ATOM 1043 N PRO 141-22. 103-49. 939 40. 505 1. 000 4. 93 ATOM 1044 CD PRO 141-21. 487-50. 799 41. 528 1. 000 0. 26 ATOM 1045 CA PRO 141-23. 230-49. 172 41. 036 1. 000 3. 17 ATOM 1046 CB PRO 141-23. 254-49. 560 42. 521 1. 000 4. 18 ATOM 1047 CG PRO 141-22. 591-50. 897 42. 556 1. 000 0. 00 ATOM 1048 C PRO 141-23. 014-47. 671 40. 890 1. 000 10. 32 ATOM 1049 0 PRO 141-21. 876-47. 203 40. 900 1. 000 17. 58 ATOM 1050 N LEU 142-24. 120-46. 942 40. 760 1. 000 9. 20 ATOM 1051 CA LEU 142-24. 079-45. 490 40. 729 1. 000 7. 44 ATOM 1052 CB LEU 142-25. 421-44. 900 40. 288 1. 000 7. 55 ATOM 1053 CG LEU 142-25. 775-45. 119 38. 812 1. 000 13. 23 ATOM 1054 CD1 LEU 142-27. 262-44. 901 38. 566 1. 000 0. 00 ATOM 1055 CD2 LEU 142-24. 932-44. 218 37. 921 1. 000 1. 85 ATOM 1056 C LEU 142-23. 711-44. 945 42. 109 1. 000 13. 38 ATOM 1057 0 LEU 142-23. 764-45. 680 43. 099 1. 000 20. 55 ATOM 1058 N ALA 143-23. 363-43. 670 42. 126 1. 000 15. 81 ATOM 1059 CA ALA 143-22. 960-42. 941 43. 322 1. 000 13. 69 ATOM 1060 CB ALA 143-21. 461-42. 676 43. 239 1. 000 3. 16 ATOM 1061 C ALA 143-23. 762-41. 656 43. 475 1. 000 16. 69

ATOM 1062 0 ALA 143-24. 500-41. 280 42. 552 1. 000 10. 61 ATOM 1063 N PRO 144-23. 668-40. 968 44. 609 1. 000 19. 19 ATOM 1064 CD PRO 144-22. 997-41. 377 45. 852 1. 000 16. 93 ATOM 1065 CA PRO 144-24. 315-39. 659 44. 745 1. 000 19. 29 ATOM 1066 CB PRO 144-23. 730-39. 076 46. 031 1. 000 17. 13 ATOM 1067 CG PRO 144-22. 904-40. 130 46. 664 1. 000 12. 97 ATOM 1068 C PRO 144-24. 009-38. 723 43. 578 1. 000 17. 14 ATOM 1069 0 PRO 144-22. 902-38. 626 43. 048 1. 000 12. 89 ATOM 1070 N MET 145-25. 049-38. 002 43. 161 1. 000 18. 09 ATOM 1071 CA MET 145-24. 925-37. 064 42. 052 1. 000 14. 70 ATOM 1072. CB MET 145-25. 912-37. 398 40. 942 1. 000 21. 06 ATOM 1073 CG MET 145-25. 711-38. 740 40. 263 1. 000 24. 88 ATOM 1074 XD MET 145-27. 259-39. 577 39. 860 1. 000 18. 47 ATOM 1075 CE MET 145-27. 956-39. 804 41. 495 1. 000 34. 91 ATOM 1076 C MET 145-25. 155-35. 645 42. 559 1. 000 11. 49 ATOM 1077 0 MET 145-26. 205-35. 342 43. 116 1. 000 18. 46 ATOM 1078 N PRO 146-24. 182-34. 763 42. 367 1. 000 6. 41 ATOM 1079 CD PRO 146-22. 909-34. 993 41. 683 1. 000 8.'62 ATOM 1080 CA PRO 146-24. 325-33. 388 42. 851 1. 000 10. 88 ATOM. 1081 CB PRO 146-22. 916-32. 814 42. 759 1. 000 10. 59 ATOM 1082 CG PRO 146-22. 064-33. 819 42. 072 1. 000 12. 17 ATOM 1083 C PRO 146-25. 292-32. 588 41. 972 1. 000 13. 13 ATOM 1084 0 PRO 146-25. 999-31. 712 42. 484 1. 000 17. 39 ATOM 1085 N HIS 147-25. 311-32. 901 40. 677 1. 000 10. 50 ATOM 1086 CA HIS 147-26. 203-32. 215 39. 758 1. 000 9. 69 ATOM 1087 CB HIS 147-25. 865-32. 480 38. 279 1. 000 14. 24 ATOM 1088 CG HIS 147-26. 441-31. 373 37. 431 1. 000 6. 69 ATOM 1089 CD2 HIS 147-25. 875-30. 297 36. 850 1. 000 5. 99 ATOM 1090 ND1 HIS 147-27. 780-31. 296 37. 134 1. 000 11. 40 ATOM 1091 CE1 HIS 147-28. 018-30. 226 36. 391 1. 000 11. 68 ATOM 1092 NE2 HIS 147-26. 871-29. 600. 36. 201 1. 000 12. 68 ATOM 1093 C HIS 147-27. 658-32. 596 40. 013 1. 000 5. 47 ATOM 1094 0 HIS 147-28. 052-33. 761 39. 960 1. 000 11. 15 ATOM 1095 N PRO 148-28. 463-31. 575 40. 291 1. 000 12. 88 ATOM 1096 CD PRO 148-28. 098-30. 148 40. 322 1. 000 12. 98 ATOM 1097 CA PRO 148-29. 877-31. 806 40. 602 1. 000 13. 30 ATOM 1098 CB PRO 148-30. 440-30. 401 40. 811 1. 000 14. 82 ATOM 1099 CG PRO 148-29. 426-29. 455 40. 267 1. 000 16. 64 ATOM 1100 C PRO 148-30. 600-32. 508 39. 456 1. 000 15. 39 ATOM 1101 0 PRO 148-31. 525-33. 290 39. 689 1. 000 15. 71 ATOM 1102 N TRP 149-30. 218-32. 263 38. 201 1. 000 21. 29 ATOM 1103 CA TRP 149-30. 909-32. 947 37. 109 1. 000 15. 64 ATOM 1104 CB TRP 149-30. 571-32. 328 35. 750 1. 000 17. 31 ATOM 1105 CG TRP 149-31. 296-33. 043 34. 639 1. 000 10. 06 ATOM 1106 CD2 TRP 149-32. 715-33. 086 34. 444 1. 000 4. 30 ATOM 1107 CE2 TRP 149-32. 952-33. 862 33. 295 1. 000 8. 55 ATOM 1108 CE3 TRP 149-33. 805-32. 541 35. 129 1. 000 4. 24

ATOM 1109 CD1 TRP 149-30. 748-33. 774 33. 629 1. 000 11. 09 ATOM 1110 NE1 TRP 149-31. 736-34. 272 32. 813 1. 000 5. 61 ATOM 1111 CZ2 TRP 149-34. 240-34. 107 32. 815 1. 000 12. 36 ATOM 1112 CZ3 TRP 149--35. 076-32. 785 34. 654 1. 000 13. 41 ATOM 1113 CH2 TRP 149-35. 286-33. 563 33. 505 1. 000 14. 13 ATOM 1114 C TRP 149-30. 566-34. 432 37. 101 1. 000 12. 85 ATOM 1115 0 TRP 149-31. 447-35. 290 37. 033 1. 000 7. 92 ATOM 1116 N PHE 150-29. 270-34. 728 37. 186 1. 000 11. 11 ATOM 1117 CA PHE 150-28. 841-36. 125 37. 305 1. 000 11. 76 ATOM 1118 CB PHE 150-27. 321-36. 192 37. 483 1. 000 8. 65 ATOM 1119 CG PHE 150-26. 581-36. 170 36. 150 1. 000 13. 44 ATOM 1120 CD1 PHE 150-25. 315-35. 623 36. 047 1. 000 14. 41 ATOM 1121 CD2 PHE 150-27. 167-36. 697 35. Q14 1. 000 12. 01 ATOM 1122 CE1 PHE 150-24. 650-35. 604 34. 838 1. 000 14. 96 ATOM 1123 CE2 PHE 150-26. 511-36. 684 33. 797 1. 000 13. 41 ATOM 1124 CZ PHE 150-25. 246-36. 136 33. 711 1. 000 18. 95 ATOM 1125 C PHE 150-29. 555-36. 813 38. 459 1. 000 10. 90 ATOM 1126 0 PHE 150-30. 059-37. 930 38. 354 1. 000 7. 95 ATOM 1127 N GLN 151-29. 606-36. 120 39. 598 1. 000 12. 36 ATOM 1128 CA GLN 151-30. 294-36. 665 40. 759 1. 000 19. 45 ATOM 1129 CB GLN 151-30. 306-35. 680 41. 932 1. 000 12. 11 ATOM 1130 CG GLN 151-28. 947-35. 446 42. 561 1. 000 16. 34 ATOM 1131 CD GLN 151-29. 048-34. 481 43. 734 1. 000 22. 05 ATOM 1132 OE1 GLN 151-29. 693-34. 803 44. 729 1. 000 39. 76 ATOM 1133 NE2 GLN 151-28. 423-33. 317 43. 598 1. 000 16. 49 ATOM 1134 C GLN 151-31. 745-37. 027 40. 441 1. 000 20. 77 ATOM 1135 0 GLN 151-32. 232-38. 044 40. 936 1. 000 19. 36 ATOM 1136 N LEU 152-32. 397-36. 183 39. 644 1. 000 11. 67 ATOM 1137 CA LEU 152-33. 818-36. 360 39. 365 1. 000 13. 95 ATOM 1138 CB LEU 152-34. 438-35. 101 38. 764 1. 000 14. 14 ATOM 1139 CG LEU 152-34. 837-33. 957 39. 688 1. 000 12. 09 ATOM 1140 CD1 LEU 152-34. 781-32. 631 38. 935 1. 000 11. 66 ATOM 1141 CD2 LEU 152-36. 225-34. 162 40. 274 1. 000 12. 14 ATOM 1142 C LEU 152-34. 053-37. 544 38. 428 1. 000 13. 07 ATOM 1143 0 LEU 152-34. 913-38. 372 38. 729 1. 000 13. 96 ATOM 1144 N ILE 153-33. 310-37. 613 37. 326 1. 000 13. 21 ATOM 1145 CA ILE 153-33. 519-38. 661 36. 334 1. 000 12. 12 ATOM 1146 CB ILE 153-32. 814-38. 377 34. 991 1. 000 9. 74 ATOM 1147 CG2 ILE 153-33. 360-37. 106 34. 355 1. 000 0. 00 ATOM 1148 CG1 ILE 153-31. 284-38. 333 35. 061 1. 000 8. 16 ATOM 1149 CD1 ILE 153-30. 635-38. 332 33. 684 1. 000 0. 00 ATOM 1150 C ILE 153-33. 054-40. 024 36. 836 1. 000 9. 56 ATOM 1151 0 ILE 153-33. 540-41. 043 36. 342 1. 000 4. 79 ATOM 1152 N PHE 154-32. 138-40. 069 37. 797 1. 000 12. 41 ATOM 1153 CA PHE 154-31. 645-41. 349 38. 301 1. 000 8. 75 ATOM 1154 CB PHE 154-30. 113-41. 372 38. 348 1. 000 8. 88 ATOM 1155 CG PHE 154-29. 456-41. 758 37. 031 1. 000 8. 38

ATOM 1156 CD1 PHE 154-28. 597-40. 887 36. 384 1. 000 9. 10 ATOM 1157 CD2 PHE 154-29. 703-42. 990 36. 458 1. 000 0. 00 ATOM 1158 CE1 PHE 154-28. 000-41. 232 35. 188 1. 000 9. 85 ATOM 1159 CE2 PHE 154-29. 119-43. 344 35. 260 1. 000 5. 02 ATOM 1160 CZ PHE 154-28. 258-42. 468 34. 624 1. 000 8. 39 ATOM 1161 C PHE 154-32. 199-41. 648 39. 690 1. 000 11. 55 ATOM 1162 0 PHE 154-31. 683.-42. 515 40. 400 1. 000 10. 77 ATOM 1163 N GLU 155-33. 246-40. 936 40. 093 1. 000 15. 11 ATOM 1164 CA GLU 155-33. 898-41. 221 41. 367 1. 000 19. 95 ATOM 1165 CB GLU 155-35. 134-40. 343 41. 542 1. 000 26. 08 ATOM 1166 CG GLU 155-35. 558-40. 107 42. 980 1. 000 33. 00 ATOM 1167 CD GLU 155-36. 339-41. 267 43. 568 1. 000 44. 51 ATOM 1168 OE1 GLU 155-37. 432-41. 585 43. 051 1. 000 49. 47 ATOM 1169 OE2 GLU 155-35. 862-41. 867 44. 558 1. 000 61. 39 ATOM 1170 C GLU 155-34. 270-42. 702 41. 449 1. 000 18. 82 ATOM 1171 0 GLU 155-34. 978-43. 212 40. 582 1. 000 14. 49 ATOM 1172 N GLY 156-33. 779-43. 376 42. 481 1. 000 12. 58 ATOM 1173 CA GLY 156-33. 993-44. 787 42. 696 1. 000 6. 50 ATOM 1174 C GLY 156-33. 061-45. 684 41. 914 1. 000 12. 22 ATOM 1175 0 GLY 156-33. 205-46. 914 41. 914 1. 000 27. 90 ATOM 1176 N GLY 157-32. 082-45. 107 41. 224 1. 000 9. 19 ATOM 1177 CA GLY 157-31. 216-45. 877 40. 358 1. 000 8. 21 ATOM 1178 C GLY 157-30. 007-46. 514 40. 991 1. 000 8. 61 ATOM 1179 0 GLY 157-29. 563-47. 579 40. 549 1. 000 17. 22 ATOM 1180 N GLU 158-29. 442-45. 887 42. 018 1. 000 7. 58 ATOM 1181 CA GLU 158-28. 299-46. 453 42. 721 1. 000 7. 50 ATOM. 1182 CB GLU 158-27. 807-45. 505 43. 814 1. 000 9. 84 ATOM 1183 CG GLU 158-26. 756-46. 097 44. 739 1. 000 11. 00 ATOM 1184 CD GLU 158-26. 031-45. 053 45. 564 1. 000 24. 40 ATOM 1185 OE1 GLU 158-26. 158-43. 845 45. 267 1. 000 33. 57 ATOM 1186 OE2 GLU 158-25. 325-45. 439 46. 523 1. 000 39. 11 ATOM 1187 C GLU 158-28. 696-47. 807 43. 302 1. 000 13. 34 ATOM 1188 0 GLU 158-27. 956-48. 787 43. 225 1. 000 29. 78 ATOM 1189 N GLN 159-29. 895-47. 840 43. 875 1. 000 10. 17 ATOM 1190 CA GLN 159-30. 481-49. 058 44. 406 1. 000 15. 50 ATOM 1191 CB GLN 159-31. 856-48. 764 45. 017 1. 000 19. 57 ATOM 1192 CG GLN 159-32. 548-49. 952 45. 647 1. 000 24. 93 ATOM 1193 CD GLN 159-31. 737-50. 676 46. 704 1. 000 30. 24 ATOM 1194 OE1 GLN 159-31. 940-50. 499 47. 909 1. 000 40. 80 ATOM 1195 NE2 GLN 159-30. 800-51. 510 46. 265 1. 000 20. 75 ATOM 1196 C GLN 159-30. 605-50. 132 43. 336 1. 000 17. 89 ATOM 1197 0 GLN 159-30. 218-51. 285 43. 544 1. 000 21. 71 ATOM 1198 N LYS 160-31. 154-49. 791 42. 168 1. 000 15. 99 ATOM 1199 CA LYS 160-31. 361-50. 855 41. 176 1. 000 6. 75 ATOM 1200 CB LYS 160-32. 314-50. 369 40. 090 1. 000 10. 24 ATOM 1201 CG LYS 160-33. 666-49. 907 40. 607 1. 000 6. 13 ATOM 1202 CD LYS 160-34. 386-49. 041 39. 581 1. 000 11. 21

ATOM 1203 CE LYS 160-35. 897-49. 190 39. 702 1. 000 9. 55 ATOM 1204 NZ LYS 160-36. 616-48. 235 38. 811 1. 000 20. 37 ATOM 1205 C LYS 160-30. 029-51. 305 40. 591 1. 000 14. 32 ATOM 1206 0 LYS 160-29. 842-52. 475 40. 257 1. 000 14. 42 ATOM 1207 N THR 161-29. 082-50. 375 40. 465 1. 000 10. 29 ATOM 1208 CA THR 161-27. 771-50. 734 39. 933 1. 000 13. 43 ATOM 1209 CB THR 161-26. 878-49. 508 39. 672 1. 000 10. 03 ATOM 1210 OG1 THR 161-27. 070-48. 557 40. 730 1. 000 30. 01 ATOM 1211 CG2 THR 161-27. 263-48. 788 38. 389 1. 000 13. 57 ATOM 1212 C THR 161-27. 057-51. 683 40. 896 1. 000 12. 06 ATOM 1213 0 THR 161-26. 160-52. 415 40. 481 1. 000 6. 51 ATOM 1214 N THR 162-27. 457-51. 664 42. 165 1. 000 8. 39 ATOM 1215 CA THR 162-26. 894-52. 551 43. 177 1. 000 9. 75 ATOM 1216 CB THR 162-27. 286-52. 130 44. 604 1. 000 12. 96 ATOM 1217 OG1 THR 162-26. 705-50. 863 44. 941 1. 000 11. 98 ATOM 1218 CG2 THR 162-26. 735-53. 132 45. 605 1. 000 20. 35 ATOM 1219 C THR 162-27. 349-53. 991 42. 956 1. 000 10. 87 ATOM 1220 0 THR 162-26. 764-54. 942 43. 471 1. 000 12. 87 ATOM 1221 N GLU 163-28. 410-54. 170 42. 174 1. 000 16. 58 ATOM 1222 CA GLU 163-28. 949-55. 496 41. 905 1. 000 20. 69 ATOM 1223 CB GLU 163-30. 486-55. 450 41. 861 1. 000 21. 36 ATOM 1224 CG GLU 163-31. 136-54. 918 43. 122 1. 000 19. 81 ATOM 1225 CD GLU 163-30. 918-55. 799 44. 332 1. 000 20. 57 ATOM 1226 OE1 GLU 163-30. 336-56. 894 44. 181 1. 000 13. 38 ATOM 1227 OE2 GLU 163-31. 340-55. 394 45. 441 1. 000 37. 36 ATOM 1228 C GLU 163-28. 455-56. 101 40. 596 1. 000 12. 31 ATOM 1229 0 GLU 163-28. 614.-57. 306 40. 384 1. 000 8. 17 ATOM 1230 N LEU 164-27. 880-55. 296 39. 710 1. 000 14. 12 ATOM 1231 CA LEU 164-27. 561-55. 746 38. 356 1. 000 8. 92 ATOM 1232 CB LEU 164-26. 960-54. 602 37. 541 1. 000 5. 54 ATOM 1233 CG LEU 164-27. 903-53. 857 36. 593 1. 000 10. 39 ATOM 1234 CD1 LEU 164-29. 295-53. 740 37. 197 1. 000 23. 43 ATOM 1235 CD2 LEU 164-27. 352-52. 485 36. 240 1. 000 2. 48 ATOM 1236 C LEU 164-26. 621-56. 943 38. 361 1. 000 6. 54 ATOM 1237 0 LEU 164-26. 847-57. 925 37. 653 1. 000 4. 26 ATOM 1238 N ALA 165-25. 562-56. 865 39. 159 1. 000 7. 24 ATOM 1239 CA ALA 165-24. 609-57. 965 39. 239 1. 000 11. 41 ATOM 1240 CB ALA 165-23. 542-57. 659 40. 276 1. 000 11. 40 ATOM 1241 C ALA 165-25. 312-59. 284 39. 551 1. 000 16. 26 ATOM 1242 0 ALA 165-24. 980-60. 302 38. 947 1. 000 18. 13 ATOM 1243 N ARG 166-26. 266-59. 245 40. 469 1. 000 20. 04 ATOM 1244 CA ARG 166-27. 014-60. 397 40. 947 1. 000 10. 10 ATOM 1245 CB ARG 166-27. 875-59. 992 42. 145 1. 000 15. 40 ATOM 1246 CG ARG 166-28. 600-61. 127 42. 843 1. 000 15. 67 ATOM 1247 CD ARG 166-29. 286-60. 640 44. 115 1. 000 20. 34 ATOM 1248 NE ARG 166-30. 097-59. 453 43. 851 1. 000 31. 99 ATOM 1249 CZ ARG 166-31. 261-59. 505 43. 202 1. 000 37. 46

ATOM 1250 NH1 ARG 166-31. 718-60. 673 42. 770 1. 000 41. 26 ATOM 1251 NH2 ARG 166-31. 974-58. 410 42. 979 1. 000 44. 85 ATOM 1252 C ARG 166-27. 899-60. 991 39. 862 1. 000 10. 33 ATOM 1253 0 ARG 166-27. 862-62. 186 39. 569 1. 000 11. 28 ATOM 1254 N VAL 167-28. 724-60. 143 39. 253 1. 000 10. 14 ATOM 1255 CA VAL 167-29. 647-60. 637 38. 231 1. 000 8. 08 ATOM 1256 CB VAL 167-30. 800-59. 642 38. 007 1. 000 12. 63 ATOM 1257 CG1 VAL 167-31. 873-60. 262 37. 129 1. 000 23. 15 ATOM 1258 CG2 VAL 167-31. 423-59. 212 39. 331 1. 000 16. 49 ATOM 125, 9 C VAL 167-28. 941-60. 943 36. 916 1. 000 8. 93 ATOM 1260 0 VAL 167-29. 342-61. 889 36. 230 1. 000 11. 00 ATOM 1261 N TYR 168-27. 906-60. 209 36. 507 1. 000 6. 53 ATOM 1262 CA TYR 168-27. 225-60. 549 35. 262 1. 000 5. 82 ATOM 1263 CB TYR 168-26. 220-59. 494 34. 815 1. 000 12. 35 ATOM 1264 CG TYR 168-26. 746-58,. 249 34. 148 1. 000 10. 53 ATOM 1265 CD1 TYR 168-25. 898-57. 415 33. 429 1. 000 4. 25 ATOM 1266 CE1 TYR 168-26. 377-56. 273 32. 816 1. 000 3. 59 ATOM 1267 CD2 TYR 168-28. 085-57. 889 34. 230 1. 000 9. 22 ATOM 1268 CE2 TYR 168-28. 565-56. 750 33. 624 1. 000 11. 67 ATOM 1269 CZ TYR 168-27. 708-55. 940 32. 912 1. 000 8. 76 ATOM 1270 OH TYR 168-28. 194-54. 801 32. 308 1. 000 13. 56 ATOM 1271 C TYR 168-26. 466-61. 863 35. 444 1. 000 9. 45 ATOM 1272 0 TYR 168-26. 398-62. 696 34. 544 1. 000 5. 20 ATOM 1273 N SER 169-25. 896-61. 972 36. 648 1. 000 5. 94 ATOM 1274 CA SER 169-25. 145-63. 174 36. 999 1. 000 11. 65 ATOM 1275 CB SER 169-24. 663-63. 109 38. 445 1. 000 12. 52 ATOM 1276 OG SER 169-23. 611-64. 024 38. 688 1. 000 13. 86 ATOM 1277 C SER 169-26. 034-64. 389 36. 740 1. 000 14. 93 ATOM 1278 0 SER 169-25. 709-65. 240 35. 912 1. 000 25. 35 ATOM 1279 N ALA 170-27. 161-64. 434 37. 448 1. 000 9. 54 ATOM 1280 CA ALA 170-28. 154-65. 483 37. 259 1. 000 7. 33 ATOM 1281 CB ALA 170-29. 397-65. 155 38. 069 1. 000 3. 12 ATOM 1282 C ALA 170-28. 495-65. 659 35. 785 1. 000 12. 27 ATOM 1283 0 ALA 170-28. 526-66. 772 35. 262 1. 000 20. 56 ATOM 1284 N LEU 171-28. 753-64. 558 35. 081 1. 000 15. 11 ATOM 1285 CA LEU 171-29. 115-64. 661 33. 665 1. 000 17. 04 ATOM 1286 CB LEU 171-29. 329-63. 272 33. 076 1. 000 13. 64 ATOM 1287 CG LEU 171-29. 846-63. 164 31. 645 1. 000 21. 08 ATOM 1288 CD1 LEU 171-28. 692-63. 043 30. 658 1. 000 45. 18 ATOM 1289 CD2 LEU 171-30. 734-64. 340 31. 270 1. 000 17. 34 ATOM 1290 C LEU 171-28. 052-65. 404 32. 868 1. 000 18. 57 ATOM 1291 0 LEU 171-28. 328-66. 409 32. 219 1. 000 17. 64 ATOM 1292 N ALA 172-26. 825-64. 890 32. 920 1. 000 22. 46 ATOM 1293 CA ALA 172-25. 735-65. 489 32. 157 1. 000 17. 47 ATOM 1294 CB ALA 172-24. 454-64. 699 32. 377 1. 000 10. 29 ATOM 1295 C ALA 172-25. 549-66. 953 32. 536 1. 000 13. 15 ATOM 1296 0 ALA 172-25. 192-67. 797 31. 713 1. 000 17. 25

ATOM 1297 N SER 173-25. 802-67. 242 33. 809 1. 000 11. 55 ATOM 1298 CA SER 173-25. 653-68. 595 34. 337 1. 000 15. 80 ATOM 1299 CB SER 173-25. 837-68. 578 35. 856 1. 000 15. 14 ATOM 1300 OG SER, 173-26. 298.-69. 837 36. 293 1. 000 15. 66 ATOM 1301 C SER 173-26. 640-69. 565 33. 691 1. 000 10. 39 ATOM 1302 0 SER 173-26. 263-70. 667 33. 284 1. 000 5. 06 ATOM 1303 N PHE 174-27. 882-69. 119 33. 601 1. 000 6. 57 ATOM 1304 CA PHE 174-28. 970-69. 778 32. 908 1. 000 4. 04 ATOM 1305 CB PHE 174-30. 288-69. 024 33. 114 1. 000 4. 43 ATOM 1306 CG PHE 174-31. 524-69. 765 32. 626 1. 000 3. 57 ATOM 1307 CD1 PHE 174-32. 219-70. 606 33. 475 1. 000 0. 40 ATOM 1308 CD2 PHE 174-31. 988-69. 615 31. 331 1. 000 11. 71 ATOM 1309 CE1 PHE 174-33. 343-71. 281 33. 051 1. 000 1. 63 ATOM 1310 CE2 PHE 174-33. 114-70. 285 30. 886 1. 000 10. 57 ATOM 1311 CZ PHE 174-33. 795-71. 119 31. 756 1. 000 10. 59 ATOM 1312 C PHE 174-28. 701-69. 872 31. 408 1. 000 8. 80 ATOM 1313 0 PHE 174-28. 846-70. 949 30. 834 1. 000 0. 14 ATOM 1314 N MET 175-28. 328-68. 751 30. 793 1. 000 7. 91 ATOM 1315 CA. MET 175-28. 058-68. 739 29. 356 1. 000 5. 97 ATOM 1316 CB MET 175-28. 103-67. 321 28. 780 1. 000 0. 00 ATOM 1317 CG MET 175-29. 492-66. 712 28. 751 1. 000 7. 42 ATOM 1318 XD MET 175-29. 573-65. 056 28. 023 1. 000 16. 37 ATOM 1319 CE MET 175-30. 064-65. 488 26. 348 1. 000 21. 02 ATOM 1320 C MET. 175-26. 715-69. 399 29. 045 1. 000 6. 31 ATOM 1321 0 MET 175-26. 332-69. 479 27. 880 1. 000 8. 17 ATOM 1322 N LYS 176-26. 020-69. 872 30. 070 1. 000 8. 77 ATOM 1323 CA LYS 176-24. 762-70. 598 29. 939 1. 000 10. 68 ATOM 1324 CB LYS 176-24. 970-71. 945 29. 239 1. 000 10. 45 ATOM 1325 CG LYS 176-25. 907-72. 900 29. 971 1. 000 3. 74 ATOM 1326 CD LYS 176-25. 133-73. 755 30. 964 1. 000 5. 05 ATOM 1327 CE LYS 176-26. 084-74. 568 31. 833 1. 000 6. 09 ATOM 1328 NZ LYS 176-26. 739-73. 721 32. 861 1. 000 24. 38 ATOM 1329 C LYS 176-23. 733-69. 760 29. 190 1. 000 12. 34 ATOM 1330 0 LYS 176-23. 084-70. 178 28. 231 1. 000 24. 85 ATOM 1331 N VAL 177-23. 601-68. 520 29. 648 1. 000 12. 09 ATOM 1332 CA VAL 177-22. 709-67. 581 28. 953 1. 000 12. 10 ATOM 1333 CB VAL 177-23. 569-66. 629 28. 106 1. 000 9. 74 ATOM 1334 CG1 VAL 177-23. 831-65. 319 28. 835 1. 000 18. 59 ATOM 1335 CG2 VAL 177-22. 921-66. 372 26. 753 1. 000 20. 30 ATOM 1336 C VAL 177-21. 848-66. 876 29. 982 1. 000 13. 62 ATOM 1337 0 VAL 177-22. 292-66. 730 31. 126 1. 000 20. 25 ATOM 1338 N PRO 178-20. 635-66. 454 29. 637 1. 000 10. 56 ATOM 1339 CD PRO 178-20. 019-66. 530 28. 312 1. 000 2. 11 ATOM 1340 CA PRO 178-19. 760-65. 842 30. 642 1. 000 10. 32 ATOM 1341 CB PRO 178-18. 433-65. 656 29. 913 1. 000 6. 70 ATOM 1342 CG PRO 178-18. 623-66. 026 28. 499 1. 000 0. 81 ATOM 1343 C PRO 178-20. 281-64. 483 31. 119 1. 000 20. 65

ATOM 1344 0 PRO 178-20. 796-63. 674 30. 351 1. 000 22. 70 ATOM 1345 N PHE 179-20. 124-64. 253 32. 412 1. 000 22. 55 ATOM 1346 CA PHE 179-20. 474-63. 025 33. 107 1. 000 19. 13 ATOM 1347 CB PHE 179-21. 518-63. 283 34. 194 1. 000 8. 91 ATOM 1348 CG PHE 179-21. 661-62. 215 35. 268 1. 000 8. 12 ATOM 1349 CD1 PHE 179-22. 433-61. 087 35. 044 1. 000 10. 36 ATOM 1350 CD2 PHE 179-21. 031-62. 337 36. 499 1. 000 2. 04 ATOM 1351 CE1 PHE 179-22. 590-60. 103 36. 004 1. 000 2. 43 ATOM 1352 CE2 PHE 179-21. 183-61. 367 37. 470 1. 000 0. 76 ATOM 1353 CZ PHE 179-21. 963-60. 248 37. 228 1. 000 2. 96 ATOM 1354 C PHE 179-19. 231-62. 400 33. 736 1. 000 13. 74 ATOM 1355 0 PHE 179-18. 309-63. 110 34. 128 1. 000'15. 60 ATOM 1356 N PHE 180-19. 214-61. 080 33. 838 1. 000 14. 28 ATOM 1357 CA PHE 180-18. 178-60. 371 34. 573 1. 000 13. 03 ATOM 1358 CB PHE 180-17. 004-59. 952 33. 686 1. 000 17. 94 ATOM 1359 CG PHE 180-15. 933-59. 164 34. 433 1. 000 21. 76 ATOM 1360 CD1 PHE 180-14. 960-59. 807 35. 176 1. 000 21. 38 ATOM 1361 CD2 PHE 180-15. 904-57. 780 34. 391 1. 000 19. 62 ATOM 1362 CE1 PHE 180-13. 979-59. 108 35. 859 1. 000 15. 07 ATOM 1363 CE2 PHE 180-14. 941-57. 064 35. 075 1. 000 21. 73 ATOM 1364 CZ PHE 180-13. 979-57. 727 35. 816 1. 000 21. 65 ATOM 1365 C PHE 180-18. 822-59. 164 35. 256 1. 000 12. 16 ATOM 1366 0 PHE 180-19. 594-58. 423 34. 648 1. 000 11. 01 ATOM 1367 N ASP 181-18. 504-58. 988 36. 536 1. 000 7. 72 ATOM 1368 CA ASP 181-19. 062-57. 864 37. 286 1. 000 10. 61 ATOM 1369 CB ASP 181-19. 521-58. 346 38. 659 1. 000 5. 77 ATOM 1370 CG ASP 181-19. 986-57. 225 39. 559 1. 000 4. 11 ATOM 1371 OD1 ASP 181-20. 116-56. 076 39. 092 1. 000 8. 61 ATOM 1372 OD2 ASP 181-20. 217-57. 508 40. 750 1. 000 11. 49 ATOM 1373 C ASP 181-18. 037-56. 743 37. 378 1. 000 15. 44 ATOM 1374 0 ASP 181-17. 023-56. 872 38. 060 1. 000 16. 84 ATOM 1375 N ALA 182-18. 293-55. 639 36. 672 1. 000 18. 65 ATOM 1376 CA ALA 182-17. 359-54. 517 36. 678 1. 000 18. 00 ATOM 1377 CB ALA 182-17. 778-53. 459 35. 668 1. 000 7. 66 ATOM 1378 C ALA 182-17. 240-53. 911 38. 075 1. 000 18. 92 ATOM 1379 0 ALA 182-16. 198-53. 340 38. 400 1. 000 8. 61 ATOM 1380 N GLY 183-18. 296-54. 044 38. 872 1. 000 15. 67 ATOM 1381 CA GLY 183-18. 374-53. 516 40. 219 1. 000 13. 53 ATOM 1382 C GLY 183-17. 444-54. 230 41. 176 1. 000 14. 96 ATOM 1383 0 GLY 183-17. 268-53. 846 42. 330 1. 000 25. 31 ATOM 1384 N SER 184-16. 830-55. 306 40. 696 1. 000 16. 38 ATOM 1385 CA SER 184-15. 940-56. 105 41. 525 1. 000 12. 32 ATOM 1386 CB SER 184-16. 009-57. 574 41. 116 1. 000 14. 55 ATOM 1387 OG SER 184-15. 237-57. 867 39. 967 1. 000 12. 36 ATOM 1388 C SER 184-14. 516-55. 572 41. 439 1. 000 13. 33 ATOM 1389 0 SER 184-13. 644-55. 986 42. 204 1. 000 12. 05 ATOM 1390 N VAL 185-14. 276-54. 640 40. 515 1. 000 9. 89

ATOM 1391 CA VAL 185-12. 902-54. 156 40. 358 1. 000 14. 54 ATOM 1392 CB VAL 185-12. 320-54. 649 39. 021 1. 000 16. 34 ATOM 1393 CG1 VAL 185-12. 034-56. 141 39. 100 1. 000 13. 09 ATOM 1394 CG2 VAL 185-13. 274-54. 346 37. 877 1. 000 20. 34 ATOM 1395 C VAL 185-12. 802-52. 642 40. 445 1. 000 20. 13 ATOM 1396 0 VAL 185-11. 718-52. 101 40. 682 1. 000 11. 67 ATOM 1397 N ILE 186'-13. 912-51. 929 40. 260 1. 000 19. 83 ATOM 1398 CA ILE 186-13. 905-50. 479 40. 381 1. 000 13. 97 ATOM 1399 CB ILE 186-13. 716-49. 752 39. 031 1. 000 8. 30 ATOM 1400 CG2 ILE 186-12. 362-50. 070 38. 428 1. 000 12. 39 ATOM 1401 CG1 ILE 186-14. 830-50. 005 38. 014 1. 000 10. 45 ATOM 1402 CD1 ILE 186-14. 956-48. 929 36. 957 1. 000 3. 60 ATOM 1403 C ILE 186-15. 209-49. 957 40. 979 1. 000 13. 38 ATOM 1404 0 ILE 186-16. 256-50. 583 40. 857 1. 000 12. 90 ATOM 1405 N SER 187-15. 120-48. 788 41. 596 1. 000 11. 99 ATOM 1406 CA SER 187-16. 287-48. 046 42. 052 1. 000 9. 16 ATOM 1407 CB SER 187-16. 110-47. 594 43. 498 1. 000 10. 88 ATOM 1408 OG SER 187-14. 889-46. 879 43. 658 1. 000 16. 58 ATOM 1409 C SER 187-16. 517-46. 839 41. 145 1. 000 11. 87 ATOM 1410 0 SER 187-15. 567-46. 304 40. 563 1. 000 16. 73 ATOM 1411 N THR 188-17. 767-46. 410 41. 015 1. 000 15. 17 ATOM 1412 CA THR 188-18. 077-45. 244 40. 189 1. 000 13. 51 ATOM 1413 CB THR 188-19. 571-45. 151 39. 848 1. 000 12. 88 ATOM 1414 OG1 THR 188-19. 969-46. 308 39. 101 1. 000 16. 33 ATOM 1415 CG2 THR 188-19. 843-43. 943 38. 961 1. 000 8. 08 ATOM 1416 C THR 188-17. 639-43. 978 40. 916 1. 000 14. 09 ATOM 1417 0 THR 188-18. 293-43. 535 41. 860 1. 000 10. 72 ATOM 1418 N ASP 189-16. 518-43. 414 40. 474 1. 000 15. 51 ATOM 1419 CA ASP 189-15. 911-42. 313 41. 210 1. 000 11. 58 ATOM, 1420 CB ASP 1. 89-14. 407-42. 594 41. 362 1. 000 12. 86 ATOM 1421 CG ASP 189-14. 158-43. 791 42. 261 1. 000 4. 55 ATOM 1422 OD1 ASP 189-14. 915-43. 960 43. 239 1. 000 13. 27 ATOM 1423 OD2 ASP 189-13. 208-44. 549 41. 989 1. 000 6. 91 ATOM 1424 C ASP 189-16. 120-40. 949 40. 567 1. 000 15. 34 ATOM 1425 0 ASP 189-15. 910-39. 948 41. 263 1. 000 18. 48 ATOM 1426 N GLY 190-16. 510-40. 918 39. 303 1. 000 19. 39 ATOM 1427 CA GLY 190-16. 710-39. 718 38. 515 1. 000 15. 08 ATOM 1428 C GLY 190-17. 385-38. 613 39. 303 1. 000 18. 57 ATOM 1429 0 GLY 190-18. 263-38. 908 40. 119 1. 000 20. 64 ATOM 1430 N VAL 191-16. 952-37. 381 39. 057 1. 000 13. 86 ATOM 1431 CA VAL 191-17. 428-36. 226 39. 806 1. 000 10. 59 ATOM 1432 CB VAL 191-16. 825-34. 905 39. 286 1. 000 17. 05 ATOM 1433 CG1 VAL 191-15. 324-34. 875 39. 559 1. 000 30. 84 ATOM 1434 CG2 VAL 191-17. 092-34. 701 37. 803 1. 000 8. 10 ATOM 1435 C VAL 191-18. 950-36. 129 39. 774 1. 000 10. 47 ATOM 1436 0 VAL 191-19. 542-35. 686 40. 761 1. 000 13. 60 ATOM 1437 N ASP 192-19. 571-36. 534 38. 668 1. 000 1. 46

ATOM 1438 CA ASP 192-21. 018-36. 447 38. 540 1. 000 0. 70 ATOM 1439 CB ASP 192-21. 387-36. 356 37. 056 1. 000 2. 10 ATOM 1440 CG ASP 192-20. 918-37. 566 36. 268 1. 000 9. 82 ATOM 1441 OD1 ASP 192-20. 296-38. 478 36. 857 1. 000 8. 20 ATOM 1442 OD2 ASP 192-21. 182-37. 597 35. 047 1. 000 6. 78 ATOM 1443 C ASP 192-21. 754-37. 622 39. 173 1. 000 7. 73 ATOM 1444 0 ASP 192-22. 988-37. 674 39. 136 1. 000 7. 10 ATOM 1445 N GLY 193-21. 027-38. 572 39. 753 1. 000 15. 10 ATOM 1446 CA GLY 193-21. 631-39. 747 40. 351 1. 000 17. 83 ATOM 1447 C GLY 193-22. 153-40. 758 39. 352 1. 000 18. 93 ATOM 1448 0 GLY 193-22. 820-41. 732 39. 718 1. 000 10. 12 ATOM 1449 N ILE 194-21. 867-40. 565 38. 062 1. 000 11. 77 ATOM 1450 CA ILE 194-22. 330-41. 546 37. 081 1. 000 7. 87 ATOM 1451 CB ILE 194-23. 401-40. 945 36. 154 1. 000 9. 95 ATOM 1452 CG2 ILE 194-23. 790-41. 927 35. 063 1. 000 0. 00 ATOM 1453 CG1 ILE 194-24. 643-40. 441 36. 896 1. 000 9. 90 ATOM 1454 CD1 ILE 194-25. 248-39. 237 36. 206 1. 000 8. 85 ATOM 1455 C ILE 194-21. 191-42. 068 36. 225 1. 000 2. 97 ATOM 1456 0 ILE 194-21. 086-43. 251 35. 924 1. 000 6. 72 ATOM 1457 N HIS 195-20. 277.-41. 195 35. 792 1. 000 6. 33 ATOM 1458 CA HIS 195-19. 256-41. 719 34. 884 1. 000 10. 76 ATOM 1459 CB HIS 195-19. 089-40. 790 33. 673 1. 000 11. 36 ATOM 1460 CG HIS 195-20. 402-40. 647 32. 958 1. 000 11. 50 ATOM 1461 CD2 HIS 195-20. 981-41. 395 31. 989 1. 000 5. 43 ATOM 1462 ND1 HIS 195-21. 283-39. 633 33. 253 1. 000 7. 30 ATOM 1463 CE1 HIS 195-22. 351-39. 753 32. 485 1. 000 9. 11 ATOM 1464 NE2 HIS 195-22. 192-40. 814 31. 711 1. 000 8. 18 ATOM 1465 C HIS 195-17. 918-41. 941 35. 577 1. 000 8. 63 ATOM 1466 0 HIS 195-17. 762-41. 602 36. 743 1. 000 13. 71 ATOM 1467 N PHE 196-17. 010-42. 529 34. 812 1. 000 6. 37 ATOM 1468 CA PHE 196-15. 725-43. 017 35. 249 1. 000 9. 06 ATOM 1469 CB PHE 196-15. 233-44. 136 34. 320 1. 000 5. 38 ATOM 1470 CG PHE 196-16. 048-45. 412 34. 451 1. 000 10. 20 ATOM 1471 CD1 PHE 196-15. 822-46. 481 33. 602 1. 000 8. 01 ATOM 1472 CD2 PHE 196-17. 027-45. 509 35. 427 1. 000 6. 21 ATOM 1473 CE1 PHE 196-16. 571-47. 637 33. 722 1. 000 11. 17 ATOM 1474 CE2 PHE 196-17. 779-46. 662 35. 546 1. 000 14. 06 ATOM 1475 CZ PHE 196-17. 549-47. 727 34. 694 1. 000 13. 03 ATOM 1476 C PHE 196-14. 663-41. 925 35. 273 1. 000 12. 92 ATOM 1477 0 PHE 196-14. 757-40. 983 34. 494 1. 000 15. 16 ATOM 1478 N THR 197-13. 694-42. 112 36. 158 1. 000 13. 17 ATOM 1479 CA THR 197-12. 477-41. 318 36. 183 1. 000 17. 95 ATOM 1480 CB THR 197-11. 886-41. 168 37. 593 1. 000 20. 94 ATOM 1481 OG1 THR 197-11. 650-42. 458 38. 173 1. 000 20. 14 ATOM 1482 CG2 THR 197-12. 882-40. 454 38. 499 1. 000 31. 55 ATOM 1483 C THR 197-11. 443-41. 978 35. 269 1. 000 10. 26 ATOM 1484 0 THR 197-11. 713-43. 037 34. 705 1. 000 14. 53

ATOM 1485 N GLU 198-10. 283-41. 362 35. 133 1. 000 9. 05 ATOM 1486 CA GLU 198-9. 192-41. 943 34. 362 1. 000 12. 89 ATOM 1487 CB GLU 198-8. 023-40. 960 34. 314 1. 000 20. 40 ATOM 1488 CG GLU 198-6. 903-41. 349 33. 362 1. 000 32. 30 ATOM 1489 CD GLU 198-5. 764-40. 346 33. 328 1. 000 35. 77 ATOM 1490 OE1 GLU 198-5. 127-40. 141 34. 385 1. 000 42. 59 ATOM 1491 OE2 GLU 198-5. 498-39. 761 32. 256 1. 000 25. 40 ATOM 1492 C GLU 198-8. 779-43. 279 34. 970 1. 000 16. 23 ATOM 1493 0 GLU 198-8. 636-44. 296 34. 292 1. 000 14. 85 ATOM 1494 N ALA 199-8. 596-43. 284 36. 291 1. 000 11. 36 ATOM 1495 CA ALA 199-8. 233-44. 489 37. 022 1. 000 5. 99 ATOM 1496 CB ALA 199-8. 047-44. 154 38. 499 1. 000 2. 34 ATOM 1497 C ALA 199-9. 273-45. 594 36. 373 1. 000 7. 89 ATOM 1498 0 ALA 199-8. 922-46. 767 36. 748 1. 000 16. 70 ATOM 1499 N ASN 200-10. 548-45. 210 36. 897 1. 000 13. 48 ATOM 1500 CA ASN 200-11. 644-46. 155 36. 715 1. 000 11. 59 ATOM 1501 CB ASN 200-13. 007-45. 474 36. 805 1. 000 4. 12 ATOM 1502 CG ASN 200-13. 492-45. 192 38. 209 1. 000 11. 67 ATOM 1503 OD1 ASN 200-13. 045-45. 767 39. 200'1. 000 6. 19 ATOM 1504 ND2 ASN 200-14. 455-44. 276 38. 330 1. 000 13. 74 ATOM 1505 C ASN 200-11. 505-46. 869 35. 366 1. 000 8. 88 ATOM 1506 0 ASN 200-11. 667-48. 084 35. 305 1. 000 9. 08 ATOM 1507 N ASN 201-11. 208-46. 111 34. 315 1. 000 14. 48 ATOM 1508 CA ASN 201-11. 074-46. 639 32. 963 1. 000 14. 27 ATOM 1509 CB ASN 201-10. 903-45. 495 31. 960 1. 000 16. 17 ATOM 1510 CG ASN 201-12. 221-44. 853 31. 570 1. 000 14. 25 ATOM 1511 OD1 ASN 201-13. 050-45. 436 30. 871 1. 000 13. 77 ATOM 1512 ND2 ASN 201-12. 441-43. 624 32. 021 1. 000 16. 01 ATOM 1513 C ASN 201-9. 908-47. 620 32. 870 1. 000 12. 95 ATOM 1514 0 ASN 201-10. 050-48. 720 32. 334 1. 000 11. 02 ATOM 1515 N ARG 202-8. 775-47. 207 33. 412 1. 000 15. 80 ATOM 1516 CA ARG'202-7. 571-48. 020 33. 532 1. 000 14. 85 ATOM 1517 CB ARG 202-6. 491-47. 250 34. 294 1. 000 17. 85 ATOM 1518 CG ARG 202-5. 109-47. 874 34. 325 1. 000 17. 66 ATOM 1519 CD ARG 202-4. 141-47. 026 35. 143 1. 000 19. 69 ATOM 1520 NE ARG 202-3. 646-45. 881 34. 388 1. 000 30. 64 ATOM 1521 CZ ARG 202-2. 410-45. 407 34. 412 1. 000 36. 54 ATOM 1522 NH1 ARG 202-1. 470-45. 972 35. 164 1. 000 35. 38 ATOM 1523 NH2 ARG 202-2. 093-44. 353 33. 669 1. 000 23. 31 ATOM 1524 C ARG 202-7. 862-49. 344 34. 229 1. 000 6. 52 ATOM 1525 0 ARG 202-7. 636-50. 401 33. 644 1. 000 9. 98 ATOM 1526 N ASP 203-8. 365-49. 285 35. 464 1. 000 3. 83 ATOM 1527 CA ASP 203-8. 597-50. 500 36. 237 1. 000 12. 72 ATOM 1528 CB ASP 203-9. 148-50. 181 37. 631 1. 000 9. 96 ATOM 1529 CG ASP 203-8. 170-49. 370 38. 458 1. 000 16. 04 ATOM 1530 OD1 ASP 203-6. 980-49. 324 38. 086 1. 000 18. 66 ATOM 1531 OD2 ASP 203-8. 584-48. 772 39. 474 1. 000 22. 09

ATOM 1532 C ASP 203-9. 548-51. 455 35. 524 1. 000 18. 07 ATOM 1533 0 ASP 203-9. 383-52. 674 35. 579 1. 000 12. 38 ATOM 1534 N LEU 204-10. 550-50. 890 34. 859 1. 000 23. 73 ATOM 1535 CA LEU 204-11. 541-51. 706 34. 169 1. 000 21. 34 ATOM 1536 CB LEU 204-12. 745-50. 872 33. 727 1. 000 26. 39 ATOM 1537 CG LEU 204-14. 123-51. 510 33. 908 1. 000 26. 92 ATOM 1538 CD1 LEU 204-15. 079-51. 066 32. 809 1. 000 10. 26 ATOM 1539 CD2 LEU 204-14. 019-53. 027 33. 942 1. 000 35. 07 ATOM 1540 C LEU 204-10. 938-52. 392 32. 948 1. 000 10. 84 ATOM 1541 0 LEU 204-11. 212-53. 567 32. 707 1. 000 16. 23 ATOM 1542 N GLY 205-10. 143-51. 649 32. 189 1. 000 8. 26 ATOM 1543 CA GLY 205-9. 534-52. 173 30. 984 1. 000 6. 27 ATOM 1544 C GLY 205-8. 472-53. 215 31. 265 1. 000 8. 34 ATOM 1545 0 GLY 205-8. 228-54. 094 30. 436 1. 000 9. 21 ATOM 1546 N VAL 206-7. 829-53. 130 32. 425 1. 000 8. 74 ATOM 1547 CA VAL 206-6. 833-54. 135 32. 796 1. 000 9. 33 ATOM 1548 CB VAL 206-5. 942-53. 653 33. 957 1. 000 16. 14 ATOM 1549 CG1 VAL 206.-5. 020-54. 754 34. 457 1. 000 6. 58 ATOM 1550 CG2 VAL 206-5. 124-52. 445 33. 514 1. 000 6. 33 ATOM 1551 C VAL 206-7. 526-55. 447 33. 154 1. 000 5. 34 ATOM 1552 0 VAL 206-7. 118-56. 498 32. 664 1. 000 5. 68 ATOM 1553 N ALA 207-8. 564-55. 384 33. 982 1. 000 4. 56 ATOM 1554 CA ALA 207-9. 349-56. 547 34. 369 1. 000 8. 39 ATOM 1555 CB ALA 207-10. 323-56. 180 35. 490 1. 000 0. 79 ATOM 1556 C-ALA 207-10. 144-57. 160 33. 219 1. 000 10. 03 ATOM 1557 0 ALA 207-10. 485-58. 346 33. 261 1. 000 13. 69 ATOM 1558 N LEU 208-10. 471-56. 382 32. 193 1. 000 14. 72 ATOM 1559 CA LEU 208-11. 278-56. 888 31. 082 1. 000 11. 49 ATOM 1560 CB LEU 208-12. 065-55. 755 30. 422 1. 000 12. 04 ATOM 1561 CG LEU 208-13. 325-55. 317 31. 175 1. 000 10. 97 ATOM 1562 CD1 LEU 208-13. 985-54. 127 30. 497 1. 000 18. 17 ATOM 1563 CD2 LEU 208-14. 302-56. 477 31. 290 1. 000 17. 03 ATOM 1564 C LEU 208-10. 391-57. 604 30. 067 1. 000 6. 10 ATOM 1565 0 LEU 208-10. 857-58. 502 29. 369 1. 000 15. 12 ATOM 1566 N ALA 209-9. 132-57. 191 30. 019 1. 000 10. 78 ATOM 1567 CA ALA 209-8. 103-57. 815 29. 203 1. 000 16. 00 ATOM 1568 CB ALA 209-6. 827-56. 992 29. 220 1. 000 18. 55 ATOM 1569 C ALA 209-7. 829-59. 238 29. 694 1. 000 19. 15 ATOM 1570 0 ALA 209-7. 639-60. 143 28. 882 1. 000 13. 89 ATOM 1571 N GLU 210-7. 822-59. 396 31. 015 1. 000 9. 97 ATOM 1572 CA GLU 210-7. 645-60. 692 31. 653 1. 000 11. 15 ATOM 1573 CB GLU 210-7. 535-60. 520 33. 168 1. 000'21. 07 ATOM 1574 CG GLU 210-6. 097-60. 365 33. 647 1. 000 39. 63 ATOM 1575 CD GLU 210-5. 696-58. 921 33. 860 1. 000 47. 94 ATOM 1576 OE1 GLU 210-5. 958-58. 391 34. 960 1. 000 64. 71 ATOM 1577 OE2 GLU 210-5. 097-58. 319 32. 949 1. 000 43. 70 ATOM 1578 C GLU 210-8. 791-61. 634 31. 308 1. 000 10. 80

ATOM 1579 0 GLU 210-8. 589-62. 787 30. 927 1. 000 10. 93 ATOM 1580 N GLN 211-10. 007-61. 120 31. 441 1. 000 10. 29 ATOM 1581 CA GLN 211-11. 190-61. 871 31. 035 1. 000 17. 12 ATOM 1582 CB GLN 211-12. 443-61. 052 31. 363 1. 000 15. 73 ATOM 1583 CG GLN 211-12. 542-60. 709 32. 844 1. 000 19. 97 ATOM 1584 CD GLN 211-12. 936-61. 923 33. 671 1. 000 20. 12 ATOM 1585 OE1 GLN 211-13. 886-62. 628 33. 331 1. 000 17. 44 ATOM 1586 NE2 GLN 211-12. 218-62. 166 34. 759 1. 000 12. 84 ATOM 1587 C GLN 211-11. 146-62. 237 29. 556 1. 000 19. 66 ATOM 1588 0 GLN 211-11. 399-63. 384 29. 170 1. 000 12. 73 ATOM 1589 N VAL 212-10. 822-61. 287 28. 679 1. 000 17. 48 ATOM 1590 CA VAL 212-10. 785-61. 612 27. 249 1. 000 19. 02 ATOM 1591 CB VAL 212-10. 426-60. 369 26. 415 1. 000 14. 47 ATOM 1592 CG1 VAL 212-10. 189-60. 744 24. 958 1. 000 15. 00 ATOM 1593 CG2 VAL 212-11. 527-59. 320 26. 523 1. 000 8. 88 ATOM 1594 C VAL 212-9. 816-62. 745 26. 936 1. 000 23. 29 ATOM 1595 0 VAL 212-10. 192-63. 735 26. 294 1. 000 25. 62 ATOM 1596 N ARG 213-8. 557-62. 645 27. 361 1. 000 21. 16 ATOM 1597 CA ARG 213-7. 617-63. 740 27. 126 1. 000 22. 08 ATOM 1598 CB ARG 213-6. 251-63. 462 27. 752 1. 000 19. 45 ATOM 1599 CG ARG 213-5. 577-62. 178 27. 300 1. 000 20. 41 ATOM 1600 CD ARG 213-4. 621-61. 690 28. 380 1. 000 26. 40 ATOM 1601 NE ARG 213-3. 847-60. 527 27. 952 1. 000 29. 86 ATOM 1602 CZ ARG 213-3. 556-59. 504 28. 745 1. 000 26. 00 ATOM 1603 NH1 ARG 213-3. 968-59. 485 30. 007 1. 000 15. 34 ATOM 1604 NH2 ARG 213-2. 847-58. 491 28. 268 1. 000 17. 74 ATOM 1605 C ARG 213-8. 157-65. 052 27. 695 1. 000 21. 76 ATOM 1606 0 ARG 213-7. 893-66. 138 27. 182 1. 000 28. 34 ATOM 1607 N SER 214-8. 924-64. 952 28. 780 1. 000 15. 76 ATOM 1608 CA SER 214-9. 486-66. 151 29. 389 1. 000 15. 09 ATOM 1609 CB SER 214-10. 043-65. 824 30. 781 1. 000 19. 35 ATOM 1610 OG SER 214-11. 053-66. 745 31. 144 1. 000 46. 77 ATOM 1611 C SER 214-10. 561-66. 790 28. 529 1. 000 15. 48 ATOM 1612 0 SER 214-10. 692-68. 016 28. 535 1. 000 24. 87 ATOM 1613 N LEU 215-11. 355-66. 030 27. 772 1. 000 21. 40 ATOM 1614 CA LEU 215-12. 367-66. 673 26. 938 1. 000 21. 52 ATOM 1615 CB LEU 215-13.. 655-65. 855 26. 860 1. 000 22. 40 ATOM 1616 CG LEU 215-14. 176-65. 153 28. 103 1. 000 20. 48 ATOM 1617 CD1 LEU 215-15. 071-63. 990 27. 697 1. 000 27. 15 ATOM 1618 CD2 LEU 215-14. 931-66. 118 29. 006 1. 000 13. 10 ATOM 1619 C LEU 215-11. 884-66. 920 25. 510 1. 000 20. 60 ATOM 1620 0 LEU 215-12. 536-67. 682 24. 789 1. 000 31. 41 ATOM 1621 N LEU 216-10. 790-66. 303 25. 077 1. 000 21. 43 ATOM 1622 CA LEU 216-10. 291-66. 503 23. 718 1. 000 19. 55 ATOM 1623 CB LEU 216-10. 114-65. 148 23. 021 1. 000 19. 47 ATOM 1624 CG LEU 216-11. 385-64. 305 22. 870 1. 000 16. 11 ATOM 1625 CD1 LEU 216-11. 095-63. 042 22. 076 1. 000 17. 60

ATOM 1626 CD2 LEU 216-12. 495-65. 108 22. 211 1. 000 4. 00 ATOM 1627 C LEU 216-8. 983-67. 283 23. 688 1. 000 24. 37 ATOM 1628 OT1 LEU 216-8. 472-67. 525 22. 571 1. 000 29. 22 ATOM 1629 OT2 LEU 216-8. 463-67. 655 24. 758 1. 000 19. 02 In addition to the above-described determinations, a carbamate-inhibited perhydrolase crystal was also produced and analyzed. In these experiments, a N- hexylcarbamate derivative of wild type perhydrolase was used. Wild-type perhydrolase (14.5 mg in 1 mL, 67mM NaP04 pH 7 buffer) was titrated at room temperature with 1.25 gL aliquots of 400 mM p-nitrophenyl-N-hexylcarbamate dissolved in DMSO.

Perhydrolase. activity was measured withp-nitrophenylbutyrate assay (See, Example 2), as a function of time after each addition of the inhibitor. Several additions over several hours were required for complete inhibition of the enzyme. After inhibition was complete, the buffer of the inhibited enzyme solution was exchanged for 10 mM HEPES pH 8.3. This solution was stored at-80'C until used for crystallization screening experiments were conducted as described above. The inhibitorp-nitrophenyl-N- hexylcarbamate was prepared by methods known in the art (See e. g., Hosie et al., J. Biol.

Chem. , 262: 260-264 [1987] ). Briefly, the carbamate-inhibited perhydrolase was crystallized by vapor diffusion using the hanging drop method known in the art. A ml solution of inhibited perhydrolase (15 mg/ml in 10 mM HEPES, pH 8. 2), was mixed with 4 gL of a reservoir solution (30% PEG-4,000 with 0.2 M lithium sulfate and 0.1 M Tris, pH 8.5) on a plastic coverslip, then inverted and sealed for a well of 6x4 Linbro plate containing 0.5 ml of the reservoir solution and allowed to equilibrate. Crystals formed within a few days. The crystals were flash frozen in liquid nitrogen and analyzed as described above.

While the native octamer was determined in space group P4 with unit cell dimensions: a= 98. 184 b= 98. 184 and c= 230. 119 a=90. 00 ß=90. 00 7=90. 00, this crystal diffracted

to about 2.0 A. The carbamate-inhibited crystal grew in the space group PI with unit cell dimensions a=67. 754, b=80.096, and c=85. 974 a=104. 10°, p=l 12. 10°, andy=97. 40° and these crystals diffract to a resolution exceeding 1.0 A.

The carbamate was bound in a manner to exploit the interactions between the keto oxygen of the carbamate and residues forming the oxyanion hole, the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobic side chain extends along the hydrophobic surface of the binding site out into the surface opening between pairs of dimers in the octamer structure. The carbamate moiety direction highlights the pivotal role of the S54V mutation. The hydrophobic moiety passes adjacent to the side chain of ser 54. Mutating the serine side to valine increased the hydrophobicity, and also served as a gatekeeper to prevent hydrophilic nucleophiles (e. g. , water) for competing with desired deacylating nucleophiles. The t residues surrounding the carbamate moiety on the same and neighboring molecules forming the extended entry are expected to influence the selection of the optimal de-acylating nucleophile.

In addition, residues with surface-accessible side chain atoms were identified using the program"AreaMol,"within the CCP4 program package. Table 15-1 lists these residues. In this Table, the residue number, residue name, number of surface-accessible side chain atoms having at least 10.0 square atoms of accessible surface area, and maximum surface area (square angstroms) for any side chain atom within that residue (or CA for GLY residues) in the octameric structure of perhydrolase are provided.

Table 15-1. Surface-Accessible Side Chain Atoms Residue Residue Number of Accessible Maximum Surface Number Name Side Chain Atoms Area (Square Angstroms) 1 ALA 1 15. 7 3 LYS'2 54. 10 17 VAL 1 29. 5 19 VAL 1 28. 0 20 GLU 4 30. 2 21 ASP 2 41. 3 24 PRO 2 23. 2 26 GLU 3 36. 3 29 ALA 1 34. 4 30 PRP 3 32. 31 ASP 3 50. 6 32 VAL 1 27. 0 39 ALA 1 27. 5 40 GLN 3 38. 7 41 GLN 2 22 1 43 GLY 1 20. 4 44 ALA 1 63. 8 45 ASP 3 52. 7 46 PHE 2 17. 1 47 GLU329. 6 61 ASP 3 S. 1 63 PRO 3 _ 28. 0 64 THR 1 15. 7 65 ASP 1 10 8 66 PRO 3 33. 5 67 ARG 2 20 3 69 ASN 1 11 0 72 SER 2 26 75 PRO 2 17. 4 83 PRO 2 15. 1 85 ASP 1 36. 80 98 ALA 1 14. 60 101 ARG 4 25. 0 102 ARG 1 19. 9 103 THR 1 43. 7 104 PRO 1 17. 90 105 LEU 1 10. 1 113 VAL 1 17. 3 116 THR 2 39. 5 117 GLN 2 15. 3 119 LEU 3 21. 4 120 THR 2 34. 1 122 ALA 1 38. 0 123 GLY 1 11. 0 126 GLY 1 11. 9 128 THR 2 18. 2 129 TYR 1 17. 6 130 PRO 3 30. 2 131 ALA 1 13. 7 133 LYS 3 46. 9 141 PRO 3 25. 3 143 ALA 1 19. 8 144 PRO 3 34. 90 146 PRO 2 24. 30 148 PRO 3 24. 1 151 GLN 3 35. 6 152 LEU112. 90 155 GLU 3 53. 0 156 GLY 1 28. 9 158 GLU 3 30. 3 159 N 4. 9 160 LYS 2 21. 5 162 THR 2 25. 0 163 GLU 2 23. 3 165 ALA 1 23. 1 169 SER 1 39. 1 173 SER 2 33. 3 174 PHE 1 11. 1 175 MET 1 18. 5 176 LYS 2 21. 4 178 PRO 1 12. 0 179 PHE 2 14. 0 180 PHE 1 13. 9 181 ASP 1 24. 9 184 SER 1 27. 0 185 VAL 1 27. 5 187 SER 2 34. 0 189 ASP 2 25. 4 191 VAL 2 24. 5 197 THR 2 21. 6 198 GLU 3 43. 5 199 ALA 1 50. 5 202 ARG 3 37. 2 203 ASP 2 30. 9 206 VAL 2 45. 2 210 GLU 3 34. 6 211 GLN 2 19. 6 213 ARG 5 30. 8 214 SER 2 20. 8 215 LEU 1 25. 80

EXAMPLE 16 Stain Removal In this Example, experiments conducted to assess the stain removal abilities of perhydrolase are described.

Individual wells of 24 well culture plates were used to mimic conditions found in ordinary washing machines. Each well was filled with commercially available detergent (e. g., Ariel [Procter & Gamble], WOB [AATCC], and WFK [WFK] ), and pre-stained cloth discs cut to fit inside of each well were added., Temperature and agitation were accomplished by attaching the plate to the inside of a common laboratory incubator/shaker. To measure bleaching effectiveness of the perhydrolase, fabric stained with tea (EMPA # 167, available commercially from Test Fabrics) was used. A single cloth disc was placed in each well, and 1 ml of detergent liquid, containing enzyme, ester substrate, and peroxide was added. After agitation at 100-300 rpm @ 20-60°C, the fabric discs were removed, rinsed with tap water, and allowed to dry overnight. The reflectance of each individual cloth disc was measured, and plotted as an"L"value.

These results are provided in Figure 21, which shows that the addition of the perhydrolase of the present invention to the detergent consistently provides a greater degree of bleaching than the detergents alone. In this Figure, "E"indicates the results for each of the detergents tested in combination with the perhydrolase of the present invention.

EXAMPLE 17 Cotton Bleaching In this Example, experiments to assess the use of the perhydrolase of the present invention for bleaching of cotton fabrics are described.

In these experiments, six cotton swatches per canister were treated at 55°C for 60 minutes in a Launder-O-meter. The substrates used in these experiments were: 3 (3"x3") 428U and 3 (3"x3") 400U per experiments. Two different types of 100% unbleached cotton fabrics from Testfabrics were tested (style 428U (desized but not bleached army carded cotton sateen); and style 400U (desized but not bleached cotton print cloth). The liquor ratio was about 26 to 1 (-7. 7 g fabric/-200 ml volume liquor). The perhydrolase enzyme was tested at 12.7 mgP/ml, with. ethyl acetate (3 % (v/v) ), hydrogen peroxide ( 1500 ppm), and Triton X-100 (0. 001%), in a sodium phosphate buffer (100 mM) for pH 7 and pH 8; as well as in a sodium carbonate (100 mM) buffer, for pH 9 and pH 10.

Bleaching effects were quantified with total color difference by taking 4 CE L*a*b* values per each swatch before and after the treatments using a Chroma Meter CR-200 (Minolta), and total color difference of the swatches after the treatments were calculated according to the following: Total color difference (hE) = j (22 + Aa2+ Ab2)

(where AL, Aa, Ab, are differences in CE L*, CIE a*, and CIE b* values respectively before and after the treatments).

Higher AE values indicate greater bleaching effects. The results (See, Figure 22) indicated that the perhydrolase showed significantly improved bleaching effects on both types of 100% cotton fabrics at pH 7 and pH 8 under the conditions tested.

It was also observed that high amounts of motes (e. g., pigmented spots) disappeared on the enzyme treated substrates.

EXAMPLE 18 Linen Bleaching In this Example, experiments conducted to assess the linen bleaching capability of the perhydrolase of the present invention are described. The same methods and conditions as describe above for cotton testing (in Example 17) were used to test linen swatches. As indicated above, experiments were conduction in a Launder-O-meter using a linen fabric (linen suiting, Style L-53; Testfabrics).

In these experiments, 3 (4"x4") linen swatches were treated with 12.7 mgP/ml of the perhydrolase enzyme with ethyl acetate (3 % v/v), hydrogen peroxide (1200 ppm), and Triton X-100 (0. 001%), in a sodium phosphate buffer (100 mM) for pH 7 and pH 8.

The bleaching effects were calculated as described above in Example 17. Figure 23 provides a graph showing the bleaching effects of the perhydrolase of the present invention tested at pH 7 and pH 8 on linen.

EXAMPLE 19 Detergent Compositions In the following Example, various detergent compositions are exemplified. In

these formulations, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions. The abbreviated component identifications therein have the following meanings : LAS Sodium linear C 11-13 alkyl benzene sulfonate.

TAS Sodium tallow alkyl sulfate.

CxyAS Sodium ClX-Cly alkyl sulfate.

CxyEz : ClX-Cly predominantly linear primary alcohol condensed with an average of z moles of ethylene oxide.

CxyAEzS : ClX-Cly sodium alkyl sulfate condensed with an average of z moles of ethylene oxide. Added molecule name in the examples.

Nonionic Mixed ethoxylatedlpropoxylated fatty alcohol e. g. Plurafac LF404 being an alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5.

QAS R2. N+ (CH3) 2 (C2H40H) with R2 = C12-C14.

Silicate Amorphous Sodium Silicate (SiO2:Na2O ratio = 1. 6-3.2 : 1).

Metasilicate Sodium metasilicate (Si02 : Na2O ratio = 1.0).

Zeolite A Hydrated Aluminosilicate of formula Nal2 (A102SiO2) 12. 27H20 SKS-6 Crystalline layered silicate of formula 8-Na2Si205.

Sulphate Anhydrous sodium sulphate.

STPP Sodium Tripolyphosphate.

MA/AA Random copolymer of 4: 1 acrylate/maleate, average molecular weight about 70,000-80, 000.

AA Sodium polyacrylate polymer of average molecular weight 4,500.

Polycarboxylate Copolymer comprising mixture of carboxylated monomers such as acrylate, maleate and methyacrylate with a MW ranging between 2, 000-80, 000 such as Sokolan commercially available from BASF, being a copolymer of acrylic acid, MW4,500.

BB1 3- (3, 4-Dihydroisoquinolinium) propane sulfonate BB2 1- (3, 4-dihydroisoquinolinium) -decane-2-sulfate PB 1 Sodium perborate monohydrate.

PB4 Sodium perborate tetrahydrate of nominal formula NaB03. 4H20.

Percarbonate Sodium percarbonate of nominal formula 2Na2C03. 3H202.

TAED Tetraacetyl ethylene diamine.

NOBS Nonanoyloxybenzene sulfonate in the form of the sodium salt.

DTPA Diethylene triamine pentaacetic acid.

HEDP : l, l-hydroxyethane diphosphonic acid.

DETPMP : Diethyltriamine penta (methylene) phosphonate, marketed by Monsanto under the Trade name Dequest 2060.

EDDS : Ethylenediamine-N, N'-disuccinic acid, (S, S) isomer in the form of its sodium salt Diamine : Dimethyl aminopropyl amine ; 1, 6-hezane diamine ; 1, 3-propane diamine; 2-methyl-1, 5-pentane diamine ; 1,3-pentanediamine ; 1- methyl-diaminopropane.

DETBCHD 5, 12-diethyl-1, 5,8, 12-tetraazabicyclo [6,6, 2] hexadecane, dichloride, Mn (ID salt PAAC Pentaamine acetate cobalt (III) salt.

Paraffin Paraffin oil sold under the tradename Winog 70 by Wintershall.

Paraffin Sulfonate A Paraffm oil or wax in which some of the hydrogen atoms have been replaced by sulfonate groups.

Aldose oxidase Oxidase enzyme sold under the tradename Aldose Oxidase by Novozymes A/S Galactose oxidase Galactose oxidase from Sigma Protease Proteolytic enzyme sold under the tradename Savinase, Alcalase, Everlase by Novo Nordisk A/S, and the following from Genencor International, Inc : "Protease A"described in US RE 34,606 in Figures 1A, 1B, and 7, and at column 11, lines 11-37;"Protease B" described in US5,955, 340 and US5,700, 676 in Figures 1A, 1B and 5, as well as Table 1; and"Protease C"described in US6, 312,936 and US 6,482, 628 in Figures 1-3 [SEQ ID 3], and at column 25, line 12, "Protease D"being the variant 1O1G/103A/1041159D/232V/236H/245R/248D/252K (BPN' numbering) described in WO 99/20723.

Amylase : Amylolytic enzyme sold under the tradename Purafact Ox AmR described in WO 94/18314, W096/05295 sold by Genencor; Katalase, Termamyl@, Fungamyl and Duramyl, all available from Novozymes A/S.

Lipase Lipolytic enzyme sold under the tradename Lipolase Lipolase Ultra by Novozymes A/S and Lipomax by Gist-Brocades.

Cellulase Cellulytic enzyme sold under the tradename Carezyme, Celluzyme and/or Endolase by Novozymes A/S.

Pectin Lyase Pectaway0 nd pectawash available from Novozymes A/S.

PVP Polyvinylpyrrolidone with an average molecular weight of 60,000 PVNO Polyvinylpyridine-N-Oxide, with an average molecular weight of 50,000.

PVPVI Copolymer of vinylimidazole and vinylpyrrolidone, with an average molecular weight of 20,000.

Brightener 1 : Disodium 4,4'-bis (2-sulphostyryl) biphenyl.

Silicone antifoam : Polydimethylsiloxane foam controller with siloxane-oxyalkylene copolymer as dispersing agent with a ratio of said foam controller to said dispersing agent of 10: 1 to 100: 1.

Suds Suppressor 12% Silicone/silica, 18% stearyl alcohol, 70% starch in granular form.

SRP 1 Anionically end capped poly esters.

PEG X Polyethylene glycol, of a molecular weight of x.

PVP K60 E) Vinylpyrrolidone homopolymer (average MW 160,000) Jeffamine # ED-2001 : Capped polyethylene glycol from Huntsman Isachem @ AS A branched alcohol alkyl sulphate from Enichem MME PEG (2000) Monomethyl ether polyethylene glycol (MW 2000) from Fluka Chemie AG.

DC3225C Silicone suds suppresser, mixture of Silicone oil and Silica from Dow Corning.

TEPAE Tetreaethylenepentaamine ethoxylate.

BTA Benzotriazole.

Betaine (CH3) 3N+CH2COO- Sugar Industry grade D-glucose or food grade sugar CFAA : C12-C14 alkyl N-methyl glucamide TPKFA : C12-C14 topped whole cut fatty acids.

Clay A hydrated aluminumu silicate in a general formula 203SiO2'H20. Types: Kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite.

MCAEM : Esters in the formula of R'Ox [(R2) m (R3) Mp pH : Measured as a 1% solution in distilled water at 20°C.

EXAMPLE 20 Liquid Laundry Detergents The following liquid laundry detergent compositions of the present invention are prepared.

I II III IV V LAS 18. 0 6. 0 C-C AE S 2. 0 8.0 11.0 5.0 C8-C1o propyl dimethyl 2.0 2.0 2.0 2.0 1.0 amine C12-C14 alkyl dimethyl - - - - 2. 0 amine oxide C-C AS 17. 0 7. 0 8.0 CFAA 5. 0 4.0 4.0 3.0 C12-C14 Fatty alcohol 12. 0 6.0 1.0 1.0 1.0 ethoxvlate C-C Fa acid 11. 0 11. 0 4.0 4.0 3. 0 Citric acid anhydrous) 5.0 1. 0 3.0 3.0 2.0 DETPMP 1. 0 1.0 1.0 1. 0 0. 5 Monoethanolamine 11. 0 8.0 5. 0 5. 0 2.0 Sodium hydroxide 1. 0 1.0 2.5 1.0 1.5 Percarbonate - 3.5 - 2.5 Propanediol 12. 7 14. 5 13.1 10.8. 0 Ethanol 1.8 1.8 4.7 5.4 1.0 Pectin Lyase - - - 0.005 Amylase - 0.002 - Cellulase 0. 0002 0. 0001 Lipase 0.1 - 0. 1 0. 1 Protease A 0. 05 0.3 0.055 0. 5 0.2 Aldose Oxidase 0.03 - 0.3 - 0.00 PAAC 0.01 0. 01 DETBCHD - - 0. 02 0. 01 SRP 1 0. 5 0. 5 - 0.3 0.3 Boric acid 2. 4 2.4 2.8 2. 8 2.4 Sodium xvlene sulfonate 3. 0 DC 3225C 1.0 1.0 1.0 1.0 1.0 2-butyl-octanol 0. 03 0. 04 0. 04 0. 03 0. 03 DTPA 0. 5 0.4 0.35 0.28 0.4 Brightener 1 0. 18 0.10 0. 11 Perhydrolase 0. 05 0. 3 0. 08 0.5 0.2 MCAEM 3.0 8. 0 12. 0 1.5 4.8 C-C E Acetate Balance to 100% perfume/dye and/or water

EXAMPLE 21 Hand-Dish Liquid Detergent Compositions The following hand dish liquid detergent compositions of the present invention are prepared. I II III IV V VI C12-C15 AE1.8S 30. 0 28. 0 25. 0 - 15. 0 10.0 LAS 5. 0 15. 0 12. 0 Paraffin Sulfonate 20. 0 Clo-Cis Alkyl Dimethyl 5.0 3.0 7. 0 Amine Oxide Betaine 3. 0 1. 0 3.0 1. 0 C12 poly-OH fatty acid - - - 3.0 - 1. 0 amide C14 poly-OH fatty acid - 1.5 amide Cl tE9 2. 0 - 4.0 - - 20. 0 DTPA 0. 2 Tri-sodium Citrate dihydrate 0. 25 - - 0.7 Diamine 1.0 5.0 7.0 1.0 5.0 7.0 MgCl2 0.25 - - 1.0 - - Protease A 0.02 0.01 0.02 0.01 0.02 0.05 Amylase 0. 001 - - 0.002 - 0. 001 Aldose Oxidase 0. 03 - 0.02 - 0.05 Sodium Cumene Sulphonate - - - 2. 0 1.5 3.0 PAAC 0.01 0. 01 0. 02 - - - DETBCHD - - - 0.01 0.02 0.01 PB1 1.5 2.8 1.2 Perhydrolase 0.02 0.01 0.03 0. 01 0. 02 0. 05 I II III IV V VI MCAEM 3.4 2.8 4.0 2.6 4.6 6.8 (C14-C15 E7 Acetate) Balance to 100% perfume/dye and/or water The pH of Compositions (I)- (VI) is about 8 to about 11

EXAMPLE 22 Liquid Automatic Dishwashing Detergent The following liquid automatic dishwashing detergent compositions of the present are prepared.

I II in IV v STPP 16 16 18 16 16 Potassium Sulfate-10 8-10 1, 2 propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid 4.0 3.0 3.0 4.0 3.0 CaCl2 dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.5 0.5 0.5 0.5 0.5 Protease B 0.03 0.03 0.03 0.03 0.03 Amylase 0.02-0. 02 0. 02 Aldose Oxidase-0. 15 0.02-0. 01 Galactose Oxidase--0. 01-0.01 PAAC 0.01--0. 01- DETBCHD-0. 01--0.01 Perhydrolase 0. 1 0.03 0.05 0.03 0.06 MCAEM 5.0 3.0 12.0 8.0 1.0 (Cl4-ClsEi2 Acetate)

I II III IV V Balance to 100% perfume/dye and/or water EXAMPLE 23 Laundry Compositions The following laundry compositions of present invention, which may be in the form of granules or tablet, are prepared.

I II III IV V Base Product Ci4-Ci5ASorTAS 8.0 5.0 3.0 3.0 3.0 LAS 8. 0-8. 0-7.0 Cl2-ClsAE3S 0.5 2.0 1. 0-- Cl2-CIsEs or E3 2.0-5. 0 2.0 2.0 QAS---1.0 1.0 Zeolite A 20.0 18.0 11.0-10. 0 SKS-6 (dry add)--9. 0-- MA/AA 2.0 2.0 2. 0-- AA----4.0 3Na Citrate 2H20-2. 0--- Citric Acid (Anhydrous) 2.0-1. 5 2. 0 DTPA 0.2 0. 2 - - - EDDS--0.5 0. 1- HEDP--0.2 0.1- PB1 3.0 4.8--4. 0 Percarbonate--3. 8 5. 2

I II III IV v NOBS 1. 9---- NACA OBS--2. 0-- TAED 0.5 2. 0 2.0 5.0 1. 00 BB1 0. 06-0. 34-0. 14 BB2-0.14-0. 20- Anhydrous Na Carbonate 15.0 18. 0 8.0 15.0 15.0 Sulfate 5.0 12.0 2.0 17.0 3.0 Silicate-1.0--8. 0 Protease B 0.033 0. 033 Protease C--0. 033 0.046 0.033 Lipase-0. 008 Amylase 0. 001---0. 001 Cellulase-0. 0014 - - - Pectin Lyase 0.001 0.001 0.001 0.001 0.001 Aldose Oxidase 0. 03-0. 05 PAAC-0.01--0. 05 Perhydrolase 0.03 0.05 1.0 0.06 0.1 MCAEM** 2.0 5.0 12.0 3.5 6.8 Balance to 100% Moisture and/or Minors* <BR> <BR> <BR> <BR> . Perfume/Dye, Brightener/SRP1/Na Carboxymethylcellulose/Photobleach/MgS04/ PVPVI/Suds suppressor/High Molecular PEG/Clay.

** MCAEM is selected from the group consisting of C9-C11E2.5 Acetate, [C12H25N (CH3) (CH2CH20Ac) 2]+ Cl-, (CH3) 2NCH2CH20CH2CH20Ac, or mixtures thereof..

EXAMPLE 24 Liquid Laundry Detergents The following liquid laundry detergent formulations of the present invention are prepared.

I I II III IV V LAS 11.5 11.5 9.0-4. 0- Cl2-CisAE2. 85S--3. 0 18.0-16. 0 C14-CisE 2. s S 11.5 11.5 3.0-16. 0 C12-C13E9 - - 3. 0 2.0 2.0 1.0 C 12-CI3E 7 3.2 3. 2- CFAA---5. 0-3. 0 TPKFA 2.0 2.0-2. 0 0.5 2.0 Citric Acid 3.2 3.2 0.5 1.2 2.0 1.2 (Anhydrous) Ca formate 0. 1 0. 1 0.06 0. 1 Na formate 0.5 0.5 0.06 0.1 0.05 0.05 Na Culmene 4.0 4.0 1.0 3.0 1. 2 Sulfonate Borate 0.6 0.6-3. 0 2.0 3.0 Na hydroxide 6.0 6.0 2.0 3.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1, 2 Propanediol 3.0 3.0 2.0 8. 0 8.0 5.0 Mono-3.0 3.0 1.5 1.0 2.5 1.0 ethanolamine TEPAE 2.0 2.0-1. 0 1.0 1.0 PB1 - 4. 5-2. 8 Protease A 0.03 0.03 0.01 0.03 0.02 0.02

III IV v<BR> Lipase---0. 002--<BR> Amylase----0. 002 Cellulase-----0. 0001 Pectin Lyase 0.005 0. 005 Aldose Oxidase 0.05--0. 05-0.02 Galactose oxidase-0.04 Perhydrolase 0.03 0.05 0.01 0.03 0.08 0.02 MCAEM 3.2 4.6 1.8 3.5 6.2 2.8 (C 12-Cis E6 Acetate) PAAC 0.03 0.03 0. 02--- DETBCHD---0.02 0. 01 SRP 1 0.2 0.2-0. 1-- DTPA---0. 3-- PVNO---0.3-0. 2 Brightener 1 0.2 0.2 0.07 0. 1 Silicone antifoam 0.04 0.04 0.02 0.1 0.1 0.1 Balance to 100% perfume dye, and/or water EXAMPLE 25 Compact High-Density Dishwashing Detergents The following compact high density dishwashing detergent of the present invention are prepared: I II III IV V VI STPP-45. 0 45. 0--40. 0 I II III IV V VI 3Na Citrate 2H20 17.0--50. 0 40. 2 Na Carbonate 17.5 14.0 20.0-8. 0 33.6 Bicarbonate---26. 0 Silicate 15.0 15.0 8.0-25. 0 3.6 Metasilicate 2.5 4.5 4. 5 - - - PB1--4. 5-- PB4 - - - 5.0 - - Percarbonate-----4. 8 BB1-0. 1 0.1-0. 5- BB2 0.2 0.05-0. 1-0.6 Nonionic 2.0 1.5 1.5 3.0 1.9 5.9 HEDP 1. 0----- DETPMP 0. 6----- PAAC 0.03 0.05 0. 02--- Paraffin 0.5 0.4 0.4 0. 6-- Protease B 0.072 0.053 0.053 0.026 0.059 0.01 Amylase 0.012-0. 012-0.021 0.006 Lipase-0.001-0. 005 Pectin Lyase 0.001 0.001 0. 001 Aldose Oxidase 0.05 0.05 0.03 0.01 0.02 0.01 Perhydrolase 0.072 0.053 0.053 0.026 0.059 0.01 MCAEM 3.5 2. 8 1.6 7.5 4.2 0. 8 (C 12-C13 E 6.5 Acetate) BTA 0. 3 0.2 0.2 0. 3 0.3 0. 3 Polycarboxylate 6.0---4. 0 0.9

I II III IV V VI Perfume 0.2 0.1 0.1 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors* <BR> <BR> <BR> <BR> *Brightener/Dye/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO4/PVPVI/Suds suppressor/High Molecular PEG/Clay.

The pH of compositions (I) through (VI) is from about 9.6 to about 11.3.

EXAMPLE 26 Tablet Detergent Compositions The following tablet detergent compositions of the present invention are prepared by compression of a granular dishwashing detergent composition at a pressure of 13KN/cm2 using a standard 12 head rotary press.

I II III IV V VI VII VIII STPP-48. 8 44.7 38.2-42. 4 46.1 36.0 3Na Citrate 2Ha0 20.0---35. 9--- Na Carbonate 20.0 5.0 14.0 15.4 8.0 23.0 20.0 28.0 Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.3 4.2 Lipase 0.001-0. 01-0. 02 Protease B 0.042 0.072 0.042 0. 031---- Protease C----0. 052 0.023 0.023 0.029 Perhydrolase 0.01 0.08 0.05 0.04 0.052 0.023 0.023 0.029 MCAEM 2.8 6.5 4.5 3.8 4.6 2. 8 2.8 2.8 (C 12-CI3 E 6. 5 Acetate) Amylase 0.012 0.012 0.012-0. 015-0.017 0.002

I II III IV V VI VII VIII Pectin Lyase 0. 005 - - 0.002 - - - - Aldose Oxidase-0. 03-0.02 0.02-0. 03 PB1--3.8-7. 8--8.5 Percarbonate 6.0--6. 0-5. 0 BB1 0.2-0. 5-0.3 0. 2-- BB2-0.2-0. 5--0.1 0.2 Nonionic 1.5 2.0 2.0 2.2 1.0 4.2 4.0 6.5 PAAC 0.01 0.01 0. 02----- DETBCHD---0.02 0. 02 - - - TAED-----2. 1-1. 6 HEDP 1. 0--0. 9 - 0. 4 0. 2- DETPMP 0. 7------- Paraffin 0.4 0.5 0.5 0. 5 - - 0.5 - BTA 0.2 0.3 0.3 0.3 0. 3 0.3 0.3 - Polycarboxylate 4.0---4. 9 0.6 0. 8, PEG 400-30,000 - - - - - 2. 0-2.0 <BR> <BR> <BR> <BR> Glycerol-----0. 4-0. 5 Perfume---0 : 05 0. 2 0. 2 0.2 0. 2 Balance to 100% Moisture and/or Minors* <BR> <BR> <BR> <BR> *Brightener/Dye/SRP 1/Na Carboxymethylcellulose/Photobleach/MgS04/PVPVI/Suds suppressor/High Molecular PEG/Clay.

The pH of Compositions (I) through 7 (Vin) is from about 10 to about 11.5.

The tablet weight of Compositions 7 (1) through 7 (Vin) is from about 20 grams to about 30 grams.

EXAMPLE 27 Liquid Hard Surface Cleaning Detergents The following liquid hard surface cleaning detergent compositions of the present invention are prepared.

I II III IV V VI VII C9-CIEs 2.4 1.9 2.5 2.5 2.5 2.4 2.5 Ci2-Cl4Es 3.6 2.9 2.5 2.5 2.5 3.6 2.5 C7-C9E6 - - - - 8.0 - - Ci2-Ci4E21 1.0 0.8 4.0 2.0 2.0 1.0 2.0 LAS---0.8 0. 8-0. 8 Sodium culmene sulfonate 1.5 2.6-1. 5 1.5 1.5 1.5 Isachem M) AS 0.6 0.6---0. 6 - Na2CO3 0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na Citrate 2H20 0.5 0.56 0.5 0.6 0.75 0.5 0.75 NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.13 0.6 0.1 0.4 0.6 0.4 2-butyl octanol 0.3 0.3-0. 3 0.3 0.3 0.3 PEG DME-20009 0.4-0. 3 0.35 0. 5-- PVP 0. 3 0.4 0.6 0.3 0. 5-- MME PEG (2000) t)-----0. 5 0. 5 Jeffamine # ED-2001 - 0. 4--0. 5 PAAC---0.03 0.03 0. 03 DETBCHD 0.03 0.05 0. 05---- Protease B 0.07 0.05 0.05 0.03 0.06 0.01 0.04 Amylase 0.12 0.01 0.01-0. 02-0.01 Lipase-0.001-0. 005-0. 005- Perhydrolase 0.07 0.05 0. 08 0.03 0.06 0.01 0.04

1 II III IV v vi VII MCAEM (Cl2-CIsEs 3.5 5.6 4.8 5.3 3.6 8.0 4.7 Acetate) Pectin Lyase 0.001-0. 001---0.002 PB1-4. 6-3. 8--- Aldose Oxidase 0. 05-0.03-0. 02 0.02 0.05 Balance to 100% perfume/dye, and/or water The pH of Compositions (I) through (VII) is from about 7.4 to about 9.5.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.

Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically

disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.