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Title:
THERMOSTABLE LIPOLYTIC ENZYME VARIANT
Document Type and Number:
WIPO Patent Application WO/2002/055679
Kind Code:
A2
Abstract:
Lipolytic enzyme variants with improved thermostability are obtained by substituting certain specified amino acid residues in a fungal lipolytic enzyme. The thermostable lipolytic enzyme variants are useful, e.g., for controlling pitch troubles in a process for the production of mechanical pulp or a paper-making process using mechanical pulp.

Inventors:
Minning, Stefan (Lykkesholms Allé 3C, 2. tv Frederiksberg C, DK-1902, DK)
Vind, Jesper (Hejrebakken 20 Værløse, DK-3500, DK)
Glad, Sanne Schrøder O. (Viggo Barfoeds Allé 59 Ballerup, DK-2750, DK)
Danielsen, Steffen (Holsteinsgade 12A, 4th København Ø, DK-2100, DK)
Borch, Kim (Vandtårnsvej 18 Birkerød, DK-3460, DK)
Application Number:
PCT/DK2002/000016
Publication Date:
July 18, 2002
Filing Date:
January 10, 2002
Export Citation:
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Assignee:
Novozymes, A/s (Krogshøjvej 36 Bagsværd, DK-2880, DK)
Minning, Stefan (Lykkesholms Allé 3C, 2. tv Frederiksberg C, DK-1902, DK)
Vind, Jesper (Hejrebakken 20 Værløse, DK-3500, DK)
Glad, Sanne Schrøder O. (Viggo Barfoeds Allé 59 Ballerup, DK-2750, DK)
Danielsen, Steffen (Holsteinsgade 12A, 4th København Ø, DK-2100, DK)
Borch, Kim (Vandtårnsvej 18 Birkerød, DK-3460, DK)
International Classes:
C12N15/09; A21D8/04; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N9/20; D06M16/00; D21C9/08; D21H21/02; (IPC1-7): C12N9/00
Domestic Patent References:
WO1992013130A11992-08-06
WO1992005249A11992-04-02
WO1992019726A11992-11-12
WO1997007202A11997-02-27
WO1998026057A11998-06-18
WO1998045453A11998-10-15
WO1998031790A11998-07-23
WO1996018729A11996-06-20
WO1996013578A11996-05-09
WO1997027276A21997-07-31
WO1992007138A11992-04-30
WO1994004035A11994-03-03
WO2000032758A12000-06-08
WO2002003805A12002-01-17
WO1988002775A11988-04-21
WO1997004079A11997-02-06
WO1997041212A11997-11-06
WO1998008939A11998-03-05
WO1997043375A11997-11-20
WO1995022615A11995-08-24
WO1998000529A11998-01-08
WO2000024883A12000-05-04
Foreign References:
EP0374700A21990-06-27
US5869438A1999-02-09
EP0258068A21988-03-02
EP0305216A11989-03-01
EP0130064A11985-01-02
JPH10155493A1998-06-16
JPH02160984A1990-06-20
EP1057415A12000-12-06
US6156548A2000-12-05
US5776741A1998-07-07
EP0792106A11997-09-03
EP0093602A21983-11-09
EP0307154A21989-03-15
US5578489A1996-11-26
US6077316A2000-06-20
GB2233665A1991-01-16
EP0505920A11992-09-30
Other References:
GIVER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 12809 - 12813
MOORE ET AL., NAT. BIOTECH, vol. 14, 1996, pages 458 - 467
YAMAGUCHI ET AL., PROTEIN ENGINEERING, vol. 9, 1996, pages 789 - 795
NEEDLEMAN, S.B.; WUNSCH, C.D., JOURNAL OF MOLECULAR BIOLOGY, vol. 48, 1970, pages 443 - 45
KYNCLOVA, E ET AL., JOURNAL OF MOLECULAR RECOGNITION, vol. 8, 1995, pages 139 - 145
HORTON ET AL., GENE, vol. 77, 1989, pages 61 - 68
MANDEL, M.; A. HIGA, J. MOL. BIOL., vol. 45, 1970, pages 154
SAMBROOK: "Molecular cloning, a laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
Attorney, Agent or Firm:
Novozymes, A/s (Patents Krogshøjvej 36 Bagsværd, DK-2880, DK)
Download PDF:
Claims:
CLAIMS
1. A variant of a parent fungal lipolytic enzyme, wherein the variant a) has an amino acid sequence which compared to the parent lipolytic enzyme comprises substitution of an amino acid residue corresponding to any of amino ac ids 21,27,29,32,3442,51,54,76,84,9097,101,105,111,118,125,131,135, 137,162,187,189,206212,216,224234,242252 and 256 of SEQ ID NO: 1, and b) is more thermostable than the parent lipolytic enzyme.
2. The variant of the preceding claim which is at least 4° C more thermostable than the parent lipolytic enzyme.
3. The variant of either preceding claim, wherein the amino acid residue is substituted with an amino acid residue different from Pro.
4. The variant of any preceding claim wherein the parent lipolytic enzyme has at least 50 % homology with SEQ ID NO: 1.
5. The variant of the preceding claim wherein the parent lipolytic enzyme is the lipase produced by Thermomyces lanuginosus DSM 4109 and having the amino acid sequence of SEQ ID NO : 1.
6. The variant of either preceding claim which comprises substitution of an amino acid residue corresponding to Y21, D27, P29, T32, A40, F51, S54, 176, R84, 190, G91, N94, N101, S105, D111, R118, R125, A131, H135, D137, N162, V187, T189, E210, G212, S216, G225, L227,1238 or P256 of SEQ ID NO : 1.
7. The variant of any preceding claim which comprises one or more substitutions corre sponding to D27N/R/S, P29S, T32S, F511/L, 176V, R84C, 90L/V, G91A/N/S/T/W, L93F, N94K/R/S, F951, D96G/N, N101D, D111A/G, R118M, A131V, H135Y, D137N, N162R, V1871, F211Y, S216P, S2241/Y, G225P, T226N, L227F/P/G/V, L227X, V228C/I, 238V and P256T of SEQ ID NO : 1. 8.
8. The variant of any preceding claim which has one, two, three, four, five, six, seven or eight of said substitutions.
9. The variant of any preceding claim which further comprises one or more substitutions of amino acid residues other than those listed in claim 1, preferably 15 such substitutions.
10. The variant of any preceding claim which comprises substitutions corresponding to the following in SEQ ID NO: 1: a) D27N b) D111G +S216P c) L227F d) L227F +V2281 e) G225P f) S2241 +G225W +T226N +L227P +V228C g) S224Y +G225W +T226N +L227P +V228C h) D27R +D111G +S216P i) D27S +D111G +S216P j) D27N +D111A k) D27R +D111 G +S216P +L227P +P256T I) D27R +D111G +S216P +L227G +P256T m) D27R +D111G +S216P +L227F +P256T n) D27R +D111G +S216P +L227V +P256T o) D27R +D111G +S216P +L227G p) D27R +D111G +S216P +L227X q) D27P+D111G+S216P+L227X r) S2241 + G225W + T226N + L227P + V228C s) W221 C + G246C t) D27R+D111G+S216P u) D27N + D111A v) D27R + D111G + S216P + L227G + P256T w) D27R + D111G + S216P + L227F + P256T x) D27R + D111G + S216P + L227G y) D27S+D111G+S216P z) D27R + D111A + S216P + L227G + P256T aa) D27R + D111G + S216P + G225P + L227G + P256T bb) D27R + T37S + D111 G + S216P + L227G + P256T cc) D27R + N39F + D111 G + S216P + L227G + P256T dd) D27R + G38C + D111G + S216P + L227G + P256T ee) D27R + D111G + S216P + L227G + T2441 + P256T ff) D27R + G91A + D111G + S216P + L227G + P256T gg) N251 +D27R + D111A + S216P + L227G + P256T hh) N25L +D27R + D111A + S216P + L227G + P256T ii) N26D +D27R + D111A + S216P + L227G + P256T jj) D27R +K46R + D111A + S216P + L227G + P256T kk) D27R + V60N +D111A + S216P + L227G + P256T II) D27R + D111A + P136A +S216P + L227G + P256T mm) D27R + D111A + S216P + L227G + P256T +1265F nn) D27R + S58Y +D111A + S216P + L227G + P256T + oo) N26D +D27R +E56Q +D111A + S216P + L227G + P256T pp) D27R +G91A +D96E +L97Q +D111A +S216P + L227G + P256T qq) D27R +G91A +D111A + S216P + L227G + P256T + rr) D27R + G91T +N94S +D111A +S216P + L227G + P256T ss) D27R +G91 S +D111A + S216P + L227G + P256T + tt) D27R +G91 N +D111A + S216P + L227G + P256T uu) D27R +D96E +D111A + S216P + L227G + P256T vv) D27R +190L +G91A +N94K +D111A + S216P + L227G + P256T ww) D27R +G91S +F95V +D111A + S216P + L227G + P256T.
11. The variant of any preceding claim having a denaturation temperature which is at least 5° C higher than the parent lipolytic enzyme, preferably measured at pH 57.
12. A DNA sequence encoding the variant of any preceding claim.
13. A vector comprising the DNA sequence of the preceding claim.
14. A transformed host cell harboring the DNA sequence of claim 12 or the vector of claim 13.
15. A method of producing the variant of any of claims 111 comprising a) cultivating the cell of claim 14 so as to express and preferably secrete the vari ant, and b) recovering the variant.
16. A method of producing a lipolytic enzyme variant comprising: a) selecting a parent fungal lipolytic enzyme, b) in the parent lipolytic enzyme substituting at least one amino acid residue corre sponding to any of 21,27,29,32,3442,51,54,76,84,9097,101,105,111,118, 125,131,135,137,162,187,189,206212,216,224234,242252 and 256 of SEQ ID NO : 1, c) optionally, substituting one or more amino acids other than b), d) preparing the variant resulting from steps a)c), e) testing the thermostability of the variant, f) selecting a variant having an increased thermostability, and g) producing the selected variant.
17. The method of the preceding claim wherein the parent lipolytic enzyme has at least 50 % homology with SEQ ID NO: 1.
18. The method of the preceding claim wherein the parent lipolytic enzyme is the lipase produced by Thermomyces lanuginosus DSM 4109 and having the amino acid sequence of SEQ ID NO : 1.
19. The method of either preceding claim which comprises substituting an amino acid residue corresponding to Y21, D27, P29, T32, A40, F51, S54, 176, R84,190, G91, N94, N101, S105, D111, R118, R125, A131, H135, D137, N162, V187, T189, E210, G212, S216, G225, L227, 1238 or P256 of SEQ ID NO : 1.
20. The method of the preceding claim which comprises substituting an amino acid resi due corresponding to D27N/R/S, P29S, T32S, F511/L, 176V, R84C, 190LN, G91A/N/S/T/W. L93F, N94K/R/S, F951, D96G/N, N101D, D111A/G, R118M, A131V, H135Y, D137N, N162R, V1871, F211Y, S216P, S2241/Y, G225P, T226N, L227F/P/G/V, L227X, V228C/I, 238V and P256T of SEQ ID NO : 1.
21. A process for hydrolyzing a carboxylic acid ester, comprising incubating the ester with the lipase of any of claims 111 in the presence of water.
22. A process for controlling pitch troubles in a process for the production of mechanical pulp or a papermaking process using mechanical pulp, which comprises adding the lipase of any of claims 111 to the pulp and incubating.
23. The process of either preceding claim wherein the incubation is done at a temperature of 6095°C, particularly 7590°C.
24. The process of any preceding claim, wherein the incubation is done at a pH in the range 4.511, particularly 56.5.
25. A process for preparing a dough or a baked product prepared from the dough, com prising adding the lipolytic enzyme of any of claims 111 to the dough.
26. A process for hydrolyzing, synthesizing or interesterifying an ester, comprising react ing the ester with water, reacting an acid with an alcohol or interesterifying the ester with an acid, an alcohol or a second ester in the presence of the lipolytic enzyme of any of claims 111.
27. A process for enzymatic removal of hydrophobic esters from fabrics, which process comprises treating the fabric with an amount of the lipolytic enzyme of any of claims 111 ef fective to achieve removal of hydrophobic esters from fabric.
Description:
LIPOLYTIC ENZYME VARIANT FIELD OF THE INVENTION The present invention relates to variants of fungal lipolytic enzymes, particularly vari- ants with improved thermostability, and to methods of producing and using such variants.

BACKGROUND OF THE INVENTION It is known to use fungal lipolytic enzymes, e. g. the lipase from Thermomyces lanugi- nosus (synonym Humicola lanuginosa), for various industrial purposes, e. g. to improve the ef- ficiency of detergents and to eliminate pitch problems in pulp and paper production. In some situations, a lipolytic enzyme with improved thermostability is desirable (EP 374700, WO 9213130).

WO 92/05249, WO 92/19726 and WO 97/07202 disclose variants of the T. lanugino- sus (H, lanuginosa) lipase.

SUMMARY OF THE INVENTION The inventors have found that the thermostability of a fungal lipolytic enzyme can be improved by certain specified substitutions in the amino acid sequence.

Accordingly, the invention provides a variant of a parent fungal lipolytic enzyme, which variant comprises substitution of one or more specified amino acid residues and is more ther- mostable than the parent lipolytic enzyme. The invention also provides a method of producing a lipolytic enzyme variant comprising: a) selecting a parent fungal lipolytic enzyme, b) in the parent lipolytic enzyme substituting at least one specified amino acid residue, c) optionally, substituting one or more amino acids other than b), d) preparing the variant resulting from steps a)-c), e) testing the thermostability of the variant, f) selecting a variant having an increased thermostability, and g) producing the selected variant.

The specified amino acid residues comprise amino acid residues corresponding to any of 21,27,29,32,34-42,51,54,76,84,90-97,101,105,111,118,125,131, 135,137, 162, 187,189,206-212,216,224-234,242-252 and 256 of SEQ ID NO: 1.

The thermostability may particularly be increased by more than 4°C. The substitutions may be with a different amino acid residue, particularly one different from Pro.

DETAILED DESCRIPTION OF THE INVENTION Parent lipolytic enzyme The lipolytic enzyme to be used in the present invention is classified in EC 3.1.1 Car- boxylic Ester Hydrolases according to Enzyme Nomenclature (available at http://www. chem. qmw. ac. uk/iubmb/enzyme). The substrate specificity may include activities such as EC 3.1.1.3 triacylglycerol lipase, EC 3.1.1.4 phospholipase A2, EC 3.1.1.5 lysophos- pholipase, EC 3.1.1.26 galactolipase, EC 3.1.1.32 phospholipase A1, EC 3.1.1.73 feruloyl es- terase.

The parent lipolytic enzyme is fungal and has an amino acid sequence that can be aligned with SEQ ID NO: 1 which is the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of US 5,869,438 for the lipase from Thermomyces lanuginosus (synonym Humicola lanuginosa), described in EP 258 068 and EP 305 216. The parent lipolytic enzyme may par- ticularly have an amino acid sequence with at least 50 % homology with SEQ ID NO: 1. In ad- dition to the lipase from T lanuginosus, other examples are a lipase from Penicillium camem- bertii (P25234), lipase/phospholipase from Fusarium oxysporum (EP 130064, WO 98/26057), lipase from F. heterosporum (R87979), lysophospholipase from Aspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), li- pase/ferulic acid esterase from A. niger (Y09330), lipase/ferulic acid esterase from A. tubin- gensis (Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipase from A. niger (WO 98/31790), lipase from F. solanii having an isoelectric point of 6.9 and an apparent mo- lecular weight of 30 kDa (WO 96/18729).

Other examples are the Zygomycetes family of lipases comprising lipases having at least 50 % homology with the lipase of Rhizomucor miehei (P19515) having the sequence shown in SEQ ID NO: 2. This family also includes the lipases from Absidia reflexa, A. sporo- phora, A. corymbifera, A. blakesleeana, A. griseola (all described in WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parentheses indicate publication or ac- cession to the EMBL, GenBank, GeneSeqp or Swiss-Prot databases.

Amino acid substitutions The lipolytic enzyme variant of the invention comprises one or more substitutions of an amino acid residue in any of the regions described above. The substitution may, e. g., be made in any of the regions corresponding to 206-208,224-228,227-228,227-231,242-243 and 245-252 of SEQ ID NO: 1. The amino acid residue to be substituted may correspond to residue Y21, D27, P29, T32, A40, F51, S54, 176, R84, 190, G91, N94, N101, S105, D111,

R118, R125, A131, H135, D137, N162, V187, T189, E210, G212, S216, G225, L227,1238 or P256 of SEQ ID NO: 1. Some particular substitutions of interest are those corresponding to D27N/R/S, P29S, T32S, F511/L, 176V, R84C, 190LN, G91A/N/S/T/W, L93F, N94K/R/S, F951, D96G/N, N101D, D111A/G, R118M, A131V, H135Y, D137N, N162R, V1871, F211Y, S216P, S2241/Y, G225P, T226N, L227F/P/G/V, L227X, V228C/I, 238V and P256T of SEQ ID NO: 1.

The total number of substitutions in the above regions is typically not more than 10, e. g. one, two, three, four, five, six, seven or eight of said substitutions. In addition, the lipolytic enzyme variant of the invention may optionally include other modifications of the parent en- zyme, typically not more than 10, e. g. not more than 5 such modifications. The variant may particularly have a total of not more than 10 amino acid modifications (particularly substitu- tions) compared to the parent lipolytic enzyme. The variant generally has a homology with the parent lipolytic enzyme of at least 80 %, e. g. at least 85 %, typically at least 90 % or at least 95 %.

Lipolytic enzyme variant The variant has lipolytic enzyme activity, i. e. it is capable of hydrolyzing carboxylic es- ter bonds to release carboxylate (EC 3.1.1). It may particularly have lipase activity (triacylglyc- erol lipase activity, EC 3.1.1.3), i. e. hydrolytic activity for carboxylic ester bonds in triglycerides, e. g. 1,3-specific activity.

Specific variants The following are some examples of variants of the T. lanuginosus lipase. Corre- sponding substitutions may be made by making corresponding amino acid substitutions in other fungal lipolytic enzymes: D27N D111G +S216P L227F L227F +V2281 G225P S2241 +G225W +T226N +L227P +V228C S224Y +G225W +T226N +L227P +V228C <BR> <BR> <BR> <BR> D27R +D111G +S216P<BR> <BR> <BR> <BR> <BR> D27S +D111G +S216P<BR> <BR> <BR> <BR> <BR> D27N +D111A D27R +D111 G +S216P +L227P +P256T D27R +D111G +S216P+L227G +P256T <BR> <BR> D27R +D111G +S216P+L227F+P256T D27R +D111G +S216P +L227V +P256T <BR> <BR> D27R +D111G +S216P+L227G<BR> <BR> D27R +D111G +S216P+L227X<BR> <BR> D27P +D111G +S216P+L227X

Thermostability The thermostability can be measured at a relevant pH for the intended application us- ing a suitable buffer. Examples of buffers and pH are: pH 10.0 (50 mM glycine buffer), pH 7.0 (50 mM HEPES Buffer) or pH 5.0 (50 mM sodium acetate as buffer).

For comparison, measurements should be made in the same buffer, at the same con- ditions and at the same protein concentration. Various methods can be used for measuring the thermostability : Differential Scanning Calorimetrv (DSC) In DSC, the heating rate may be 90 degrees per hour. The sample may be purified to homogeneity, and the melting temperature (TM) may be taken as an expression of the thermo- stability.

Residual enzyme activity Alternatively, the thermostability can be determined by measuring residual lipolytic enzyme activity after incubation at selected temperatures. p-nitrophenyl ester in 10 mM Tris- HCI, pH 7.5 may be used as the substrate, as described in Giver et al., Proc. Natl. Acad. Sci.

USA 95 (1998) 12809-12813 and Moore et al. Nat. Biotech. 14 (1996) 458-467. Samples may be added periodically, or only one sample may be used with or without different additives to pre- vent or enhance denaturing, e. g. in a 96 well format.

CD spectroscopy CD spectroscopy as described e. g. in Yamaguchi et al. Protein engineering 9 (1996) 789-795. Typical enzyme concentration is around 1 mg/ml, Temperature between 5-80 degrees Use of variant The lipolytic enzyme variants may be used in various processes, and some particular uses are described below. The variant is typically used at 60-95°C (particularly 75-90°C, 70- 90°C or 70-85°C) and pH 4.5-11 (particularly 4.5-8 or 5-6.5).

Use in the paper and pulp industry The lipase may be used in a process for avoiding pitch troubles in a process for the production of mechanical pulp or a paper-making process using mechanical pulp, which com- prises adding the lipase to the pulp and incubating. The lipase addition may take place in the so-called white water (recycled process water). It may also be used to remove ink from used paper. The improved thermostability allows the variant to be used at a higher temperature, generally preferred in the industry. This may be done in analogy with WO 9213130, WO 9207138, JP 2160984 A, EP 374700.

Use in cereal-based food products The lipolytic enzyme variant may be added to a dough, and the dough may be used to prepare a baked product (particularly bread), pasta or noodles. The improved thermostability of the variant allows it to remain active for a longer time during the heating step (baking, boiling or frying). This may be done in analogy with WO 94/04035, WO 00/32758, PCT/DK 01/00472, EP 1057415.

The addition of the variant may lead to improved dough stabilization, i. e. a larger loaf volume of the baked product and/or a better shape retention during baking, particularly in a stressed system, e. g. in the case of over-proofing or over-mixing. It may also lead to a lower initial firmness and/or a more uniform and fine crumb, improved crumb structure (finer crumb, thinner cell walls, more rounded cells), of the baked product, and it may further improve dough properties, e. g. a less soft dough, higher elasticity, lower extensibility.

Use in the fat and oil industry The lipolytic enzyme variant may be used as a catalyst in organic synthesis, e. g. in a process for hydrolyzing, synthesizing or interesterifying an ester, comprising reacting the ester with water, reacting an acid with an alcohol or interesterifying the ester with an acid, an alcohol or a second ester in the presence of the lipolytic enzyme variant. Favorably, the improved thermostability allows the process to be conducted at a relatively high temperature which may be favorable to increase the rate of reaction and to process high-melting substrates.

The ester may be a carboxylic acid ester, e. g. a triglyceride. The interesterification may be done in the presence or absence of a solvent. The enzyme may be used in immobi- lized form. The process may be conducted in analogy with WO 8802775, US 6156548, US 5776741, EP 792106, EP 93602, or EP 307154.

Use in textile industry The variant may be used in a process for enzymatic removal of hydrophobic esters from fabrics, which process comprises treating the fabric with an amount of the lipolytic en- zyme effective to achieve removal of hydrophobic esters from fabric. The treatment may be done at a temperature of 75°C or above, e. g. for a period of 1-24 hours. The treatment may be preceded by impregnating the fabric with an aqueous solution of the lipase variant to a liquor pick-up ratio of 50-200%, and may be followed by washing and rinsing to remove the fatty ac- ids.

The process may be conducted in analogy with US 5578489 or US 6077316.

Use in detergents The variant may be used as a detergent additive, e. g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e. g. 0.01-1) mg per gram of detergent or 0.001-100 (e. g.

0.01-10) mg per liter of wash liquor. This may be done in analogy with WO 97/04079, WO 97/07202, WO 97/41212, WO 98/08939 and WO 97/43375.

Use for leather The variants of the invention can also be used in the leather industry in analogy with GB 2233665 or EP 505920.

Nomenclature for amino acid substitutions The nomenclature used herein for defining amino acid substitutions uses the single- letter code, as described in WO 92/05249.

Thus, D27N indicates substitution of D in position 27 with N. D27N/R indicates a sub- stitution of D27 with N or R. L227X indicates a substitution of L227 with any other amino acid.

D27N +D111A indicates a combination of the two substitutions.

Homology and alignment For purposes of the present invention, the degree of homology may be suitably de- termined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Ge- netics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

In the present invention, corresponding (or homologous) positions in the lipase se- quences of Rhizomucor miehei (rhimi), Rhizopus delemar (rhidl), Thermomyces lanuginosa

(former; Humicola lanuginosa) (SP400), Penicillium camembertii (Pcl) and Fusarium ox- ysporum (FoLnp11), are defined by the alignment shown in Figure 1 of WO 00/32758.

To find the homologous positions in lipase sequences not shown in the alignment, the sequence of interest is aligned to the sequences shown in Figure 1. The new sequence is aligned to the present alignment in Fig. 1 by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is provided in the GCG program package (Pro- gram Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-45). The following settings are used for polype- tide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

Procedure for obtaining thermostable variants Variants of a lipolytic enzyme can be obtained by methods known in the art, such as site-directed mutagenesis, random mutagenesis or localized mutagenesis, e. g. as described in WO 9522615 or WO 0032758.

Thermostable variants of a given parent lipolytic enzyme can be obtained by the fol- lowing standard procedure: Mutagenesis (error-prone, doped oligo, spiked oligo) Primary Screening Identification of more temperature stable mutants Maintenance (glycerol culture, LB-Amp plates, Mini-Prep) Streaking out on another assay plate-secondary screening (1 degree higher then primary screening) 'DNA Sequencing Transformation in Aspergillus Cultivation in 100 ml scale, purification, DSC Primary screening Assay The following assay method is used to screen lipolytic enzyme variants and identify variants with improved thermostability.

E. coli cells harboring variants of a lipolytic enzyme gene are prepared, e. g. by error- prone PCR, random mutagenesis or localized random mutagenesis or by a combination of beneficial mutants and saturation mutagenesis.

The assay is performed with filters on top of a LB agar plate. E. coli cells are grown on cellulose acetate filters supplied with nutrients from the LB agar plate and under the selection pressure of ampicillin supplied with the LB agar. Proteins including the desired enzyme are col-

lected on a nitrocellulose filter between LB agar and cellulose acetate filter. This nitrocellulose filter is incubated in a buffer of desired pH (generally 6.0) and at the desired temperature for 15 minutes (e. g. 78 degrees for the T. lanuginosus lipase). After quenching the filters in ice- water, the residual lipase activity is determined through the cleavage of indole acetate and the subsequent coloration of the reaction product with nitro-blue tetrazolium chloride as described by Kynclova, E et al. (Journal of Molecular Recognition 8 (1995) 139-145).

The heat treatment applied is adjusted so that the parent generation is slightly active, approximately 5-10 % compared to samples incubated at room temperature. This facilitates the identification of beneficial mutants.

EXAMPLES Example 1: Expression of lipase Plasmid pMT2188 The Aspergillus oryzae expression plasmid pCaHj 483 (WO 98/00529) consists of an expression cassette based on the Aspergillus niger neutral amylase 11 promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the A. niger amyloglycosidase terminater (Tamg). Also present on the plasmid is the As- pergillus selective marker amdS from A. nidulans enabling growth on acetamide as sole nitro- gen source. These elements are cloned into the E. coli vector pUC19 (New England Biolabs).

The ampicillin resistance marker enabling selection in E. coli of this plasmid was replaced with the URA3 marker of Saccharomyces cerevisiae that can complement a pyrF mutation in E. coli, the replacement was done in the following way: The pUC19 origin of replication was PCR amplified from pCaHj483 with the primers 142779 (SEQ ID NO: 3) and 142780 (SEQ ID NO: 4).

Primer 142780 introduces a Bbul site in the PCR fragment. The Expand PCR system (Roche Molecular Biochemicals, Basel, Switserland) was used for the amplification following the manufacturers instructions for this and the subsequent PCR amplifications.

The URA3 gene was amplified from the general S. cerevisiae cloning vector pYES2 (Invitrogen corporation, Carlsbad, Ca, USA) using the primers 140288 (SEQ ID 5) and 142778 (SEQ ID 6).

Primer 140288 introduces an EcoRl site in the PCR fragment. The two PCR frag- ments were fused by mixing them and amplifying using the primers 142780 and 140288 in the splicing by overlap method (Horton et al (1989) Gene, 77,61-68).

The resulting fragment was digested with EcoRl and Bbul and ligated to the largest fragment of pCaHj 483 digested with the same enzymes. The ligation mixture was used to

transform the pyrF E. coli strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa (Mandel, M. and A. Higa (1970) J. Mol. Biol. 45,154). Transformants were selected on solid M9 medium (Sambrook et. al (1989) Molecular cloning, a laboratory manual, 2. edition, Cold Spring Harbor Laboratory Press) supplemented with 1 gui casaminoacids, 500 Rg/i thiamine and 10 mg/l kanamycin.

A plasmid from a selected transformant was termed pCaHj 527. ThePna2/tpi pro- moter present on pCaHj527 was subjected to site directed mutagenises by a simple PCR ap- proach.

Nucleotide 134-144 was altered from SEQ ID NO: 7 to SEQ ID NO: 8 using the mutagenic primer 141223 (SEQ ID NO: 9).

Nucleotide 423-436 was altered from SEQ ID NO: 10 to SEQ ID NO: 11 using the mutagenic primer 141222 (SEQ ID 12).

The resulting plasmid was termed pMT2188.

Plasmid pEN11849 Plasmid pEN11849 was made in order to truncate the pyrG gene to the essential se- quences for pyrG expression, in order to decrease the size of the plasmid, thus improving transformation frequency. A PCR fragment (app. 1800 bp) was made using pEN11299 (de- scribed in WO 00/24883) as template and the primers 270999J8 (SEQ ID 13) and 270999J9 (SEQ ID 14).

The PCR-fragment was cut with the restriction enzymes Stul and Sphl, and cloned into pEN11298 (described in WO 0024883), also cut with Stul and Sphl ; the cloning was veri- fied by sequencing.

Plasmid pEN11861 Plasmid pEN11861 was made in order to have the state of the art Aspergillus promoter in the expression plasmid, as well as a number of unique restriction sites for cloning.

A PCR fragment (app. 620 bp) was made using pMT2188 (see above) as template and the primers 051199J1 (SEQ ID 15) and 1298TAKA (SEQ ID 16).

The fragment was cut BssHII and Bgl II, and cloned into pEN11849 which was also cut with BssHll and Bgl II. The cloning was verified by sequencing.

Plasmid pEN11902 Plasmid pEN11902 was made in order to have a promoter that works in both E. coli and Aspergillus. This was done by unique site elimination using the"Chameleon double stranded site-directed mutagenesis kit"as recommended by Stratagene@.

Plasmid pEN11861 was used as template and the following primers with 5'phosphory- lation were used as selection primers: 177996 (SEQ ID 17), 135640 (SEQ ID 18) and 135638 (SEQ ID 19).

The 080399J19 primer (SEQ ID NO: 20) with 5'phosphorylation was used as mutagenic primer to introduce a-35 and-10 promoter consensus sequence (from E. coll) in the Aspergillus expression promoter. Introduction of the mutations was verified by sequencing.

Plasmid pSMin001 Plasmid pSMinO01 was made in order to permit the expression of the T. lanuginosus lipase in E. coli and Aspergillus.

Plasmid pAHL (described in WO 9205249) was used as template for PCR to amplify the T lanuginosus lipase gene with the following Primers: 19671 (SEQ ID NO: 21) and 991213J5 (SEQ ID NO: 22). Primer 991213J5 introduced a Sacll site into the PCR fragment.

The PCR fragment (appr. 1100 bp) was cut with BamHl and Sacll and cloned into pEni1902 cut with the same enzymes. The cloning was verified by DNA sequencing. The plasmid was transformed in E. coli DH5a, and lipase expression was detected by using the described filter assay.

Using this newly developed plasmid it was possible to express the desired enzyme in Aspergillus without any modification. The achieved expression rates in E. coli were quite low, but sufficient for the screening assay.

Example 2: Production of thermostable lipase variants Several techniques were used to create diversity in the T lanuginosus lipase gene: error-prone PCR, localized random mutagenesis with the aid of doped oligonucleotides, and site-directed mutagenesis.

Variants exhibiting higher temperature stability were selected by the primary assay described above, and were cultivated in LB media and streaked out again on assay plates as described above for a secondary screening. The assay in the secondary screening was per- formed with a 1-1.5 degrees higher temperature. The DNA of mutants still active under these conditions were sequenced and transformed into Aspergillus to obtain a higher amount of pro- tein, followed by a chromatographic purification. The purified enzyme was used for DSC analy- sis to prove the enhancement of the stability.

Next, amino acid substitutions found in the beneficial variants were combined, and saturation mutagenesis was used to ensure that all 20 amino acids were introduced in the de- sired positions.

Example 3: Thermostability of lipase variants All samples identified as more thermostable in the primary and secondary screening in Example 2 were purified to homogeneity, and their stability was checked by differential scanning calorimetry (DSC) at pH 5.0 and/or 7.0 to determine the stability of the protein, given by its melting temperature (TM) The parent lipase from T. lanuginosus was included for com- parison.

Eight variants were found to have increased thermostability at pH 5.0, four variants showing an increase of more than 4°C. Two variants were tested at pH 7.0 and found to have improved thermostability.

Example 4: Thermostability of lipase variants by DSC A number of variants of the T. lanuginosus lipase were prepared and purified, and the thermostability was checked by differential scanning calorimetry (DSC) at pH 5.0 to determine the stability of the protein, given by its melting temperature (TM). The parent lipase from T. la- nuginosus was included for comparison.

The following variants were found to be more thermostable than the parent lipase : D111G+S216P D27N L227F S2241 + G225W + T226N + L227P + V228C L227F + V2281 G225P W221C + G246C The following variants were found to be more thermostable than the parent lipase with at least 4°C increase of the melting temperature.

D27R + D111G + S216P<BR> <BR> D27N + D111A<BR> <BR> D27R + D111G + S216P +L227G +P256T D27R + D111G + S216P + L227F + P256T D27R + D111 G + S216P + L227G D27S + D111G + S216P D27R + D111A + S216P + L227G + P256T D27R + D111G + S216P + G225P + L227G + P256T D27R + T37S + D1211G + S216P + L227G + P256T D27R + N39F+ D111G + S216P +L227G + P256T D27R + G38C + D111G + S216P + L227G + P256T D27R + D111G + S216P + L227G + T2441 + P256T D27R + G91A + D111G + S216P + L227G + P256T N251 +D27R + D111A + S216P + L227G + P256T N25L +D27R + D111A + S216P + L227G + P256T N26D +D27R + D111A + S216P + L227G + P256T D27R +K46R + D111A + S216P + L227G + P256T D27R + V60N +D111A + S216P + L227G + P256T D27R + D111A + P136A +S216P + L227G + P256T D27R + D111A + S216P + L227G + P256T +1265F D27R + S58Y +D111A + S216P + L227G + P256T + N26D +D27R +E56Q +D111A + S216P + L227G + P256T D27R +G91A +D96E +L97Q +D111A +S216P + L227G + P256T D27R +G91A +D111A + S216P + L227G + P256T + D27R + G91T +N94S +D111A +S216P + L227G + P256T D27R +G91 S +D111A + S216P + L227G + P256T + D27R +G91 N +D111A + S216P + L227G + P256T D27R +D96E +D111A + S216P + L227G + P256T D27R +190L +G91A +N94K +D111A + S216P + L227G + P256T D27R +G91 S +F95V +D111A + S216P + L227G + P256T

Example 5: Thermostability by plate assay A number of variants of the T. lanuginosus lipase were prepared and tested for ther- mostability as described above under"primary screening assay". The parent lipase from T. la- nuginosus was included for comparison.

The following variants were found to be more thermostable than the parent lipase : D27R +190V +G91 S +D111A + S216P + L227G + P256T D27R +G91 N +N94R +D111A + S216P + L227G + P256T D27R +190L +L93F +D96N +D111A + S216P + L227G + P256T D27R +190L +G91A +D96E +D111A + S216P + L227G + P256T D27R +G91 S +L93F +D111A + S216P + L227G + P256T D27R +G91T +N94K + D111A + S216P + L227G + P256T D27R +G91T +D111A + S216P + L227G + P256T D27R +L93F + D111A +D137N + S216P + L227G + P256T D27R +G91S +D96N +D111A + S216P + L227G + P256T D27R +G91W +D111A + S216P +L227G + P256T D27R +I90L +G91T +D111A + S216P + L227G + P256T D27R +G91S +L93F +N94R +D96G +D111A +S216P + L227G + P256T D27R +G91T +D96N +D111A +S216P + L227G + P256T D27R +190V +G91T +L93F +N94K +D111A +S216P + L227G + P256T D27R +L93V +D111A +S216P + L227G + P256T D27R +G91S +N94K +D111A +S216P + L227G + P256T D27R +I90L +G91T +D111A +S216P + L227G + P256T D27R +G91 S +L93F +F951 +D96N +D111 A +S216P + L227G + P256T D27R + D111A +V1871 + S216P + L227G + P256T D27R + D111A +F211Y + S216P + L227G + P256T D27R + R118M +D111A +A131V +S216P + L227G + P256T D27R +P29S +R84C +D111A + H135Y +S216P + L227G + P256T D27R +T32S +D111A + H135Y +S216P + L227G + P256T D27R +G91R +D111A +1238V +S216P + L227G + P256T D27R +F571I+I76V +N101D +D111A + N162R +S216P + L227G + P256T D27R +F51 L +D111A + S216P + L227G + P256T