This application is a continuation-in-part of 5 U.S. application 08/044,621 filed April 8, 1993.

> BACKGROUND AND PRIOR ART

1

The present invention is directed to a modified 10 xylanase, which shows an improved stability when compared to the naturally occurring xylanase. Specifically the present invention is directed to a modified xylanase, wherein said xylanase has increased thermostability, increased temperature optimum and/or increased pH optimum, 15 and wherein said xylanase is modified through either the introduction of at least one non-native disulfide bridge, the introduction of at least one N-terminal mutation, or combinations thereof.

20 Current strategies for the production of paper use a chemical bleaching step, which is essential for the colour and quality of the paper. Chemical bleaching uses chlorine or chlorine dioxide and produces substantial amounts of by-products, which are environmental pollutants.

25 The bleaching process can be enhanced by using an enzymatic pre-treatment with xylanase (Paice and Jurasek, 1984 Journal of Wood Chemistry and Technology, 4(2) :187-198) , which lowers the chlorine charge needed to affect bleaching, thereby reducing pollutants. In addition there

30 is less bleaching chemical used, which lowers the chemical costs. New bleaching technology using, oxygen or peroxides with xylanase, are also as effective in brightening the pulp.

« 35 The step in the process where the enzyme is applied is after a hot alkali treatment, so that the pulp is very basic and hot. Both of these conditions are sub-optimal for xylanase enzymatic activity. Many pulp mills have the capability to acidify the pulp to a pH which

is closer to the pH optimum for the enzyme and the pulp is adjusted from pH 10-11 to pH 6-8. Due to corrosion risk the pH should preferably be higher than 6.5. A xylanase with a higher pH optimum would be advantageous, especially if the pH-adjustment step could be completely eliminated. Cooling the pulp is too energy intensive (expensive) to be used in the mill setting. Therefore, a xylanase, which is active at a higher temperature would be useful in the bio-bleaching processes. According to Nissen et al. (Xylanases for the Pulp and Paper Industry in: Xylans and Xylanases, Ed. J. Visser et al., 1992, Elsevier Science publishers B.V.) the ideal xylanase for the bleaching process would have a temperature optimum of 70°C and a pH optimum of 9.

Xylanase also has uses in non-pulp applications. Xylanases have been reported to be useful in clarifying juice and wine (Zeikus. J.G., Lee, Y.-E., and Saha, B.C. 1991. ACS Symp. Ser. 460:36-51; Beily, P. 1991. ACS Sy p. Ser. 460:408-416; Woodward J. 1984. Top Enzyme Ferment. Biotechnol. 8:9-30), extracting coffee, plant oils and starch (McCleary, B.V. 1986. Int. J. Biol. Macromol. 8:349-354; Beily, P. 1991. ACS Symp. Ser. 460:408-416; Woodward J. 1984. Top Enzyme Ferment. Biotechnol. 8:9-30), for the production of food thickeners (Zeikus. J.G., Lee, Y.-E., and Saha, B.C. 1991. ACS Symp. Ser. 460:36-51), altering texture in bakery products (Maat, J. , Roza. M. , Verbakel, J. , Stam, H. , Santos da Silva, M.J., Bosse, M. , Egmond, M.R., Hagemanε, M.L.D., v.Gorcom, R.F.M., Hessing, J.G.M., v.d.Hodel, C.A.M.J.J. , and Rotterdam,C. 1992. In Xylans and xylanases. Visser, J. , Beld an, G., Kusters-van Someren, M.A. and Voragen, A.G.J., eds. Elsevier Sci pub., Amsterdam. ISBN 0-444-894-772; McCleary, B.V. 1986. Int. J. Biol. Macromol. 8:349-354), and in the washing of super precision devices and semiconductors (Takayuki, I., Shoji, S. US patent number 5078802, issue date 92-01-07). Several of these application could benefit from a thermostable xylanase, for example, food processing at elevated temperatures.

A thermostable xylanase from Thermoascus aurantiacus was produced in US Patent 4,966,850 (Yu et al.) from a particular strain of T. aurantiacus, while culturing the strain at high temperature culturing conditions, however this enzyme is not a member of the family G xylanases (Gilkes et al. 1991, Microbiol. Reviews 55(2) :303-315) , which is the subject of this patent.

Arase et al. (FEBS 316:123-127, 1993) report improvements in thermostability of Bacillus pumilus xylanase through random mutagenesis of the gene by chemical mutagens. Their improvements in thermostability are minor in comparison to that of the present invention. The prior art most stable mutant maintained 40% residual activity after a short period of 20 minutes at 57°C. This mutant had a low specific activity, equivalent to 19% of the wild type B. pumilus xylanase. It is noted that the temperature optimum and pH optimum of these prior art mutants were not studied.

Site directed mutagenesis has been used to produce more stable proteins. Disulfide (SS) bonds in proteins restrict the degree of freedom for the unfolded state and thereby stabilize the folded state. The first type of protein stabilization performed by genetic manipulation was the introduction of disulfide bonds. One or two amino acids in the protein are replaced with cysteines; a disulfide bond forms in vivo or in vitro. If the introduced disulfide bond causes no or little tertiary structural change, the cross-links stabilizes the protein. Disulfide bonds have been engineered into T4 lysozyme (T4L) (Perry, L.J. and Wetzel, R. (1984) Science 226, 555-557; Wetzel, R. , Perry, L.J. Baase, W.A. and Becktel, W.J. (1988) Proc. Nat. Acad. Sci. USA 85, 401-405), subtilisin (Wells, J.A. and Powers, D.B. (1986) J. Biol. Chem. 261,

6564-6570; Mitchinson, C. and Wells, J.A. (198 ), dihydrofolate reductase (DHFR) (Villafranca, J.E. , Howell, E.E., Oatley, S.J., Xuong, N. and Kraut, J. (1987) Biochemistry 26, 2182-2189) , and the Phage λ repressor (Cl) (Sauer, R.T,, Kehir, K. , Stearman, R.S. et al. (1986) Biochemistry 25, 5992-5998) with stabilization occurring in some cases but not others.

For example, in T4L the introduction of SS bonds showed an increase in thermostability of between 6 and 11°C based on reversible denaturation at pH 2 (Matsumura et al, 1989, Nature 342:291-293). This data does not show how much activity remains after heating the sample, which in a functional sense is what is important for an industrial enzyme. The RNASE H SS bond mutant is also stabilized by 11.8 ^β C as measured by reversible thermal denaturation, but has no enzymatic activity (Kanaya et al, 1991, Journal of Biological Chemistry 226(10) :6038-6044) . In DHFR the artificial SS bond contributes to stability of the protein against chemical denaturation, but does not confer thermostability (Villafranca et al, 1987, Biochemistry 26:2182-2189) . A similar situation occurs with subtilisin, where 5 different engineered SS bonds do not confer thermostability (Mitchinson and Wells, 1989, Biochemistry 28:4807-4815).

SUMMARY OF INVENTION

According to the present invention there is provided a modified xylanase, wherein said xylanase has increased stability and wherein said xylanase is modified through either the introduction of at least one non-native disulfide bridge, the introduction of at least one N- terminal mutation, or combinations thereof. The modified xylanases of the present invention exhibit at least one of the following traits: increased thermostability, increased temperature optimum or increased pH optimum.

In one embodiment of the present invention, the modified xylanase has been modified by the introduction of an intra-molecular disulfide bridge between a cysteine amino acid, which has been introduced on the last strand of sheet III, and a cysteine amino acid, which has been introduced on the alpha helix.

In a further embodiment of the present invention, the modified xylanase has been modified by the introduction of an inter-molecular disulfide bridge between two xylanase molecules, wherein a cysteine amino acid has been introduced in each of said two molecules.

In a further embodiment of the present invention, the modified xylanase has been modified by the introduction of a mutations at the N-terminus of the xylanase.

In one embodiment of the present invention there is provided a modified family G xylanase essentially having the structure of the B. circulans enzyme or mutated to essentially have this structure, wherein said xylanase has increased stability and wherein said xylanase is modified through either the introduction of at least one non-native disulfide bridge, the introduction of at least one N- terminal mutation, or combinations thereof; wherein the disulfide bridge is an intra- molecular bridge between a cysteine amino acid, which has been introduced on the last strand of beta-sheet III, and a cysteine amino acid, which has been introduced on the alpha helix, or on either side, adjacent to the alpha helix or the disulphide bridge is an inter-molecular bridge between two xylanase molecules, wherein a cysteine amino acid has been introduced in each of said two molecules, on the external region; and wherein the N-terminal mutation is selected from at least one mutation of the group consisting of the introduction of tyrosine at amino acid position 8,

introduction of phenylalanine at amino acid position 8, introduction of proline at amino acid position 22, and introduction of an N-terminal to C-terminal disulfide bridge, and wherein these N-terminal mutations can be used in combination with other N-terminal mutation introduced at amino acid position 1 to 25 of the N-terminal region, based on the amino acid numbering from B . circulans xylanase.

In a further embodiment of the present invention there is provided a modified family G xylanase essentially having the structure of the B . circulans enzyme or mutated to essentially have this structure, wherein said xylanase has increased stability and wherein said xylanase is modified through either the introduction of at least one non-native disulfide bridge, the introduction of at least one N-terminal mutation, or combinations thereof; and wherein said modified xylanase is produced from clones selected from the group consisting of TS1, TS2, TS3, TS3a,

TS5a, TS6a, TS7a, TSlOa, TS14a, TS15a, TS17a, TS19a, TS20a and TS21a.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the multiple sequence alignment among low molecular weight xylanases.

Figure 2 shows a stereodiagram of the overall fold of the three-dimensional crystal structure of the Bacillus circulans xylanase. The strands of beta-sheet are represented by arrows and the alpha-helix is represented by a cylinder. The three beta-sheets referred to in the text are indicated by I, II, and III.

Figure 3 shows a stereodiagram of the superimposed three-dimensional crystal structures of the xylanases from B. circulans and T. harziamim. The structures are drawn such that the positions of the alpha-carbons of each

amino acid are linked by a straight line. The B. circulans structure is drawn in a thick line and the T. harzianwn structure is drawn in a thin line. The T. harzianum xylanase has a slightly more open active site due to different intermolecular crystal contacts.

Figure 4 shows a stereodiagram of the superimposed structures of the wild-type and disulfide- containing mutant (TS1) of the B. circulans xylanase in the vicinity of the mutation (residues 100 and 148) . The wild- type enzyme is drawn in thick lines and the mutant is drawn in thin lines.

Figure 5 shows a stereodiagram of the superimposed structures of the xylanases from B. circulans and

T. harzianum in the vicinity of the disulfide bond shown in

Figure 4. The B. circulans structure is drawn in a thick line and the T. harzianum structure is drawn in a thin line.

Figure 6 shows the native sequence (SEQ ID NO:l) of the B. subtilis xylanase gene from plasmid pBSX. The binding sites for the PCR primers and the S100C mutagenic primer are underlined.

Figure 7 shows the semi-synthetic gene sequence

(SEQ ID NO:2) for B. circulans xylanases. The sites for UDNA mutagenesis to produce TS2 are shown with underlining.

Figure 8 shows the SDS PAGE results of monomer and dimer fraction from TS4a, TS4M and TS4D. Lane 1 is TS4a monomer fraction, reduced; Lane 2 is as in lane 1 but non-reduced; Lane 3 is TS4a dimer fraction, reduced; Lane 4 is as lane 3 but non-reduced; Lane 5 is TS4M monomer fraction, reduced; Lane 6 is as in lane 5 but non-reduced; Lane 7 is TS4D dimer fraction reduced; Lane 8 is as lane 7 but non-reduced; Lane 9 is BCX wild type reduced; Lane 10

is as in lane 9 but non-reduced. For lanes 1-4, approximately 1 μg of protein was loaded and for the other lanes, approximately 2 μg was loaded.

Figure 9 shows the complete synthetic gene sequence (SEQ ID NO:3) encoding the B. circulans xylanase in the plasmid pXYbc.

Figure 10 shows the electrophoretic mobility of the disulfide bridge containing mutants of B. circulans xylanase. Lane 1 shows the molecular weight standard, lane 2 is the BCX wild-type, reduced, lane 3 is as in lane 2 but non-reduced, lane 4 is the TSl mutant, reduced, lane 5 is the TSl mutant, non-reduced, lane 6 is the TS2 mutant, reduced and lane 7 is a TS2 mutant, non-reduced.

Figure 11 shows a comparison of the thermostability at 58 ^β C of various mutants of B. circulans xylanase. The curve shown for TS4 is for the mixture of both monomer and dimer.

Figure 12 shows a comparison of the thermostability at 61°C of various mutants of the B. circulans xylanases. The curve shown for TS4 is for the mixture of both monomer and dimer.

Figure 13 shows the thermostability of the TS4a (S179C) dimer at 58°C.

Figure 14 shows the thermostability at 58 ^β C of B. circulans xylanase mutants TS3 and TS4D, which are combinations of mutants shown in Figures 11 and 12.

Figure 15 shows the thermostability at 62°C of B. circulans xylanase mutants TS3 and TS4D, which are combinations of mutants shown in Figures 11 and 12.

Figure 16 shows the thermostability at 6 °C of B. circulans xylanase mutants TS3 and TS4D, which are combinations of mutations shown in Figures 11 and 12.

Figure 17 illustrates the effect of temperature on enzyme activity. With the thermostable xylanase mutants shown, an increase of 18 ^β C resulted in between 2.6 and 4 fold increase in activity.

Figure 18 shows the thermostability of xylanase

BCX and mutants wherein xylanase from the wild-type or mutant strains were heated at various temperatures for 30 minutes. After cooling to 20 ^β C a residual enzymatic activity of the heated samples were determined via the HBAH assay at 40°C.

Figure 19 shows the molecular structure of residue 6-10 and 16-20 of the N-terminus of the wild-type BCX. CA represents the alpha carbon atom of each residue. CB is the beta-carbon atom of the side-chain. OD1 is the delta oxygen atom in the side-chain of asparagine-8. The number next to CA, CB and OD1 designates the residue to which these atoms belong. The two asterisks are the two buried water molecules. The broken lines represent the hydrogen bonds.

Figure 20 shows the molecular structure of the same region as Figure 19, illustrating the postulated effect of the asparagine to tyrosine mutation at residue-8 (TS5a) .

Figure 21 shows the average main-chain B-factors for the xylanase from B. circulans. The B-factor is a measure of the spreading out of electron density. It is thus a measure of the mobility of the atoms in a structure. This plot shows the B-factors (or mobility) averaged over all main-chain atoms in each amino acid. Thus the regions near

the amino-terminus and near residue 120 are the most mobile parts of the structure.

Figure 22 shows the average main-chain B-factors for the xylanase from T. harzianum.

Figure 23 shows the average main-chain B-factors for the disulfide-containing mutant (TSl) of the xylanase from B. circulans.

Figure 24 shows a Ramachandran plot for the xylanase from B. circulans. The two residues (D121 and A165) with main-chain dihedral angles outside of normal limits are labelled. Glycines are shown with circles, while all other amino acids are shown with "plus" signs.

Figure 25 shows a Ramachandran plot for the xylanase from T. harzianum. The three residues (T2, Nil and

S181) with main-chain dihedral angles outside of normal limits are labelled. Glycines are shown with circles, while all other amino acids are shown with "plus" signs.

Figure 26 shows enzymatic activity of selected xylanase mutants in a thirty minute assay on soluble xylan at temperatures ranging from 40 ^β C to 80°C.

Figure 27 shows the enzymatic activity of selected xylanase mutants at a pH ranging from 4 to 10.

Figure 28 demonstrates the bleaching of pulp at pH 8 by selected xylanase mutants at temperatures ranging from 40°C to 70 ^C C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to modified xylanases belonging to the family G xylanases (Henrissat

and Bairoch, Bioche , J. 293: 781-788, 1993 and Gebler et al., J. Biol. Chem. 266: 12559-12561, 1992; incorporated herewith by reference) recently renamed to family 11 (Henrissat and Bairoch, Biochem. J. 293: 7681-788, 1993). The original family G classification was based on the primary sequence comparison and hydrophobic cluster analysis of 4 protein sequences that were in the database at the time the paper, regarding the original classification, was published. Since that time, an additional 16 sequences have been added to this group. In addition to the sequence comparisons, the group definition, also included the stereochemical outcome of the hydrolysis reaction (catalytic mechanism) . The grouping by sequence analysis predicts the protein folding will be similar, and the examination of the stereochemistry of the reaction is an indirect measure of how the active site residues are oriented (the prediction is enzymes with similar structure give the same stereochemistry in the reaction products) .

The complete amino acid sequence of the low molecular weight xylanase has been determined from a number of different bacterial and fungal sources (M. Yaguchi et al. , Amino Acid Sequence of the Low-Molecular-Weight Xylanase from Trichoderma viride, Xylans and Xylanases, edited by J. Visser et al, 1992, 149-154). The sequence comparison reported in the reference referred above has been extended to show the sequence similarities and differences between the bacteria and fungal family G xylanases as listed below.

BACTERIAL Bacillus pumilus

Fukusaki , E. , Panbangred, W. , Shinmyo, A. , Okada , H. FEBS Letters 171: 197-201 (1984) Clostridium acetobutylicum , XYN B

Zappe, H. , Jones, W.A. , Woods, D.R. Nucleic Acids Research 18:2179 (1990)

Ruminococcus flavefaciens

Zhang, J. , Flint, H.J. EMBL database accession number Z11127 (1992)

Strepwmyces sp. No. 36a

Nagashima, M. , Okumoto, Y., Okanishi, M. Trends in Actinomycetoligia, 91-96 (1989) Strepwmyces lividans, XYN B and XYN C

Shareck, F., Roy, C. , Yaguchi, M. , Morosoli, R. , Kluepfel, D.Gene, 107:75-82 (1991)

Bacillus circulans

Yang, R.C.A., MacKenzie, C.R., Narang, R.A. Nucleic Acids Res. 16:7187 (1988) Bacillus subtilis Paice, M.G., Bourbonnais, R. , Desrochers, M. ,

Jurasek, L. , Yaguchi, M. Arch. Microbiol. 144:201-206 (1986)

FUNGAL Trichoderma reesei, XYN I and XYN II

Torronene, A., Mach, R.L. , Messner, R. , Gonzalez, R. , Kalkkinen, N. , Harkki, A., Kubicek, C.P. Bio/Technology 10:1461-1465 (1992) Trichoderma viride, 20KD Yaguchi, M. , Roy, C, Ujlie, M. , Watson, D.C.,

Wakarchuk, W. Xylans and Xylanases. ed. by J. Visser et al., Elsevier, pp. 149-154 (1992) Trichoderma harzianum , 20KD

Yaguchi, M. , Roy, C. , Watson, D.C., Rollin, F. , Tan. L.U.L., Senior, D. J. , Saddler, J.N. Xylans and Xylanases _f ed. by J. Visser et al., Elsevier, pp. 435-438 (1992) Schizophyϊlum commune, Xylanase A

Oku, T., Roy, C, Watson, D.C., Wakarchuk, W. , Yaguchi, M. , Jurasek, L. , Paice, M.G.

(unpublished)

Aspergillus niger var. awamori

Maat, J. , Roza, M. , Verbakel, J. , Sta , H. ,

Santos da Silva, M.J., Egmond, M.R. , Hagemans,

M.L.D., Gorco , R.F.M.v., Hessing, J.G.M., Hondel, C.A.M.J.J.v.d. , Rotterdam, C.v. Xylans and Xylanases. ed. by J. Visser et al.,

Elsevier, pp. 349-360 (1992) Aspergillus tubigensis, XYL A de Graaff, L.H. , van den Broeck, H.C., van Ooijan, A.J.J. , Visser, J. Xylans and Xylanases. ed. by J. Visser et al., Elsevier, pp. 235-246

(1992)

Figure 1 shows a multiple sequence alignment among low molecular mass xylanases obtained with GeneWorks version 2.2.1 (IntelliGeneticε, Inc., Mountain View, CA) , which has been manually edited to conform to the structural homology between the xylanases from B. circulans and T. harzianum. The extended N-terminal sequence of the C. acetobutylicum xylanase B (residues 1-31) and the c-terminal sequences of S. lividans, xylanase B (residues 216-293) are not shown.

Table 1 shows the amino acid sequence identity (percentage) between different groups of family G xylanases, when compared to B pumilus , B . circulans, T. harzianum and Schizo . commune as references. Due to the sequence homology among related xylanases, one could expect that mutations for the introduction of SS bonds or for the introduction of mutations at the N-terminal end of the xylanase, as demonstrated in the examples below for B. circulans xylanase, can be extended to other xylanases from bacterial or fungal sources to produce similar effects.

Table l

Amino Acid Sequence Identity (%) between the Family G

Xylanases

The xylanase from B. circulans is functionally identical to that from B. subtilis, which will be seen later in

the Examples. These proteins differ at only one residue and are among the shortest xylanases, with their N-terminus lacking at least 10 amino acid residues compared to other xylanases.

The three-dimensional crystal structure of the B. circulans xylanase is represented in Figure 2. This ribbon representation shows that the structure is composed of three beta-sheets and one alpha-helix. The first two beta- sheets are roughly parallel, while sheet III is at about a 90 ^* angle to sheet II. Sheets I and II are each composed of five strands, while sheet III contains 6 strands. The alpha-helix lies across the back of sheet III and the last two strands of sheet III fold over one edge of the alpha- helix. The active site lies in the cleft between sheets II and III. The x-ray co-ordinates of the structure is given below.

32.404 42.580 22.419 34.898 42.615 22.344

35.540 41.613 22.023

33.610 42.325 20.269

33.627 42.997 21.599

35.283 43.440 23.308 36.455 43.158 24.114

37.000 44.436 24.753

37.614 45.262 23.783

36.062 42.184 25.210

34.967 42.270 25.786 36.930 41.215 25.440

36.715 40.235 26.475

37.469 38.943 26.145

38.849 39.247 25.891

ATOM 23

1.00 17.34 ATOM 24

1.00 17.67 ATOM 25

1.00 18.99 ATOM 26

1.00 16.43 ATOM 28

1.00 16.02 ATOM 29

1.00 16.80 ATOM 30

1.00 19.10 ATOM 31

1.00 17.29 ATOM 32

1.00 21.94 ATOM 33

1.00 15.35 ATOM 34

1.00 16.73 ATOM 35

1.00 13.80 ATOM 37

1.00 12.53 ATOM 38

1.00 12.90 ATOM 39

1.00 14.19 ATOM 40

1.00 15.98 ATOM 41

1.00 17.93 ATOM 42

1.00 14.54 ATOM 43

1.00 16.97 ATOM 44

1.00 17.23 ATOM 45

1.00 21.34 ATOM 47

1.00 12.09 ATOM 48

1.00 11.58 ATOM 49

1.00 11.81 ATOM 51

1.00 12.62 ATOM 52

1.00 12.94 ATOM 53

1.00 12.88 ATOM 54

1.00 12.72

ATOM 55 CE2 TRP 6 44.595 33.232 31.184

1.00 13.51

ATOM 56 CE3 TRP 6 42.879 33.570 29.515 1.00 13 01 ATOM 57 CD1 TRP 6 43.982 34.661 32.791

1.00 13.74

ATOM 58 NE1 TRP 6 44.912 33.718 32.425

1.00 14.48 ATOM 60 CZ2 TRP 6 45.236 32.271 30.404 1.00 13.34 ATOM 61 CZ3 TRP 6 43.518 32.607 28.740 1.00 13.00 ATOM 62 CH2 TRP 6 44.681 31.974 29.187 1.00 12.55 ATOM 63 C TRP 6 40.292 34.122 32.670 1.00 12.15 ATOM 64 0 TRP 6 40.399 34.289 33.890 1.00 12.46 ATOM 65 N GLN 7 39.918 32.974 32.118 1.00 12.04 ATOM 67 CA GLN 7 39.656 31.763 32.877 1.00 12.76 ATOM 68 CB GLN 7 38.380 31.105 32.332 1.00 13.89 ATOM 69 CG GLN 7 38.105 29.698 32.821 1.00 16.24 ATOM 70 CD GLN 7 37.879 29.624 34.314 1.00 17.99 ATOM 71 OE1 GLN 7 37.712 30.644 34.993 1.00 19.93 ATOM 72 NE2 GLN 7 37.867 28.411 34.838 1.00 20.16 ATOM 75 C GLN 7 40.843 30.818 32.726 1.00 12.58 ATOM 76 O GLN 7 41.211 30.455 31.612 1.00 11.22 ATOM 77 N ASN 8 41.471 30.451 33.837 1.00 12.68 ATOM 79 CA ASN 8 42.615 29.550 33.784 1.00 13.88 ATOM 80 CB ASN 8 43.924 30.351 33.687 1.00 15.02 ATOM 81 CG ASN 8 45.166 29.462 33.674 1.00 16.98 ATOM 82 OD1 ASN 8 46.279 29.935 33.916 1.00 20.52

ATOM 83 ND2 ASN 8 44.990 28.192 33.377 1.00 17 00 ATOM 86 C ASN 8 42.604 28.712 35.045

1.00 14.62 ATOM 87 O ASN 8 43.215 29.075 36.057 1.00 15.63 ATOM 88 N TRP 9 41.894 27.594 34.978 1.00 14.02 ATOM 90 CA TRP 9 41.766 26.703 36.113 1.00 14.57

46.913 20.547 39.287 47.616 19.394 38.593 48.841 19.251 38.699 46.862 18.568 37.881 47.466 17.443 37.202 48.141 17.771 35.887 47.608 18.530 35.070 49.319 17.187 35.687 50.062 17.366 34.456 50.476 18.783 34.128 50.581 19.642 35.009 50.707 19.026 32.845 51.145 20.327 32.387 52.415 20.196 31.469 52.750 21.523 30.777 53.615 19.720 32.291 53.595 18.263 32.618 50.054 21.032 31.604 49.447 20.438 30.715 49.780 22.276 31.984 48.811 23.124 31.291 47.501 23.332 32.103 46.539 24.263 31.332 46.833 21.989 32.368 49.515 24.464 31.084 49.699 25.225 32.028 50.019 24.692 29.879 50.698 25.945 29.566

51.909 25.681 28.660 52.558 26.962 28.153 52.808 27.102 26.954 52.840 27.888 29.049 49.674 26.840 28.871 49.446 26.720 27.663 49.038 27.706 29.654 48.014 28.616 29.143 46.831 28.682 30.114 48.601 29.999 28.941 49.201 30.566 29.856 48.415 30.551 27.751 48.925 31.871 27.429 49.800 31.839 26.167 50.307 33.240 25.850 50.964 30.862 26.357 47.790 32.865 27.207 46.901 32.643 26.384 47.827 33.949 27.965 46.843 35.016 27.877 46.747 35.697 29.247 45.715 36.812 29.294 45.222 37.266 28.275 45.390 37.255 30.501 47.395 35.971 26.813 48.271 36.786 27.107 46.941 35.814 25.571 47.432 36.642 24.481

46.799 38.013 24.356 46.039 38.438 25.223 47.115 38.719 23.271 46.560 40.051 23.061 47.327 40.815 21.970 47.248 40.141 20.732 45.083 39.974 22.692 44.634 39.007 22.060 44.339 41.001 23.090 42.925 41.053 22.791 42.198 39.826 23.305 42.338 39.444 24.461 41.429 39.199 22.425 40.682 38.019 22.812 41.371 36.717 22.458 40.736 35.663 22.451 42.675 36.766 22.203 43.413 35.559 21.846 44.532 35.893 20.833 45.356 34.680 20.455 46.499 34.512 20.908 44.790 33.823 19.620 44.048 34.839 23.033 44.522 35.469 23.976 44.030 33.513 22.991 44.696 32.715 24.016 43.802 32.457 25.243 42.670 31.472 25.023

ATOM 264 O ASN 29 49.648 23.078 27.620

1.00 12.04

ATOM 265 N TRP 30 48.787 21.392 26.430 1.00 9.27 ATOM 267 CA TRP 30 48.589 20.575 ^' 27.619 1.00 7.76 ATOM 268 CB TRP 30 47.115 20.598 28.066 1.00 7.93 ATOM 269 CG TRP 30 46.123 19.856 27.209 1.00 7.36 ATOM 270 CD2 TRP 30 45.383 20.374 26.090 1.00 7.38 ATOM 271 CE2 TRP 30 44.448 19.387 25.719 1.00 6.82 ATOM 272 CE3 TRP 30 45.411 21.580 25.379 1.00 8.62 ATOM 273 CD1 TRP 30 45.632 18.610 27.444 1.00 6.99 ATOM 274 NE1 TRP 30 44.618 18.321 26.562 1.00 7.92 ATOM 276 CZ2 TRP 30 43.542 19.568 24.657 1.00 9.07 ATOM 277 CZ3 TRP 30 44.510 21.759 24.330 1.00 7.97 ATOM 278 CH2 TRP 30 43.592 20.757 23.982 1.00 7.80 ATOM 279 C TRP 30 49.112 19.166 27.431 1.00 7.73 ATOM 280 O TRP 30 49.151 18.641 26.305 1.00 8.50 ATOM 281 N SER 31 49.532 18.553 28.530 1.00 8.50 ATOM 283 CA SER 31 50.095 17.218 28.466 1.00 9.39 ATOM 284 CB SER 31 51.615 17.333 28.268

1.00 11.08 ATOM 285 OG SER 31 52.197 16.069 28.032 1.00 16.62

ATOM 287 C SER 31 49.799 16.425 29.735 1.00 8.65 ATOM 288 0 SER 31 50.040 16.912 30.846 1.00 9.67 ATOM 289 N ASN 32 49.246 15.224 29.556 1.00 9.23 ATOM 291 CA ASN 32 48.934 14.290 30.648 1.00 9.24 ATOM 292 CB ASN 32 50.215 13.581 31.076

1.00 11.30 ATOM 293 CG ASN 32 50.856 12.828 29.931 1.00 13.54 ATOM 294 OD1 ASN 32 50.311 11.835 29.448 1.00 15.18 ATOM 295 ND2 ASN 32 52.010 13.300 29.487 1.00 14.28 ATOM 298 C ASN 32 48.306 14.979 31.844 1.00 9.76

ATOM 299 O ASN 32 48.778 14.870 32.975 1.00 10.47

ATOM 300 N THR 33 47.187 15.634 31.589 1.00 .79 ATOM 302 CA THR 33 46.510 16.413 32.613 1.00 .75 ATOM 303 CB THR 33 45.734 17.580 31.943

1.00 10.19 ATOM 304 OG1 THR 33 44.705 17.033 31.099 1.00 10.42 ATOM 306 CG2 THR 33 46.668 18.456 31.117 1.00 10.04

ATOM 307 C THR 33 45.471 15.679 33.450 1.00 9.02 ATOM 308 O THR 33 45.203 14.490 33.268 1.00 9.65 ATOM 309 N GLY 34 44.930 16.436 34.397 1.00 9.55 ATOM 311 CA GLY 34 43.804 15.992 35.187 1.00 9.51 ATOM 312 C GLY 34 42.716 16.743 34.416 1.00 9.70 ATOM 313 O GLY 34 42.654 16.646 33.183 1.00 9.59 ATOM 314 N ASN 35 41.924 17.563 35.098 1.00 9.04 ATOM 316 CA ASN 35 40.873 18.355 34.450 1.00 9.43 ATOM 317 CB ASN 35 39.534 18.158 35.187

1.00 10.15 ATOM 318 CG ASN 35 38.361 18.897 34.531 1.00 12.88 ATOM 319 OD1 ASN 35 37.501 19.427 35.232 1.00 15.72 ATOM 320 ND2 ASN 35 38.301 18.905 33.204 1.00 13.33 ATOM 323 C ASN 35 41.290 19.823 34.496 1.00 9.12 ATOM 324 0 ASN 35 41.876 20.282 35.476 1.00 10.17

ATOM 325 N PHE 36 41.061 20.545 33.406 1.00 8.58 ATOM 327 CA PHE 36 41.396 21.966 33.357 1.00 7.71 ATOM 328 CB PHE 36 42.832 22.193 32.856 1.00 9.56 ATOM 329 CG PHE 36 43.018 21.898 31.389 1.00 9.71 ATOM 330 CD1 PHE 36 42.835 22.898 30.434 1.00 9.80 ATOM 331 CD2 PHE 36 43.322 20.618 30.959 1.00 9.86 ATOM 332 CE1 PHE 36 42.947 22.618 29.082

1.00 10.32 ATOM 333 CE2 PHE 36 43.437 20.335 29.600 1.00 11.13

29.132 38.479 33.658 28.429 39.746 34.170 29.088 40.195 35.361 26.973 39.451 34.497 28.474 37.989 32.377 28.435 38.692 31.364 28.034 36.742 32.416 27.381 36.161 31.267 26.024 36.785 31.013 25.426 37.413 31.888 25.528 36.587 29.803 24.233 37.110 29.405 24.421 38.433 28.649 23.221 38.819 27.993 23.556 36.106 28.484 24.219 35.472 27.660 22.232 35.919 28.641 21.363 36.467 29.703 21.500 34.985 27.788 20.190 34.774 28.559 19.978 36.084 29.224 21.264 35.599 26.395 20.771 34.915 25.492 21.644 36.872. 26.223 21.461 37.576 24.949 20.830 38.952 25.189 19.587 38.903 26.020 18.507 38.142 25.610

19.527 39.558 27.239 17.379 38.028 26.410 18.400 39.448' 28.043 17.332 38.683 27.627 22.751 37.772 24.154 22.724 38.313 23.054 23.876 37.324 24.699 25.152 37.512 24.019 26.306 37.267 24.992 27.663 37.691 24.421 28.794 37.437 25.386 28.529 37.971 26;716 28.663 39.245 27.065 29.062 40.153 26.188 28.418 39.606 28.314 25.349 36.630 22.800 24.889 35.491 22.773 25.986 37.184 21.772 26.325 36.426 20.574 25.873 37.143 19.293 24.453 37.318 19.334 26.236 36.330 18.071 27.849 36.345 20.636 28.535 37.373 20.662 28.358 35.128 20.762 29.789 34.895 20.881 30.074 33.748 21.897 31.567 33.462 21.984

ATOM 473 CGI ILE 51 29.548 34.118 23.297

1.00 11.54

ATOM 474 CD1 ILE 51 28.097 33.689 23.565

1.00 11.67

ATOM 475 C ILE 51 30.422 34.577 19.535 1.00 9.84 ATOM 476 O ILE 51 29.891 33.784 18.753 1.00 9.89 ATOM 477 N ASN 52 31.541 35.231 19.252 1.00 9.72 ATOM 479 CA ASN 52 32.265 35.007 18.002 1.00 9.63 ATOM 480 CB ASN 52 32.512 36.323 17.268

1.00 12.27 ATOM 481 CG ASN 52 31.239 37.060 16.956 1.00 16.92 ATOM 482 OD1 ASN 52 30.579 36.770 15.960 1.00 20.42 ATOM 483 ND2 ASN 52 30.869 38.004 17.817 1.00 18.67

ATOM 486 C ASN 52 33.618 34.430 18.359 1.00 8.99 ATOM 487 0 ASN 52 34.216 34.813 19.369 1.00 8.30 ATOM 488 N TYR 53 34.101 33.502 17.546 1.00 8.54 ATOM 490 CA TYR 53 35.417 32.938 17.805 1.00 8.38 ATOM 491 CB TYR 53 35.383 31.888 18.931 1.00 8.90 ATOM 492 CG TYR 53 34.816 30.545 18.510 1.00 8.55 ATOM 493 CD1 TYR 53 35.652 29.541 18.013 1.00 8.13 ATOM 494 CE1 TYR 53 35.142 28.318 17.595 1.00 7.11 ATOM 495 CD2 TYR 53 33.446 30.286 18.582 1.00 8.30 ATOM 496 CE2 TYR 53 32.924 29.062 18.164 1.00 7.39 ATOM 497 CZ TYR 53 33.771 28.085 17.670 1.00 7.17 ATOM 498 OH TYR 53 33.270 26.875 17.235 1.00 8.09 ATOM 500 C TYR 53 35.995 32.333 16.549 1.00 8.13 ATOM 501 O TYR 53 35.289 32.095 15.567 1.00 8.60 ATOM 502 N ASN 54 37.305 32.146 16.568 1.00 7.89 ATOM 504 CA ASN 54 38.011 31.525 15.466 1.00 8.58 ATOM 505 CB ASN 54 38.628 32.583 14.532 1.00 8.69 ATOM 506 CG ASN 54 39.273 31.967 13.291 1.00 9.70

39.001 32.391 12.157 40.133 30.989 13.490 39.106 30.705 16.137 39.984 31.262 16.807 39.010 29.386 16.031 40.028 28.510 16.607 39.406 27.201 17.109 41.090 28.235 15.529 40.897 27.380 14.656 42.176 29.000 15.569 43.254 28.829 14.606 43.912 27.459 14.705 44.430 26.928 13.716 43.933 26.900 15.910 44.483 25.572 16.150 45.869 25.628 16.856 46.378 24.211 17.158 46.873 26.378 15.998 43.530 24.837 17.086 43.121 25.388 18.118 43.104 23.644 16.682 42.258 22.797 17.520 40.771 22.855 17.137 39.950 21.927 17.984 39.906 21.883 19.421 39.101 20.778 19.781 40.478 22.662 20.434 39.177 20.883 17.550

38.670 20.189 18.620 38.854 20.434 21.113 40.234 22.321 21.756 39.428 21.214 22.083 42.841 21.418 17.283 42.496 20.741 16.323 43.732 21.011 18.177 44.443 19.750 18.031 45.850 20.038 17.520 44.503 18.893 19.295 45.574 18.702 19.893 43.345 18.417 19.763 41.964 18.696 19.313 43.355 17.582 20.963 41.878 17.587 21.386 41.157 17.657 20.069 43.827 16.165 20.610 43.472 15.638 19.559 44.655 15.580 21.470 45.150 14.208 21.309 46.663 14.142 21.519 47.422 14.081 20.239 46.846 14.130 19.159 48.733 13.960 20.342 44.502 13.396 22.418 45.193 12.805 23.265 43.181 13.442 22.478 42.460 12.724 23.505

41.195 13.489 23.824 40.695 14.229 22.976 40.733 13.379 25.064 39.501 14.033 25.508 38.994 13.326 26.769 37.598 13.776 27.201 37.040 13.221 28.147 37.033 14.768 26.529 39.673 15.531 25.764 40.365 15.937 26.697 39.040 16.339 24.919 39.092 17.787 25.045 37.887 18.376 24.327 37.430 17.794 23.339 37.366 19.496 24.830 36.198 20.172 24.242 35.032 20.281 25.249 34.628 19.022 25.992 34.618 17.780 25.369 34.220 16.645 26.048 34.222 19.090 27.324 33.816 17.959 28.012 33.817 16.739 27.370 33.412 15.599 28.045 36.520 21.619 23.866 37.244 22.316 24.588 35.970 22.063 22.743 36.083 23.445 22.283

1

1

2

2

3

3

4

4

5

5

24.837 30.066 38.389 25.246 28.926 38.670 24.362 30.914 39.289 24.156 30.555 40.687 24.932 31.488 41.621 26.430 31.290 41.596 27.103 32.107 42.682 28.538 31.835 42.766 29.466 32.495 42.079 29.118 33.465 41.247 30.748 32.209 42.247 22.658 30.745 40.901 22.001 31.435 40.114 22.125 30.124 41.949 20.701 30.215 42.283 20.370 31.620 42.798 21.282 31.998 43.817 19.759 29.838 41.126 18.998 30.673 40.625 19.793 28.572 40.693 18.753 28.012 39.810 20.653 27.508 41.224 19.828 26.253 40.969 19.175 26.565 39.666 21.999 27.423 40.515 22.231 28.081 39.492 22.875 26.608 41.080 24.207 26.389 40.547

ATOM 713 CB LEU 76 25.063 25.722 41.623 1.00 16.02 ATOM 714 CG LEU 76 26.575 25.774 41.487 1.00 18.13 ATOM 715 CD1 LEU 76 27.047 27.219 41.526 1.00 18.22 ATOM 716 CD2 LEU 76 27.197 24.970 42.626 1.00 18.98 ATOM 717 C LEU 76 24.112 25.488 39.312 1.00 12.79 ATOM 718 0 LEU 76 23.722 24.323 39.412 1.00 12.48 ATOM 719 N ILE 77 24.482 26.025 38.151 1.00 11.37 ATOM 721 CA ILE 77 24.431 25.266 36.902 1.00 10.64 ATOM 722 CB ILE 77 23.188 25.655 36.054 1.00 11.91 ATOM 723 CG2 ILE 77 23.253 24.994 34.686 1.00 11.77 ATOM 724 CGI ILE 77 21.898 25.234 36.769 1.00 12.91 ATOM 725 CD1 ILE 77 20.656 25.787 36.160 1.00 14.88

ATOM 726 C ILE 77 25.676 25.555 36.068 1.00 8.64 ATOM 727 O ILE 77 26.084 26.712 35.948 1.00 8.92 ATOM 728 N GLU 78 26.319 24.502 35.574 1.00 7.66 ATOM 730 CA GLU 78 27.503 24.621 34.720 1.00 7.21 ATOM 731 CB GLU 78 28.536 23.573 35.134 1.00 7.73 ATOM 732 CG GLU 78 29.868 23.635 34.372 1.00 9.08 ATOM 733 CD GLU 78 30.835 22.544 34.805

1.00 10.20 ATOM 734 OE1 GLU 78 32.050 22.814 34.932 1.00 10.78

ATOM 735 OE2 GLU 78 30.395 21.407 35.026

1.00 10.70

ATOM 736 C GLU 78 26.961 24.315 33.322 1.00 7.93 ATOM 737 O GLU 78 26.391 23.240 33.112 1.00 9.13 ATOM 738 N TYR 79 27.119 25.229 32.369 1.00 7.30 ATOM 740 CA TYR 79 26.562 24.982 31.046 1.00 6.18 ATOM 741 CB TYR 79 25.422 25.963 30.737 1.00 6.50 ATOM 742 CG TYR 79 25.844 27.414 30.631 1.00 6.96 ATOM 743 CD1 TYR 79 26.466 27.896 29.479 1.00 6.30

32.812 12.325 22.450 31.717 13.277 22.278 30.852 12.918 21.063 29.751 13.920 20.805 28.867 13.540 19.623 27.824 14.550 19.439 26.565 14.414 19.857 26.170 13.300 20.460 25.729 15.435 19.750 30.886 13.218 23.563 30.381 12.157 23.936 30.766 14.348 24.276 31.298 15.695 23.987 29.984 14.337 25.514 30.311 15.695 26.139 30.543 16.570 24.965 28.486 14.136 25.294 27.890 14.725 24.385 27.890 13.263 26.096 26.459 13.001 25.995 26.144 11.723 25.167 26.655 10.563 25.841 26.757 11.814 23.754 25.885 12.875 27.406 26.626 12.903 28.392 24.566 12.783 27.505 23.915 12.659 28.798 22.488 12.240 28.541

1.00 8.72

21.513 19.802 14.014 20.972 20.313 12.788 20.379 19.211 14.873 22.887 20.429 16.020 23.919 19.741 15.948 22.312 20.766 17.167 22.869 20.382 18.453 23.138 21.636 19.296 24.168 22.602 18.740 23.853 23.459 17.693 24.777 24.367 17.210 25.447 22.677 19.292 26.379 23.585 18.818 26.036 24.425 17.779 26.940 25.358 17.306 21.907 19.505 19.240 20.703 19.742 19.233 22.436 18.470 19.877 21.621 17.629 20.744 22.232 16.242 20.895 22.135 15.434 19.635 23.134 14.788 19.269 21.051 15.468 19.011 21.650 18.319 22.100 22.678 18.897 22.489 20.545 18.254 22.830 20.456 18.862 24.154 19.151 19.675 24.297

ATOM 1049 25.316 14.558 31.483 1.00 10.74 ATOM 1050 23.972 16.094 34.792 1.00 9.98 ATOM 1051 23.632 17.264 34.990 1.00 10.66 ATOM 1052 24.451 15.312 35.747 1.00 10.72 ATOM 1054 24.620 15.777 37.114 1.00 11.19 ATOM 1055 23.582 15.125 38.047 1.00 13.02 ATOM 1056 22.270 15.388 37.539 1.00 15.05 ATOM 1058 23.695 15.684 39.476 1.00 13.84 ATOM 1059 26.015 15.372 37.566 1.00 11.62 ATOM 1060 26.505 14.306 37.199 1.00 11.90 ATOM 1061 26.674 16.269 38.290 1.00 10.81 ATOM 1063 28.002 16.016 38.839 1.00 11.90 ATOM 1064 28.930 17.201 38.558 1.00 14.38 ATOM 1065 29.291 17.330 37.103 1.00 16.14 ATOM 1066 30.426 16.404 36.731 1.00 19.66 ATOM 1067 31.707 17.057 36.981 1.00 23.60 ATOM 1069 32.767 16.477 37.542 1.00 25.66 ATOM 1070 32.724 15.204 37.927 1.00 27.05 ATOM 1073 33.867 17.193 37.756 1.00 27.17 ATOM 1076 27.819 15.843 40.344 1.00 11.36 ATOM 1077 27.071 16.595 40.978 1.00 11.70 ATOM 1078 28.465 14.829 40.900 1.00 11.50 ATOM 1080 28.366 14.530 42.323 1.00 12.45 ATOM 1081 27.883 13.090 42.519 1.00 12.53 ATOM 1082 26.550 12.818 41.855 1.00 13.06 ATOM 1083 25.356 13.014 42.547 1.00 13.72 ATOM 1084 24.139 12.792 41.945 1.00 13.98 ATOM 1085 26.489 12.389 40.536 1.00 13.33

31.158 24.930 43.385 33.022 25.379 41.436 34.129 25.553 41.960 32.328 26.348 40.853 32.837 27.703 40.728 31.813 28.600 39.976 30.488 28.670 40.731 32.392 29.993 39.741 31.520 30.875 38.868 33.206 28.321 42.080 34.183 29.055 42.174 32.455 27.974 43.123 32.674 28.507 44.472 31.415 28.308 45.327 30.283 29.248 44.952 30.568 30.400 44.566 29.106 28.830 45.071 33.834 27.866 45.226 34.165 28.300 46.339 34.450 26.844 44.640 35.519 26.148 45.332 34.867 24.995 46.081 33.639 24.838 46.041 35.663 24.198 46.780 35.151 23.043 47.515 34.292 23.489 48.709 35.128 24.177 49.781 36.279 23.733 49.983

ATOM 1153 34.660 25.169 50.381

1.00 15.66

ATOM 1154 34.431 22.078 46.565

1.00 17.56

ATOM 1155 34.707 22.097 45.360

1.00 20.27

ATOM 1156 33.541 21.233 47.071

1.00 15.49

ATOM 1158 32.868 20.264 46.211

1.00 14.33

ATOM 1159 33.468 18.871 46.417

1.00 15.98

ATOM 1160 34.987 18.856 46.370

1.00 22.01

ATOM 1161 35.555 17.493 46.647

1.00 25.64

ATOM 1162 35.516 16.674 45.446

1.00 30.29

ATOM 1164 36.541 15.960 44.996

1.00 32.24

ATOM 1165 37.699 15.959 45.650

1.00 33.69

ATOM 1168 36.411 15.255 43.880

1.00 34.20

ATOM 1171 31.386 20.210 46.513

1.00 12.98

ATOM 1172 30.978 20.389 47.657

1.00 13.04

ATOM 1173 30.579 19.961 45.492

1.00 11.93

ATOM 1175 29.144 19.878 45.705

1.00 11.52

ATOM 1176 28.517 21.287 45.856

1.00 12.34

ATOM 1177 27.219 21.171 46.455

1.00 13.52

ATOM 1179 28.408 22.004 44.503

1.00 12.64

ATOM 1180 28.507 19.108 44.558

1.00 11.60

ATOM 1181 29.212 18.577 43.694

1.00 12.00

ATOM 1182 27.187 18.988 44.592

1.00 10.11

ATOM 1184 26.451 18.283 43.548

1.00 11.46

ATOM 1185 25.470 17.273 44.172

1.00 11.44

ATOM 1186 26.212 16.222 44.818

1.00 13.54

ATOM 1188 24.564 16.679 43.105

1.00 12.97

ATOM 1189 25.691 19.343 42.738

1.00 11.64

ATOM 1190 25.013 20.201 43.307

1.00 12.44

ATOM 1226 128 23.193 21.747 30.725 1.00 7.08 ATOM 1227 128 22.446 23.076 30.678 1.00 7.17 ATOM 1228 128 21.337 23.250 31.683 1.00 7.55 ATOM 1229 128 20.883 22.185 32.458 1.00 8.48 ATOM 1230 128 19.866 22.370 33.389 1.00 9.54 ATOM 1231 128 20.748 24.495 31.859 1.00 8.26 ATOM 1232 128 19.737 24.686 32.784 1.00 8.94 ATOM 1233 128 19.304 23.625 33.543 1.00 8.68 ATOM 1234 OH TYR 128 18.303 23.828 34.475

1.00 10.00

ATOM 1236 TYR 128 24.143 21.720 29.539 1.00 6.99 ATOM 1237 TYR 128 25.161 22.422 29.550 1.00 7.10 ATOM 1238 N TRP 129 23.786 20.956 28.511 1.00 6.30 ATOM 1240 CA TRP 129 24.626 20.849 27.320 1.00 6.02 ATOM 1241 CB TRP 129 25.266 19.455 27.203 1.00 6.55 ATOM 1242 CG TRP 129 26.025 18.930 28.374 1.00 76.122, 1.00 9.09 ATOM 1211 THR 126 24.701 21.857 36.305 1.00 9.18 ATOM 1212 N GLN 127 24.389 20.028 35.005 1.00 8.52 ATOM 1214 CA GLN 127 25.075 20.560 33.832 1.00 7.83 ATOM 1215 CB GLN 127 26.264 19.683 33.444 1.00 7.94 ATOM 1216 CG GLN 127 27.314 19.489 34.527 1.00 8.42 ATOM 1217 CD GLN 127 28.410 18.547 34.080 1.00 9.41 ATOM 1218 OEl GLN 127 28.173 17.364 33.849

1.00 11.46

ATOM 1219 NE2 GLN 127 29.599 19.072 33.903 1.00 9.24 ATOM 1222 GLN 127 24.082 20.546 32.671 1.00 8.00 ATOM 1223 GLN 127 23.335 19.570 32.500 1.00 9.01 ATOM 1224 N TYR 128 24.050 21.637 31.915 1.00 7.47 ATOM 1226 CA TYR 128 23.193 21.747 30.725 1.00 7.08 ATOM 1227 CB TYR 128 22.446 23.076 30.678 1.00 7.17

ATOM 1339 CA PRO 137 35.172 22.877 11.193 1.00 11.19

ATOM 1340 CB PRO 137 34.916 23.225 9.728

1.00 12.07

ATOM 1341 CG PRO 137 33.665 22.488 9.420

1.00 11.96

ATOM 1342 C PRO 137 35.810 24.072 11.917

1.00 10.74

ATOM 1343 0 PRO 137 35.110 25.007 12.320

1.00 12.54

ATOM 1344 N THR 138 37.114 24.023 12.139

1.00 10.87

ATOM 1346 CA THR 138 37.795 25.116 12.811

1.00 11.05

ATOM 1347 CB THR 138 38.652 24.604 13.982

1.00 11.69

ATOM 1348 OGl THR 138 39.583 23.616 13.512

1.00 12.49

ATOM 1350 CG2 THR 138 37.746 24.000 15.070

1.00 11.85

ATOM 1351 C THR 138 38.650 25.904 11.826

1.00 10.93

ATOM 1352 O THR 138 38.816 25.493 10.675

1.00 12.46

ATOM 1353 N GLY 139 39.132 27.061 12.259

1.00 10.76

ATOM 1355 CA GLY 139 39.954 27.889 11.397

1.00 11.16

ATOM 1356 C GLY 139 39.157 29.004 10.743

1.00 12.00

ATOM 1357 O GLY 139 39.730 29.873 10.085

1.00 14.10

ATOM 1358 N SER 140 37.839 28.981 10.930

1.00 11.25

ATOM 1360 CA SER 140 36.954 29.996 10.375

1.00 11.51

ATOM 1361 CB SER 140 35.857 29.343 9.538

1.00 13.27

ATOM 1362 OG SER 140 36.404 28.701 8.403

1.00 17.64

ATOM 1364 C SER 140 36.293 30.823 11.468

1.00 9.96

ATOM 1365 SER 140 36.369 30.485 12.657

1.00 10.71

ATOM 1366 N ASN 141 35.697 31.937 11.064

1.00 9.76

ATOM 1368 CA ASN 141 34.988 32.815 11.988

1.00 10.29

ATOM 1369 CB ASN 141 34.774 34.193 11.357

1.00 11.11

ATOM 1370 CG ASN 141 36.068 34.965 11.187

1.00 12.89

ATOM 1371 ODl ASN 141 37.056 34.714 11.885

1.00 11.87

ATOM 1372 ND2 ASN 141 36.066 35.914 10.270

1.00 14.96

ATOM 1586 ASN 163 23.816 34.580 38.219

1.00 14.67

ATOM 1587 ASN 163 24.280 33.454 38.320

1.00 15.00

ATOM 1588 N TRP 164 24.144 35.402 37.233

1.00 12.77

ATOM 1590 CA TRP 164 25.106 35.017 36.205

1.00 12.67

ATOM 1591 CB TRP 164 24.802 35.764 9.997 29.674 33.152 1 00 13.40 ATOM 1559 CG LEU 160 20.564 28.838 32.002

1.00 13.30

ATOM 1560 CDl LEU 160 20.954 29.73.9 30.855

1.00 14.40

ATOM 1561 CD2 LEU 160 21.758 28.020 32.482

1.00 13.55

ATOM 1562 LEU 160 19.007 29.596 35.480

1.00 14.33

ATOM 1563 LEU 160 17.882 30.024 35.763

1.00 14.67

ATOM 1564 N GLY 161 20.060 29.740 36.278

1.00 14.47

ATOM 1566 CA GLY 161 19.953 30.465 37.529

1.00 15.47

ATOM 1567 GLY 161 19.784 31.954 37.290

1.00 16.15

ATOM 1568 GLY 161 19.999 32.452 36.171

1.00 16.60

ATOM 1569 N SER 162 19.445 32.683 38.347

1.00 17.37

ATOM 1571 CA SER 162 19.239 34.121 38.230

1.00 17.84

ATOM 1572 CB SER 162 18.170 34.576 39.223

1.00 19.00

ATOM 1573 OG SER 162 18.500 34.145 40.530

1.00 21.73

ATOM 1575 SER 162 20.505 34.953 38.413

1.00 17.19

ATOM 1576 SER 162 20.538 36.123 38.041

1.00 17.92

ATOM 1577 N ASN 163 21.541 34.362 38.995

1.00 16.21

ATOM 1579 CA ASN 163 22.784 35.087 39.219

1.00 15.87 ATOM 1580 CB ASN 163 23.260 34.871 40.661

1.00 19.24

ATOM 1581 CG ASN 163 24.477 35.718. 41.020

1.00 23.45

ATOM 1582 ODl ASN 163 25.295 35.326 41.860

1.00 27.11

ATOM 1583 ND2 ASN 163 24.590 36.894 40.410

1.00 25.66

ATOM 1586 ASN 163 23.816 34.580 38.219

1.00 14.67

ATOM 1587 ASN 163 24.280 33.454 38.320

1.00 15.00

ATOM 1588 N TRP 164 24.144 35.402 37.233 1.00 12.77 ATOM 1590 CA TRP 164 25.106 35.017 36.205 1.00 12.67 ATOM 1591 CB TRP 164 24.802 35.764' 34.902 1.00 12.97 ATOM 1592 CG TRP 164 23.523 35.331 34.250 1.00 13.93 ATOM 1593 CD2 TRP 164 23.393 34.489 33.098 1.00 13.58 ATOM 1594 CE2 TRP 164 22.015 34.297 32.871 1.00 14.09 ATOM 1595 CE3 TRP 164 24.308 33.876 32.238 1.00 13.32 ATOM 1596 CDl TRP 164 22.253 35.620 34.659 1.00 15.26 ATOM 1597 NEl TRP 164 21.340 34.996 33.837 1.00 15.80 ATOM 1599 CZ2 TRP 164 21.534 33.518 31.825 1.00 14.29 ATOM 1600 CZ3 TRP 164 23.829 33.107 31.204 1.00 13.95 ATOM 1601 CH2 TRP 164 22.456 32.932 31.004 1.00 13.84 ATOM 1602 C TRP 164 26.576 35.218 36.569 1.00 12.17 ATOM 1603 O TRP 164 26.990 36.300 37.001 1.00 12.76 ATOM 1604 N ALA 165 27.365 34.173 36.360 1.00 10.88

ATOM 1606 CA ALA 165 28.796 34.233 36.611 1.00 9.93 ATOM 1607 CB ALA 165 29.275 32.979 37.333

1.00 12.00

ATOM 1608 C ALA 165 29.486 34.396 35.246 1.00 9.94 ATOM 1609 O ALA 165 28.846 34.822 34.275

1.00 10.07

ATOM 1610 N TYR 166 30.759 34.027 35.145 1.00 8.78 ATOM 1612 CA TYR 166 31.474 34.207 33.881 1.00 8.27 ATOM 1613 CB TYR 166 32.982 34.002 34.069 1.00 8.85 ATOM 1614 CG TYR 166 33.384 32.610 34.503

CDl TYR 166 33.438 31.567 33.588

CEl TYR 166 33.821 30.302 33.964

CD2 TYR 166 33.726 32.344 35.827

CE2 TYR 166 34.111 31.073 36.220

CZ TYR 166 34.157 30.061 35.283

34.553 28.803 35.642 30.941 33.364 32.717 30.242 32.355 32.915 31.327 33.765 31.509 30.894 33.122 30.272 29.687 33.897 29.742 29.059 33.379 28.465 27.822 34.189 28.116 27.896 35.405 27.941 26.674 33.531 28.081 32.084 33.233 29.322 32.463 34.340 28.940 32.684 32.103 28.961 33.871 32.126 28.109 35.152 31.861 28.970 35.294 32.912 30.062 35.093 30.475 29.599 33.857 31.097 26.992 33.087 30.141 27.034 34.671 31.325 25.962 34.822 30.353 24.875 35.078 31.020 23.527 35.016 30.028 22.389 33.355 29.295 22.243 33.759 27.805 21.363 36.066 29.618 25.357 37.175 30.167 25.347 35.871 28.375 25.750

38.603 12.811 32.115 38.619 11.926 33.181 39.236 14.586 33.579 39.258 13.709 34.648 38.948 12.383 34.443 38.970 11.506 35.508 41.311 14.466 30.552 41.107 13.773 29.553 42.359 14.308 31.359 43.354 13.248 31.184 42.837 11.937 31.787 42.566 12.065 33.283 42.419 10.732 34.001 42.573 10.665 35.228 42.110 9.673 33.258 43.781 13.073 29.720 43.726 11.976 29.151 44.279 14.155 29.137 44.680 14.133 27.74.6 43.467 14.513 26.879 43.042 15.847 27.130 45.840 15.101 27.489 46.427 15.654 28.426 46.178 15.264 26.216 47.240 16.164 25.776 48.507 15.375 25.461 48.928 14.633 26.596 46.745 16.829 24.508

ATOM 1794 CZ3 TRP 185 37.025 35.883 16.266 1.00 11.87 ATOM 1795 CH2 TRP 185 36.346 35.944 15.040 1.00 11.03 ATOM 1796 TRP 185 33.611 38.581 20.364

1.00 9.55 ATOM 1797 TRP 185 32.974 37.509 20.372 1.00 9.75 ATOM 1798 OXT TRP 185 33.062 39.698 20.362

1.00 12.79

The three-dimensional crystal structure of the T. harzianum xylanase shows a great degree of similarity to the structure of the B. circulans xylanase . (Figure 3) , although the T. harzianum xylanase contains two extra strands at the beginning of sheets I and II, and a few small insertions and deletions (see also Figure 1) . While the structures are similar at 89% of the residues of the B. circulans xylanase, the sequences are identical at only 51% of the 185 residues. The x-ray co-ordinates of the structure is given below.

ATOM 14 CG2 THR 2 21.104 14.549 7.600 1.00 27.32 ATOM 15 C THR 2 17.847 15.388 7.733 1.00 21.71 ATOM 16 O THR 2 17.768 16.039 6.692 1.00 20.85

ATOM 17 N ILE 3 17.002 15.542 8.751

1.00 18.32

ATOM 18 CA ILE 3 16.196 16.735 8.873

1.00 17.14 ATOM 19 CB ILE 3 14.723 16.216 8.640

1.00 17.94 ATOM 20 CG2 ILE 3 13.680 16.289 9.744

1.00 16.71 ATOM 21 CGI ILE 3 14.269 17.095 7.557

1.00 18.00 ATOM 22 CDl ILE 3 14.803 16.716 6.179

1.00 20.15 ATOM 23 C ILE 3 16.568 17.281 10.229

1.00 16.13 ATOM 24 O ILE 3 17.085 16.584 11.119

1.00 16.34 ATOM 25 N GLY 4 16.451 18.588 10.370

1.00 14.88 ATOM 26 CA GLY 4 16.674 19.198 11.659

1.00 11.55 ATOM 27 C GLY 4 15.299 19.349 12.251

1.00 12.16 ATOM 28 O GLY 4 14.314 18.938 11.628

1.00 11.77 ATOM 29 N PRO 5 15.134 19.926 13.445

1.00 11.94 ATOM 30 CD PRO 5 16.159 20.593 14.231

1.00 12.27 ATOM 31 CA PRO 5 13.887 19.803 14.160

1.00 12.67 ATOM 32 CB PRO 5 14.208 20.405 15.490

1.00 12.77 ATOM 33 CG PRO 5 15.703 20.207 15.627

1.00 12.89 ATOM 34 C PRO 5 12.754 20.463 13.391

1.00 13.39 ATOM 35 0 PRO 5 12.974 21.493 12.764

1.00 14.17 ATOM 36 N GLY 6 11.583 19.867 13.377

1.00 12.31 ATOM 37 CA GLY 6 10.494 20.387 12.606

1.00 13.71 ATOM 38 C GLY 6 9.260 19.487 12.636

1.00 14.99 ATOM 39 O GLY 6 9.172 18.391 13.233

1.00 14.55 ATOM 40 N THR 7 8.292 19.986 11.875

1.00 15.50 ATOM 41 CA THR 7 6.963 19.390 11.753

1.00 15.99

ATOM 70 1.00 29.54 ATOM 71 1.00 31.69 ATOM 72 1.00 34.14 ATOM 73 1.00 33.69 ATOM 74 1.00 34.10 ATOM 75 1.00 27.90 ATOM 76 1.00 28.07 ATOM 77 1.00 24.30 ATOM 78 1.00 22.09 ATOM 79 1.00 19.23 ATOM 80 1.00 19.87 ATOM 81 1.00 16.68 ATOM 82 1.00 13.96 ATOM 83 1.00 14.86 ATOM 84 1.00 16.63 ATOM 85 1.00 18.73 ATOM 86 1.00 19.94 ATOM 87 1.00 16.53 ATOM 88 1.00 17.13 ATOM 89 1.00 18.35 ATOM 90 1.00 19.27 ATOM 91 1.00 12.85 ATOM 92 1.00 11.47 ATOM 93 1.00 14.13 ATOM 94 1.00 12.57 ATOM 95 1.00 13.51 ATOM 96 1.00 15.53 ATOM 97 1.00 16.07

ATOM 98 CEl TYR 14

1.00 15.89

ATOM 99 CD2 TYR 14

1.00 16.13

ATOM 100 CE2 TYR 14

1.00 16.08

ATOM 101 CZ TYR 14

1.00 16.73

ATOM 102 OH TYR 14

1.00 18.42

ATOM 103 C TYR 14

1.00 13.04

ATOM 104 O TYR 14

1.00 11.98

ATOM 105 N TYR 15

1.00 11.94

ATOM 106 CA TYR 15

1.00 12.73

ATOM 107 CB TYR 15

1.00 15.06

ATOM 108 CG TYR 15

1.00 17.13

ATOM 109 CDl TYR 15

1.00 17.27

ATOM 110 CEl TYR 15

1.00 18.57

ATOM 111 CD2 TYR 15

1.00 18.44

ATOM 112 CE2 TYR 15

1.00 18.53

ATOM 113 CZ TYR 15

1.00 19.40

ATOM 114 OH TYR 15

1.00 21.86

ATOM 115 C TYR 15

1.00 12.96

ATOM 116 0 TYR 15

1.00 10.33

ATOM 117 N SER 16

1.00 11.29

ATOM 118 CA SER 16

1.00 11.41

ATOM 119 CB SER 16

1.00 9.89

ATOM 120 OG SER 16

1.00 10.30

ATOM 121 C SER 16

1.00 12.00

ATOM 122 O SER 16

1.00 13.12

ATOM 123 N TYR 17

1.00 10.82

ATOM 124 CA TYR 17

1.00 8.67

ATOM 125 CB TYR 17

1.00 7.15

17.864 17.941 17.903 15.396 19.550 21.166 14.989 20.636 20.741 16.131 19.441 22.251 16.504 20.540 23.135 16.797 19.893 24.518 18.025 18.968 24.621 18.718 18.720 23.650 18.310 18.428 25.678 17.695 21.403 22.637 18.103 22.388 23.291 18.234 21.045 21.466 19.380 21.652 20.875 20.648 20.856 21.103 21.648 21.119 20.438 20.741 19.936 22.053 21.960 19.155 22.258 21.741 18.177 23.358 22.979 17.601 23.957 23.428 18.016 25.187 23.803 16.685 23.489 24.747 16.526 24.386 24.505 17.334 25.395 22.310 18.377 20.984 21.423 17.986 20.218 23.598 18.178 20.750 24.077 17.456 19.601 25.543 17.722 19.414

ATOM 210 CZ TYR 27

1.00 10.33

ATOM 211 OH TYR 27

1.00 11.41

ATOM 212 C TYR 27

1.00 10.52

ATOM 213 O TYR 27 1.00 9.97 ATOM 214 N THR 28

1.00 10.67

ATOM 215 CA THR 28 1.00 13 02 ATOM 216 CB THR 28

1.00 12.93

ATOM 217 OGl THR 28

1.00 14.75 ATOM 218 CG2 THR 28 1.00 12.92 ATOM 219 C THR 28 1.00 13.28 ATOM 220 O THR 28 1.00 11.76 ATOM 221 N ASN 29 1.00 15.11 ATOM 222 CA ASN 29 1.00 17.11 ATOM 223 CB ASN 29 1.00 20.17 ATOM 224 CG ASN 29 1.00 23.95 ATOM 225 ODl ASN 29 1.00 25.19 ATOM 226 ND2 ASN 29 1.00 26.04 ATOM 227 C ASN 29 1.00 17.45 ATOM 228 O ASN 29 1.00 13.40 ATOM 229 N GLY 30 1.00 17.19 ATOM 230 CA GLY 30 1.00 20.59 ATOM 231 C GLY 30 1.00 20.60 ATOM 232 0 GLY 30 1.00 21.82 ATOM 233 N GLY 31 1.00 20.69 ATOM 234 CA GLY 31 1.00 21.04 ATOM 235 C GLY 31 1.00 20.17

ATOM 236 0 GLY 31

1.00 21.45

ATOM 237 N GLY 32

1.00 17.60

ATOM 266 CG2 THR 36

1. 00 10 . 22

ATOM 267 C THR 36 1.00 9.84 ATOM 268 O THR 36 1.00 9.61 ATOM 269 N VAL 37 1.00 9.80 ATOM 270 CA VAL 37 1.00 9.70 ATOM 271 CB VAL 37

1.00 10.77 ATOM 272 CGI VAL 37 1.00 10.77 ATOM 273 CG2 VAL 37 1.00 10.75 ATOM 274 C VAL 37 1.00 10.19

ATOM 275 O VAL 37

1.00 12.44 ATOM 276 N ASN 38 1.00 9.51 ATOM 277 CA ASN 38 1.00 10.83 ATOM 278 CB ASN 38 1.00 13.08 ATOM 279 CG ASN 38 1.00 18.12 ATOM 280 ODl ASN 38 1.00 18.42 ATOM 281 ND2 ASN 38 1.00 18.73 ATOM 282 C ASN 38 1.00 10.89 ATOM 283 O ASN 38 1.00 10.36 ATOM 284 N TRP 39 1.00 8.76 ATOM 285 CA TRP 39 1.00 10.30

ATOM 286 CB TRP 39 1.00 8.23 ATOM 287 CG TRP 39 1.00 7.73 ATOM 288 CD2 TRP 39 1.00 7.65 ATOM 289 CE2 TRP 39 1.00 7.27 ATOM 290 CE3 TRP 39 1.00 8.18 ATOM 291 CDl TRP 39 1.00 5.84 ATOM 292 NEl TRP 39 1.00 5.45 ATOM 293 CZ2 TRP 39 1.00 6.19

ATOM 350 47

1. 00 14 . 35 ATOM 351 47

1. 00 11 . 87

ATOM 352 47

1.00 11.08 ATOM 353 48 1.00 9.25 ATOM 354 48 1.00 11.87 ATOM 355 48 1.00 11.00 ATOM 356 48 1.00 11.18

ATOM 357 49 1.00 9.47 ATOM 358 49 1.00 8.49 ATOM 359 49 1.00 7.23 ATOM 360 49 1.00 7.71 ATOM 361 49 1.00 8.83 ATOM 362 49 1.00 9.61 ATOM 363 49

1.00 11.96

ATOM 364 49 1.00 8.57 ATOM 365 49 1.00 9.28 ATOM 366 50 1.00 8.33 ATOM 367 50 1.00 8.35 ATOM 368 50

1.00 10.55 ATOM 369 50 1.00 10.22 ATOM 370 51 1.00 11.01

ATOM 371 51

1.00 12.06

ATOM 372 51

1.00 11.79

ATOM 373 51

1.00 13.30

ATOM 374 51

1.00 13.13

ATOM 375 51

1.00 14.19

ATOM 376 51

1.00 12.35

ATOM 377 51

1.00 13.99

ATOM 434 NZ LYS 58

1.00 19.60

ATOM 435 C LYS 58

1.00 13.93

ATOM 436 O LYS 58

1.00 15.02

ATOM 437 N VAL 59

1.00 13.40

ATOM 438 CA VAL 59

1.00 12.92

ATOM 439 CB VAL 59

1.00 14.48

ATOM 440 CGI VAL 59

1.00 16.35

ATOM 441 CG2 VAL 59

1.00 13.23

ATOM 442 C VAL 59

1.00 11.86

ATOM 443 O VAL 59

1.00 12.38

ATOM 444 N ILE 60

1.00 10.27

ATOM 445 CA ILE 60

1.00 9.01

ATOM 446 CB ILE 60

1.00 8.58

ATOM 447 CG2 ILE 60

1.00 8.04

ATOM 448 CGI ILE 60

1.00 7.60

ATOM 449 CDl ILE 60

1.00 9.90

ATOM 450 C ILE 60

1.00 9.88

ATOM 451 O ILE 60

1.00 9.11

ATOM 452 N ASN 61

1.00 11.11 ATOM 453 CA ASN 61 1.00 10.36

ATOM 454 CB ASN 61

1.00 12.63

ATOM 455 CG ASN 61

1.00 14.76 ATOM 456 ODl ASN 61 1.00 16.78 ATOM 457 ND2 ASN 61 1.00 15.45

ATOM 458 C ASN 61 1.00 9.66 ATOM 459 O ASN 61 1.00 9.13 ATOM 460 N PHE 62 1.00 9.56 ATOM 461 CA PHE 62

1.00 10.44

ATOM 490 CG TYR 66 1.00 5.88 ATOM 491 CDl TYR 66 1.00 3.88 ATOM 492 CEl TYR 66 1.00 3.78 ATOM 493 CD2 TYR 66 1.00 5.67 ATOM 494 CE2 TYR 66 1.00 3.65 ATOM 495 CZ TYR 66 1.00 4.38 ATOM 496 OH TYR 66 1.00 5.96 ATOM 497 C TYR 66 1.00 8.49 ATOM 498 O TYR 66 1.00 8.59 ATOM 499 N ASN 67

1.00 10.09 ATOM 500 CA ASN 67 1.00 11.81 ATOM 501 CB ASN 67 1.00 15.94 ATOM 502 CG ASN 67 1.00 20.43 ATOM 503 ODl ASN 67 1.00 21.28 ATOM 504 ND2 ASN 67 1.00 22.48 ATOM 505 C ASN 67 1.00 11.16 ATOM 506 0 ASN 67 1.00 8.26 ATOM 507 N PRO 68 1.00 10.94 ATOM 508 CD PRO 68 1.00 10.82 ATOM 509 CA PRO 68 1.00 11.97 ATOM 510 CB PRO 68 1.00 11.53 ATOM 511 CG PRO 68 1.00 10.89 ATOM 512 C PRO 68 1.00 11.57 ATOM 513 O PRO 68 1.00 10.78 ATOM 514 N ASN 69 1.00 9.77 ATOM 515 CA ASN 69 1.00 11.79 ATOM 516 CB ASN 69 1.00 13.60 ATOM 517 CG ASN 69 1.00 17.40

ATOM 574 N TYR 77 1.00 6.57 ATOM 575 CA TYR 77 1.00 5.67 ATOM 576 CB TYR 77 1.00 4.54 ATOM 577 CG TYR 77 1.00 4.19 ATOM 578 CDl TYR 77 1.00 4.75 ATOM 579 CEl TYR 77 1.00 4.71 ATOM 580 CD2 TYR 77 1.00 4.76 ATOM 581 CE2 TYR 77 1.00 3.95 ATOM 582 CZ TYR 77 1.00 5.09 ATOM 583 OH TYR 77 1.00 6.11 ATOM 584 C TYR 77 1.00 5.81 ATOM 585 O TYR 77 1.00 6.39 ATOM 586 N GLY 78 1.00 5.96 ATOM 587 CA GLY 78 1.00 6.69 ATOM 588 C GLY 78 1.00 7.28 ATOM 589 O GLY 78 1.00 7.84 ATOM 590 N TRP 79 1.00 7.76 ATOM 591 CA TRP 79 1.00 7.51 ATOM 592 CB TRP 79 1.00 6.56 ATOM 593 CG TRP 79 1.00 5.50 ATOM 594 CD2 TRP 79 1.00 5.87 ATOM 595 CE2 TRP 79 1.00 5.84 ATOM 596 CE3 TRP 79 1.00 5.83 ATOM 597 CDl TRP 79 1.00 6.72 ATOM 598 NEl TRP 79 1.00 5.73 ATOM 599 CZ2 TRP 79 1.00 5.70 ATOM 600 CZ3 TRP 79 1.00 5.50 ATOM 601 CH2 TRP 79 1.00 5.81

ATOM 630 CD PRO 83

1. 00 12 .28 ATOM 631 CA PRO 83

1. 00 10 . 88 ATOM 632 CB PRO 83

1. 00 11. 28 ATOM 633 CG PRO 83

C PRO 83

0 PRO 83

N LEU 84

CA LEU 84

ATOM 638 CB LEU 84 1. 00 9.09 ATOM 639 CG LEU 84 1.00 9.70 ATOM 640 CDl LEU 84 1. 00 8.76 ATOM 641 CD2 LEU 84

1.00 10.04 ATOM 642 C LEU 84 1.00 9.91 ATOM 643 O LEU 84 1.00 10.98 ATOM 644 N ILE 85 1.00 10.87 ATOM 645 CA ILE 85 1.00 11.93 ATOM 646 CB ILE 85 1.00 11.72 ATOM 647 CG2 ILE 85 1.00 12.89 ATOM 648 CGI ILE 85 1.00 11.57 ATOM 649 CDl ILE 85 1.00 11.61 ATOM 650 C ILE 85 1.00 10.50 ATOM 651 O ILE 85 1.00 12.37 ATOM 652 N GLU 86 1.00 10.70 ATOM 653 CA GLU 86 1.00 10.27

ATOM 654 CB GLU 86 1.00 8.12 ATOM 655 CG GLU 86 1.00 6.80 ATOM 656 CD GLU 86 1.00 8.51 ATOM 657 OEl GLU 86 1.00 9.55

ATOM 686 CA ILE 89 1.00 8.75 ATOM 687 CB ILE 89

1.00 11.11 ATOM 688 CG2 ILE 89 1.00 11.61 ATOM 689 CGI ILE 89 1.00 14.51 ATOM 690 CDl ILE 89 1.00 14.55

ATOM 691 C ILE 89 1.00 7.56 ATOM 692 O ILE 89 1.00 7.22 ATOM 693 N VAL 90 1.00 5.27 ATOM 694 CA VAL 90 1.00 7.56 ATOM 695 CB VAL 90 1.00 8.16 ATOM 696 CGI VAL 90

1.00 11.52

ATOM 697 CG2 VAL 90 1.00 9.08 ATOM 698 C VAL 90 1.00 7.15 ATOM 699 O VAL 90 1.00 5.67 ATOM 700 N GLU 91 1.00 7.73 ATOM 701 CA GLU 91 1.00 6.96 ATOM 702 CB GLU 91 1.00 6.72 ATOM 703 CG GLU 91 1.00 6.68 ATOM 704 CD GLU 91 1.00 7.56 ATOM 705 OEl GLU 91 1.00 6.34 ATOM 706 OE2 GLU 91 1.00 7.24 ATOM 707 C GLU 91 1.00 8.03 ATOM 708 O GLU 91 1.00 8.58 ATOM 709 N ASN 92 1.00 6.40 ATOM 710 CA ASN 92 1.00 5.56 ATOM 711 CB ASN 92 1.00 7.20 ATOM 712 CG ASN 92 1.00 4.55 ATOM 713 ODl ASN 92 1.00 8.31

ATOM 714 ND2 ASN 92 1.00 4.54 ATOM 715 C ASN 92 1.00 6.87 ATOM 716 O ASN 92 1.00 6.30 ATOM 717 N PHE 93 1.00 6.43 ATOM 718 CA PHE 93 1.00 7.21 ATOM 719 CB PHE 93 1.00 5.14 ATOM 720 CG PHE 93 1.00 5.75 ATOM 721 CDl PHE 93 1.00 5.51 ATOM 722 CD2 PHE 93 1.00 5.97 ATOM 723 CEl PHE 93 1.00 6.76 ATOM 724 CE2 PHE 93 1.00 6.85 ATOM 725 CZ PHE 93 1.00 6.45 ATOM 726 C PHE 93 1.00 7.46 ATOM 727 O PHE 93 1.00 8.45 ATOM 728 N GLY 94 1.00 9.27 ATOM 729 CA GLY 94 1.00 9.67 ATOM 730 C GLY 94 1.00 10.54 ATOM 731 O GLY 94 1.00 11.63 ATOM 732 N THR 95 1.00 8.02 ATOM 733 CA THR 95 1.00 9.56 ATOM 734 CB THR 95 1.00 10.30 ATOM 735 OGl THR 95 1.00 12.79 ATOM 736 CG2 THR 95 1.00 11.07 ATOM 737 C THR 95 1.00 9.93 ATOM 738 O THR 95 1.00 7.89 ATOM 739 N TYR 96 1.00 9.40 ATOM 740 CA TYR 96 1.00 7.32 ATOM 741 CB TYR 96 1.00 8.01

ATOM 854 CA GLY 112

1. 00 10 . 73

ATOM 855 C GLY 112 1.00 9.91 ATOM 856 O GLY 112 1.00 9.84 ATOM 857 N SER 113 1.00 9.51 ATOM 858 CA SER 113 1.00 8.66 ATOM 859 CB SER 113 1.00 6.40 ATOM 860 OG SER 113 1.00 5.69 ATOM 861 C SER 113 1.00 8.88 ATOM 862 0 SER 113

1.00 11.46 ATOM 863 N VAL 114 1.00 10.48 ATOM 864 CA VAL 114 1.00 9.89 ATOM 865 CB VAL 114 1.00 11.18 ATOM 866 CGI VAL 114 1.00 10.85 ATOM 867 CG2 VAL 114 1.00 10.22 ATOM 868 C VAL 114 1.00 10.04

ATOM 869 0 VAL 114 1.00 9.80 ATOM 870 N TYR 115 1.00 9.29 ATOM 871 CA TYR 115 1.00 9.53 ATOM 872 CB TYR 115 1.00 8.60 ATOM 873 CG TYR 115 1.00 8.75 ATOM 874 CDl TYR 115 1.00 7.26 ATOM 875 CEl TYR 115 1.00 7.31 ATOM 876 CD2 TYR 115 1.00 8.71 ATOM 877 CE2 TYR 115 1.00 9.10 ATOM 878 CZ TYR 115 1.00 8.99 ATOM 879 OH TYR 115 1.00 9.97 ATOM 880 C TYR 115

1.00 10.03 ATOM 881 0 TYR 115 1.00 10.65

ATOM 882 N ASP 116

1.00 10.05

ATOM 883 CA ASP 116

1.00 10.95

ATOM 884 CB ASP 116

1.00 10.88

ATOM 885 CG ASP 116

1.00 12.59

ATOM 886 ODl ASP 116

1.00 12.09

ATOM 887 OD2 ASP 116

1.00 13.31

ATOM 888 C ASP 116

1.00 10.73

ATOM 889 O ASP 116 1.00 9.98 ATOM 890 N ILE 117

1.00 10.31

ATOM 891 CA ILE 117

1.00 11.24 ATOM 892 CB ILE 117 1.00 11.80 ATOM 893 CG2 ILE 117 1.00 12.99 ATOM 894 CGI ILE 117 1.00 11.14 ATOM 895 CDl ILE 117 1.00 11.30 ATOM 896 C ILE 117 1.00 11.21 ATOM 897 O ILE 117 1.00 11.16

ATOM 898 N TYR 118 1.00 9.67 ATOM 899 CA TYR 118

1.00 10.44

ATOM 900 CB TYR 118 1.00 8.83 ATOM 901 CG TYR 118 1.00 9.45 ATOM 902 CDl TYR 118 1.00 9.25 ATOM 903 CEl TYR 118

1.00 10.55

ATOM 904 CD2 TYR 118 1.00 9.83 ATOM 905 CE2 TYR 118

1.00 11.19 ATOM 906 CZ TYR 118 1.00 11.73 ATOM 907 OH TYR 118 1.00 11.26 ATOM 908 C TYR 118 1.00 10.46 ATOM 909 O TYR 118 1.00 9.48

ATOM 910 N ARG 119

1.00 11.66

ATOM 911 CA ARG 119

1.00 14.61

ATOM 912 CB ARG 119 1.00 17 40 ATOM 913 CG ARG 119

1.00 23.37

ATOM 914 CD ARG 119

1.00 26.18

ATOM 915 NE ARG 119

1.00 30.66

ATOM 916 CZ ARG 119

1.00 33.03

ATOM 917 NHl ARG 119

1.00 34.14

ATOM 918 NH2 ARG 119

1.00 32.49 ATOM 919 C ARG 119 1.00 14.29

ATOM 920 O ARG 119

1.00 12.19

ATOM 921 N THR 120

1.00 15.13

ATOM 922 CA THR 120

1.00 16.87

ATOM 923 CB THR 120

1.00 19.15

ATOM 924 OGl THR 120

1.00 22.72

ATOM 925 CG2 THR 120

1.00 21.25

ATOM 926 C THR 120

1.00 15.72 ATOM 927 O THR 120 1.00 11.42 ATOM 928 N GLN 121 1.00 18.27 ATOM 929 CA GLN 121 1.00 18.87 ATOM 930 CB GLN 121 1.00 20.46 ATOM 931 CG GLN 121 LOO 22.49

ATOM 932 CD GLN 121

1.00 23.15

ATOM 933 OEl GLN 121 1.00 24 23 ATOM 934 NE2 GLN 121

1.00 25.37 ATOM 935 C GLN 121 1.00 17.45 ATOM 936 O GLN 121 1.00 17.70 ATOM 937 N ARG 122 1.00 16.86

ATOM 966 CG GLN 125

1.00 20.97

ATOM 967 CD GLN 125

1.00 23.57

ATOM 968 OEl GLN 125

1.00 24.89

ATOM 969 NE2 GLN 125

1.00 24.87

ATOM 970 C GLN 125

1.00 14.36

ATOM 971 0 GLN 125

1.00 13.68

ATOM 972 N PRO 126

1.00 13.24

ATOM 973 CD PRO 126

1.00 13.21

ATOM 974 CA PRO 126

1.00 11.98

ATOM 975 CB PRO 126

1.00 12.53

ATOM 976 CG PRO 126

1.00 12.93 ATOM 977 C PRO 126 1.00 11.75 ATOM 978 O PRO 126 1.00 11.75 ATOM 979 N SER 127 1.00 11.03 ATOM 980 CA SER 127 1.00 10.71 ATOM 981 CB SER 127 1.00 10.39 ATOM 982 OG SER 127 1.00 11.05 ATOM 983 C SER 127 1.00 12.20 ATOM 984 O SER 127 1.00 12.63 ATOM 985 N ILE 128 1.00 10.76 ATOM 986 CA ILE 128 1.00 12.75 ATOM 987 CB ILE 128 1.00 11.99 ATOM 988 CG2 ILE 128 1.00 13.03 ATOM 989 CGI ILE 128 1.00 13.86 ATOM 990 CDl ILE 128 1.00 11.79 ATOM 991 C ILE 128 1.00 12.49 ATOM 992 O ILE 128 1.00 11.61 ATOM 993 N ILE 129 1.00 11.55

ATOM 1022 C THR 133

1.00 12.16 ATOM 1023 O THR 133

1.00 12.10 ATOM 1024 N PHE 134

1.00 12.04 ATOM 1025 CA PHE 134

1.00 10.26 ATOM 1026 CB PHE 134

1.00 9.74 ATOM 1027 CG PHE 134

1.00 10.10 ATOM 1028 CDl PHE 134

1.00 9.16 ATOM 1029 CD2 PHE 134

1.00 9.83 ATOM 1030 CEl PHE 134

1.00 10.74 ATOM 1031 CE2 PHE 134 1.00 10.39 ATOM 1032 CZ PHE 134 1.00 11.34 ATOM 1033 C PHE 134 1.00 10.97 ATOM 1034 O PHE 134 1.00 9.19 ATOM 1035 N TYR 135 1.00 10.76 ATOM 1036 CA TYR 135 1.00 11.10 ATOM 1037 CB TYR 135 1.00 11.88 ATOM 1038 CG TYR 135 1.00 15.19 ATOM 1039 CDl TYR 135 1.00 17.24 ATOM 1040 CEl TYR 135 1.00 17.77 ATOM 1041 CD2 TYR 135 1.00 17.57 ATOM 1042 CE2 TYR 135 1.00 18.71 ATOM 1043 CZ TYR 135 1.00 18.40 ATOM 1044 OH TYR 135 1.00 21.11 ATOM 1045 C TYR 135 1.00 11.69 ATOM 1046 O TYR 135 1.00 9.54 ATOM 1047 N GLN 136 1.00 10.85 ATOM 1048 CA GLN 136 1.00 10.50 ATOM 1049 CB GLN 136 1.00 10.66

ATOM 1078 CZ3 TRP 138 1.00 9.01 ATOM 1079 CH2 TRP 138 1.00 8.58 ATOM 1080 C TRP 138 1.00 9.46 ATOM 1081 O TRP 138 1.00 8.88 ATOM 1082 N SER 139

1.00 11.22 ATOM 1083 CA SER 139 1.00 11.52 ATOM 1084 CB SER 139 1.00 11.35 ATOM 1085 OG SER 139 1.00 13.26 ATOM 1086 C SER 139 1.00 10.74 ATOM 1087 O SER 139 1.00 11.43 ATOM 1088 N VAL 140 1.00 9.74 ATOM 1089 CA VAL 140

1.00 9.29 ATOM 1090 CB VAL 140 1.00 9.45 ATOM 1091 CGI VAL 140 1.00 9.67 ATOM 1092 CG2 VAL 140 1.00 9.25 ATOM 1093 C VAL 140 1.00 9.37 ATOM 1094 O VAL 140 1.00 8.14 ATOM 1095 N ARG 141

1.00 10.69

ATOM 1096 CA ARG 141

1.00 11.96

ATOM 1097 CB ARG 141

1.00 11.92

ATOM 1098 CG ARG 141

1.00 12.57

ATOM 1099 CD ARG 141

1.00 11.72

ATOM 1100 NE ARG 141

1.00 15.48

ATOM 1101 CZ ARG 141

1.00 13.58

ATOM 1102 NHl ARG 141

1.00 12.83

ATOM 1103 NH2 ARG 141

1.00 13.32

ATOM 1104 C ARG 141 1.00 9.52 ATOM 1105 O ARG 141 1.00 8.68

ATOM 1162 N SER 149 1.00 9.40 ATOM 1163 CA SER 149 1.00 10.95 ATOM 1164 CB SER 149 1.00 12.62 ATOM 1165 OG SER 149 1.00 20.18 ATOM 1166 C SER 149 1.00 11.07 ATOM 1167 O SER 149 1.00 10.99 ATOM 1168 N VAL 150 1.00 10.64 ATOM 1169 CA VAL 150 1.00 10.28 ATOM 1170 CB VAL 150 1.00 10.28 ATOM 1171 CGI VAL 150 1.00 8.88 ATOM 1172 CG2 VAL 150 1.00 10.04 ATOM 1173 C VAL 150 1.00 11.39 ATOM 1174 O VAL 150 1.00 11.90 ATOM 1175 N ASN 151 1.00 11.61 ATOM 1176 CA ASN 151 1.00 13.38 ATOM 1177 CB ASN 151 1.00 15.35 ATOM 1178 CG ASN 151 1.00 19.10 ATOM 1179 ODl ASN 151 1.00 21.76 ATOM 1180 ND2 ASN 151 1.00 19.23 ATOM 1181 C ASN 151 1.00 13.32 ATOM 1182 O ASN 151 1.00 13.06 ATOM 1183 N THR 152 1.00 12.70 ATOM 1184 CA THR 152 1.00 10.80

ATOM 1185 CB THR 152 1.00 9.08 ATOM 1186 OGl THR 152 1.00 9.66 ATOM 1187 CG2 THR 152 1.00 8.63 ATOM 1188 C THR 152

1.00 12.10 ATOM 1189 O THR 152 1.00 12.41

ATOM 1218 CD2 PHE 156

1.00 15.81

ATOM 1219 CEl PHE 156 1.00 16.11 ATOM 1220 CE2 PHE 156

1.00 15.84

ATOM 1221 CZ PHE 156

1.00 15.88 ATOM 1222 C PHE 156 1.00 15.07 ATOM 1223 O PHE 156 1.00 14.39 ATOM 1224 N ASN 157 1.00 15.95 ATOM 1225 CA ASN 157 1.00 16.78 ATOM 1226 CB ASN 157 1.00 19.91 ATOM 1227 CG ASN 157 1.00 21.97 ATOM 1228 ODl ASN 157 1.00 23.02 ATOM 1229 ND2 ASN 157 1.00 22.77 ATOM 1230 C ASN 157 1.00 16.15 ATOM 1231 O ASN 157 1.00 16.04 ATOM 1232 N ALA 158 1.00 16.31 ATOM 1233 CA ALA 158 1.00 15.97 ATOM 1234 CB ALA 158 1.00 16.14

ATOM 1235 C ALA 158

1.00 15.64

ATOM 1236 O ALA 158

1.00 15.58

ATOM 1237 N TRP 159

1.00 15.76

ATOM 1238 CA TRP 159

1.00 15.15 ATOM 1239 CB TRP 159 1.00 14.73 ATOM 1240 CG TRP 159 1.00 15.51 ATOM 1241 CD2 TRP 159 1.00 15.24 ATOM 1242 CE2 TRP 159 1.00 15.37 ATOM 1243 CE3 TRP 159 1.00 14.42 ATOM 1244 CDl TRP 159 1.00 16.38 ATOM 1245 NEl TRP 159 1.00 14.73

ATOM 1246 CZ2 TRP 159

1.00 12.93

ATOM 1247 CZ3 TRP 159

1.00 14.15

ATOM 1248 CH2 TRP 159

1.00 14.30

ATOM 1249 C TRP 159

1.00 14.08

ATOM 1250 O TRP 159

1.00 13.42

ATOM 1251 N ALA 160

1.00 13.53

ATOM 1252 CA ALA 160

1.00 15.69

ATOM 1253 CB ALA 160

1.00 16.17

ATOM 1254 C ALA 160

1.00 17.30

ATOM 1255 0 ALA 160

1.00 16.76

ATOM 1256 N SER 161

1.00 19.40 ATOM 1257 CA SER 161 1.00 21.24 ATOM 1258 CB SER 161 1.00 21.88 ATOM 1259 OG SER 161 1.00 24.44

ATOM 1260 C SER 161

1.00 21.71

ATOM 1261 0 SER 161

1.00 24.49 ATOM 1262 N HIS 162 1.00 23.09 ATOM 1263 CA HIS 162 1.00 24.28 ATOM 1264 CB HIS 162 1.00 26.20 ATOM 1265 CG HIS 162 1.00 27.93 ATOM 1266 CD2 HIS 162 1.00 29.19

ATOM 1267 ND1 HIS 162

1.00 27.81

ATOM 1268 CEl HIS 162

1.00 29.60 ATOM 1269 NE2 HIS 162 1.00 30.08 ATOM 1270 C HIS 162 1.00 24.43 ATOM 1271 O HIS 162 1.00 25.01 ATOM 1272 N GLY 163 1.00 24.39 ATOM 1273 CA GLY 163 1.00 24.50

-9.860 10.930 34.735 -9.693 12.118 35.003 -9.231 10.416 33.695 -8.277 11.191 32.910 -7.044 10.365 32.732 -5.870 10.394 33.748 -6.313 10.578 35.218 -5.109 9.096 33.505 -8.824 11.631 31.569 -8.570 11.052 30.519 -9.577 12.737 31.598 -10.232 13.318 30.432 -11.074 14.530 30.985 -11.760 15.097 29.861 -10.227 15.686 31.634 -9.218 13.719 29.337 -8.017 13.858 29.623 -9.641 13.898 28.072 -8.746 14.319 26.983 -8.422 13.284 25.835 -7.481 12.109 26.084 -7.435 11.307 24.774 -6.070 12.560 26.494 -9.593 15.334 26.271 -10.797 15.041 26.120 -8.932 16.364 25.727 -9.610 17.412 24.968 -9.629 17.078 23.482

ATOM 1302 -9.760 15.889 23.095

1.00 22.34

ATOM 1303 -9.432 18.098 22.645

1.00 19.20

ATOM 1304 -9.390 17.997 21.191

1.00 17.79

ATOM 1305 -9.760 19.453 20.735

1.00 19.15

ATOM 1306 -10.323 19.282 19.441

1.00 21.89

ATOM 1307 -8.613 20.458 20.690

1.00 18.08

ATOM 1308 -7.986 17.461 20.832

1.00 15.71

ATOM 1309 -6.987 17.903 21.384

1.00 14.65

ATOM 1310 -7.843 16.481 19.953

1.00 14.44

ATOM 1311 -6.576 15.814 19.662

1.00 14.83

ATOM 1312 -6.836 14.483 18.935

1.00 15.04

ATOM 1313 -7.727 13.517 19.684

1.00 16.43

ATOM 1314 -7.005 13.060 21.275

1.00 20.33

ATOM 1315 -6.344 11.447 20.893

1.00 18.80

ATOM 1316 -5.637 16.648 18.797

1.00 14.85

ATOM 1317 -6.024 17.252 17.775

1.00 17.21

ATOM 1318 -4.409 16.773 19.220

1.00 13.83

ATOM 1319 -3.401 17.330 18.307

1.00 13.73

ATOM 1320 -2.670 16.197 17.581

1.00 10.96

ATOM 1321 -3.383 15.283 17.156

1.00 12.91

ATOM 1322 -2.365 18.025 18.072

1.00 14.89

ATOM 1323 -1.803 19.443 18.160

1.00 17.35

ATOM 1324 -2.595 20.457 18.060

1.00 20.00

ATOM 1325 -0.538 19.626 18.333

1.00 20.00

ATOM 1326 -1.371 16.150 17.418

1.00 10.61

ATOM 1327 -0.722 15.072 16.692

1.00 11.47

ATOM 1328 0.658 15.560 16.280

1.00 10.30

ATOM 1329 1.776 15.763 17.280

1.00 10.95

ATOM 1330 CDl TYR 171

1. 00 11.20

ATOM 1331 CEl TYR 171

1. 00 11.22

ATOM 1332 CD2 TYR 171

1. 00 10. 25

ATOM 1333 CE2 TYR 171

1. 00 11.78

ATOM 1334 CZ TYR 171

1. 00 10.98

ATOM 1335 OH TYR 171 1. 00 9.29 ATOM 1336 C TYR 171

1.00 11.01

ATOM 1337 O TYR 171

1.00 10.53

ATOM 1338 N GLN 172

1. 00 10.87

ATOM 1339 CA GLN 172 1. 00 9.53 ATOM 1340 CB GLN 172

1. 00 10. 18

ATOM 1341 CG GLN 172 1.00 9.79 ATOM 1342 CD GLN 172 1.00 9.76 ATOM 1343 OEl GLN 172

1.00 10.99

ATOM 1344 NE2 GLN 172 1.00 9.74 ATOM 1345 C GLN 172 1.00 8.72 ATOM 1346 O GLN 172 1.00 9.11 ATOM 1347 N ILE 173 1.00 6.96 ATOM 1348 CA ILE 173 1.00 6.99 ATOM 1349 CB ILE 173 1.00 9.21 ATOM 1350 CG2 ILE 173 1.00 8.02 ATOM 1351 CGI ILE 173

1.00 10.65

ATOM 1352 CDl ILE 173

1.00 11.75

ATOM 1353 C ILE 173 1.00 5.19 ATOM 1354 O ILE 173 1.00 5.97 ATOM 1355 N VAL 174 1.00 5.30 ATOM 1356 CA VAL 174 1.00 5.81 ATOM 1357 CB VAL 174 1.00 5.74

ATOM 1358 CGI VAL 174 1.00 6.61 ATOM 1359 CG2 VAL 174 1.00 4.99 ATOM 1360 C VAL 174 1.00 4.60 ATOM 1361 0 VAL 174 1.00 6.58 ATOM 1362 N ALA 175 1.00 6.06 ATOM 1363 CA ALA 175 1.00 4.93 ATOM 1364 CB ALA 175 1.00 6.89 ATOM 1365 C ALA 175 1.00 3.97 ATOM 1366 O ALA 175 1.00 8.33 ATOM 1367 N VAL 176 1.00 3.82 ATOM 1368 CA VAL 176 1.00 6.89 ATOM 1369 CB VAL 176 1.00 9.34 ATOM 1370 CGI VAL 176 1.00 7.08 ATOM 1371 CG2 VAL 176

1.00 10.56

ATOM 1372 C VAL 176 1.00 8.28 ATOM 1373 O VAL 176 1.00 7.87 ATOM 1374 N GLU 177 1.00 9.10 ATOM 1375 CA GLU 177 1.00 8.39 ATOM 1376 CB GLU 177 1.00 8.89 ATOM 1377 CG GLU 177

1.00 10.47 ATOM 1378 CD GLU 177 1.00 12.55 ATOM 1379 OEl GLU 177 1.00 14.06 ATOM 1380 OE2 GLU 177 1.00 11.52 ATOM 1381 C GLU 177 1.00 9.30 ATOM 1382 O GLU 177 1.00 10.91

ATOM 1383 N GLY 178 1.00 7.47 ATOM 1384 CA GLY 178 1.00 6.20 ATOM 1385 C GLY 178 1.00 8.83

ATOM 1414 C SER 181

1. 00 11. 07 ATOM 1415 O SER 181

1. 00 10 . 95

ATOM 1416 N SER 182 1.00 8.87 ATOM 1417 CA SER 182 1.00 9.70 ATOM 1418 CB SER 182 1.00 8.44 ATOM 1419 OG SER 182

1.00 11.70

ATOM 1420 C SER 182 1.00 8.32 ATOM 1421 SER 182 1.00 8.91 ATOM 1422 N GLY 183 1.00 8.19 ATOM 1423 183 1.00 9.01 ATOM 1424 183 1.00 11.09 ATOM 1425 183

1.00 11.68

ATOM 1426 184

1.00 11.79

ATOM 1427 184

1.00 11.40

ATOM 1428 184

1.00 11.41

ATOM 1429 184

1.00 15.69

ATOM 1430 184

1.00 10.12

ATOM 1431 184

1.00 11.52

ATOM 1432 185 1.00 8.63 ATOM 1433 185 1.00 6.71 ATOM 1434 185 1.00 5.28 ATOM 1435 185 1.00 8.30 ATOM 1436 185 1.00 8.44 ATOM 1437 186 1.00 8.21 ATOM 1438 186 1.00 7.45 ATOM 1439 186 1.00 8.66 ATOM 1440 186 1.00 8.84 ATOM 1441 186 1.00 9.02

ATOM 1442 O SER 186 1.00 7.60 ATOM 1443 N ILE 187 1.00 8.36 ATOM 1444 CA ILE 187 1.00 9.26 ATOM 1445 CB ILE 187

1.00 12.57

ATOM 1446 CG2 ILE 187

1.00 10.66

ATOM 1447 CGI ILE 187

1.00 11.46

ATOM 1448 CDl ILE 187

1.00 12.68

ATOM 1449 C ILE 187 1.00 9.78 ATOM 1450 O ILE 187

1.00 10.18

ATOM 1451 N THR 188 1.00 7.46 ATOM 1452 CA THR 188 1.00 8.18 ATOM 1453 CB THR 188 1.00 6.41 ATOM 1454 OGl THR 188 1.00 5.82 ATOM 1455 CG2 THR 188 1.00 5.98 ATOM 1456 C THR 188 1.00 6.88 ATOM 1457 O THR 188 1.00 7.67 ATOM 1458 N VAL 189 1.00 7.06 ATOM 1459 CA VAL 189 1.00 9.10 ATOM 1460 CB VAL 189 1.00 9.32 ATOM 1461 CGI VAL 189 1.00 9.82 ATOM 1462 CG2 VAL 189 1.00 7.64 ATOM 1463 C VAL 189 1.00 8.74 ATOM 1464 O VAL 189 1.00 9.63 ATOM 1465 N SER 190

1.00 10.99

ATOM 1466 CA SER 190

1.00 13.10

ATOM 1467 CB SER 190

1.00 15.33

ATOM 1468 OG SER 190

1.00 15.84

ATOM 1469 C SER 190

1.00 14.43

ATOM 1470 O SER 190 -1.439 2.580 9.354 1.00 17.68

ATOM 1471 OXT SER 190 -0.392 1.975 11.150 1.00 12.62

A portion of the three-dimensional crystal structure of a mutant of the B. circulans xylanase containing an intramolecular disulfide bond is shown in Figure 4. In this mutant serine 100 and asparagine 148 have been mutated to cysteine. The disulfide which is formed links the last strand of sheet III to the alpha-helix. The structure of this mutant is identical to the wild-type enzyme except for the side chains of residues 100 and 148 and some very minor shifts of nearby atoms. The same region of the superimposed structures of the B. circulans and T. harzianum xylanases is shown in Figure 5. Further details of the analyses will be providedin the examples, however, one can see that although there are some sequence differences between the B. circulans and T. harzianum xylanases near the site of the manufactured disulfide and some small position differences at other nearby residues, the structure of the T. harzianum xylanase is virtually identical to the structure of the B. circulans xylanase at the positions of the mutations. The sequence alignment (Figure 1) shows that the sequence homology of the various xylanases in the vicinity of residues 98 to 100 and 148 to 152 is very good. One would therefore expect that the structures of other related xylanases would be similar enough that they could be modified with the introduction of an intramolecular disulfide bond linking sheet III to the alpha-helix and that this disulfide bond would stabilize them in a simliar manner as taught in the present invention.

Similarly one would expect that sequence changes , made at the N-terminal end of the B. circulans xylanase, which produced a more stable xylanase, could be made at corresponding positions in other family G xylanases, with a reasonable expectation of success. For example, the

strategy to substitute the Asn residue N-8 of B. circulans xylanasebyTyr and Phe to yield more stable mutants, can be readily applied to fungal xylanases of A. niger var. aw amor i, A. tubigensiε A and T. ressei I. These fungal xylanses, like the bacterial B . circulans xylanase, possesses the same target Asn residue, available for the same mutation.

For some xylanases with reduced sequence homology to the B. circulans xylanase it may be necessary to make other mutations to increase the homology in the vicinity of the introduced intramolecular disulfide bond or N-terminal mutation. In other words it may be necessary to make another xylanase more like the B. circulans xylanase before the disulfide bond could be formed successfully or before a useful N-icerminal mutation is made. Throughout the application reference is made to amino acid positions based on the B. circulans xylanase, as a reference. Figure 1 is provided in order to determine the corresponding position in other family G xylanases.

In the modification of family G xylanases, the B. circulans xylanase (BCX) . was chosen to exemplify the principles of the present invention. This enzyme is only moderately thermostable at temperatures up to 55°C. For application of this enzyme in pretreatment of kraft-pulp to enhance bleaching, a higher temperature stability is desirable. As noted previously, a xylanase which has a higher pH optimum, would also be benificial in the bleaching step. Such a modified enzyme would also be useful in the food industry.

As noted previously, the chief application of the modified xylanase is for pulp biobleaching in the production of paper. Before bleaching process, the pulp is hot at a temperature range of 55-70°C and in an alkaline

state. Many commercial wild-type xylanases (T. reesee and B . pummilus) only function at 55 ^β C and some require acidic pH. Therefore the desirable improvements in a mutant xylanase are higher functional temperature (temperature optimum) and pH (pH optimum) , with the former characteristic as the most important. Xylanase of higher temperature optimum can be used to treat pulp at high temperature without idle period of cooling. With higher functional pH (pH optimal) , the mutant enzyme would require less or no acid to neutralize the pulp which is basic at that stage. The economic benefits include saving in time, acid for neutralization and no acidic corrosion of the equipments. Therefore a desirable xylanase mutant would have a temperature optimum approaching 70°C and a pH optimum approaching 9 in pulp bleaching. Of course any measurable improvement over the wild type xylanase, BCX (54°C, pH 7) would be beneficial.

In the parent application (U.S. Serial No. 08/044,621), the improvement of mutant was measured in terms of thermostability, ie, its ability to survive incubation at high temperature in buffer without any substrate. Increased thermostability means an increase of 2°C to 15 ^β C, while maintaining at least 60% of activity, compared to wild type xylanase, after incubation at an elevated temperature. Such a pursuit of improved thermostability or passive heat-resistance was the sole objective in another publication involving xylanase (Arase et al, 1993). Although this characteristic may be useful and possibly related to the shelf-life of the enzyme, it is not a reliable indicator of the ability of the enzyme to function at that temperature. At the elevated temperature, the enzyme molecule may have been deactivated or unfolded but managed to be quickly refolded or reactivated before assayed at 40 ^β C. Therefore the gain in thermostability or heat-resistance (T _1/2 ) does not guarantee that the enzyme remains intact or can function at higher temperature.

An alternative assay was used to determine if the mutant enzyme can function at high temperature. Instead of incubation in buffer without xylan, xylanase is incubated in the same manner with xylan in the buffer. The modified process is designed to evaluate the hydrolysis of soluble xylan by the mutant enzyme at different temperatures. The reducing sugar released was then determined by the HBAH reagent. With addition of xylan in the mixture, the protocol remained essentially the same. The temperature optimum is defined as the highest temperature at which 150% relative enzymatic activity was maintained as compared to performance at 40 ^β C.

A further measure of increased stability, according to the present invention, is an increase in pH optimum in the hydrolysis of xylan. In the present invention, pH optimum has been defined as the highest pH at which the enzyme still maintains 50% of the maximal activity.

In addition to the assay based on the direct hydrolysis of xylan, the mutant xylanases were also tested for their ability to bleach pulp. The temperature optimum and pH optimum in pulp bleaching are defined as the highest temperature and pH at which the brightness gain by the treated pulp was not less than 0.4.

In one aspect of the present invention, the starting point was the solution of the three-dimensional structure of the enzyme (Figure 2) . By inspection of the structure, a prediction of the residues, which could be mutated to cysteine, and which would possibly oxidize to form an intra-molecular SS bridge was made. This was accomplished by searching for pairs of residues for which the inter-C-alpha distance was less than 6.6 and the inter-C-beta distance was less than 4.5 A. If either member of the pair was a glycine this pair was ignored

because glycines are important for the backbone conformation. Some candidates were excluded because they were involved in the active site of the enzyme. In addition the distance algorithm had selected pairs of residues which were too close, as they were on adjacent strands of one of the beta sheets.

Two areas for the introduction of a cysteine residue were selected. These areas are the beta-sheet III and the alpha-helix. Although the alpha-helix itself ranges from amino acids 147 to 155, some residues on either side could potentially be used to form intra-molecular disulfide linkages. Accordingly, the amino acids in the alpha-helix ranging from 143 to 158 and the amino acids on the beta-sheet III ranging from 95 to 109 are potential sites for the introduction of cysteine residues for the formation of an intra-molecular disulphide bond between these two areas. Two pairs of residues were found (details will be provided in the Examples) and the corresponding mutants were constructed, and in both cases the intra- molecular SS bond formed spontaneously.

In addition to the intra-molecular SS bond mutant, a second type of disulfide mutant was constructed by joining two protein molecules with an inter-molecular SS bond. For the production of this type of disulfide bond, a cysteine is introduced in each of the two xylanase protein molecules such that the cysteine is on the exterior of the molecule, thus the two molecules can be joined by an inter-molecular disulphide bond. Potential sites of interest include amino acid 15 to 31 of the beta-sheet I; amino acid 43 to 61 of the beta-sheet I; amino acid 87 to 104 of the loop and beta-sheet III; amino acid 133 to 163 of the alpha-helix and surrounding loops; and amino acids 177 to 185 of the beta-sheet I.

A third type of mutant unrelated to SS bond formation has also been found to confer stability. These mutants were generated by specific mutagenesis of amino acids at the N-terminus of the xylanase molecule. In one embodiment of the present invention, amino acids 1 to 25 of the xylanase protein are selected for specific mutagenesis. As discussed above, the amino acid numbering is based on the B. circulans xylanase. Corresponding amino acids from other family G xylanases can be determined by making reference to Figure 1.

In one example of the present invention, the introduction of tyrosine or phenylalanine by substitution resulted in mutants with increased thermostability, temperature optimum and pH optimum, when compared to the wild type protein. In another embodiment of the present invention, the introduction of proline at amino acid position 22, by substitution resulted in a more stable mutant. In a further example of the present invention, the stability of the mutant could be increased by combining two or mutations in a single molecule. In some examples the effect of two or more mutants was additive and in other examples the effect was co-operative.

The selected amino acids referred to above are based on the amino acid sequence of the B. circulans xylanase sequence. The present invention is not limited to producing a thermostable xylanase from this bacterial source, but includes the production of a thermostable xylanase for other sources, as listed in Table 1 for example. The choice of suitable amino acids to target for mutagenesis in xylanases from other sources will be obvious to those skilled in the art by comparing the sequences and choosing the amino acids corresponding to those identified for the B. circulans xylanase.

The present invention will be further illustrated by way of the following examples, which are not to be construed as limiting. In these examples the xylanase mutants, which were constructed, are summarized in Table 2.

Table 2 Xylanase Mutants

EXAMPLE 1

CONSTRUCTION OF THE MUTANT TSl

The gene encoding the B. subtilis xylanase (see Figure 6) , is identical to the B. circulans xylanase except at position 147, where a serine (BSX) rather than threonine (BCX) is encoded, (Yang et al, 1988, Nucleic Acid Research 16:7187 and Paice et al, 1986, Archives of Microbiology 144:201-206; all references incorporated herein by reference) . The xylanase gene encoded by pBSX was mutated by the uracil containing DNA (UDNA) method (Kunkle et al, 1987, Methods in Enzymology, 154:367-382, which is incorporated herein by reference) to produce the mutant S100C. The coding sequence for this mutant gene was then removed from the vector using PCR. The 5' portion of the resulting PCR product (codons 1-103) was combined with a synthetic gene fragment from the B. circulans gene (codons 104-185) to produce the plasmid pCWBCX: :S100C. The region containing codons 136-153 of pCWBCX: :S100C was replaced with synthetic oligonucleotides to introduce the second cysteine codon at position 148 (N148C) . The resultant plasmid contained the S100C and N148C mutations and was called TSl.

All liquid cultures in this and other Examples were grown in either 2YT medium (16 g yeast extract, 10 g bacto-tryptone, 5 g NaCl, 1 L of H ₂ 0) , or TB medium (24 g yeast extract, 12 g bacto-tryptone, 10 ml 1 M potassium phosphate buffer pH 7.5, 5 ml of 80% glycerol, 1 L H ₂ 0) . The antibiotic ampicillin was added at 150 μg/ml to all cultures of plasmid containing strains. The cultures were grown with shaking at 30 ^β C for protein and plasmid production, and 37°C for the production of single stranded DNA containing particles.

Basic recombinant DNA methods like plasmid DNA isolation, restriction enzyme digestions, the purification of DNA fragments for cloning, ligations, transformations and DNA sequencing were performed as recommended by the enzyme supplier, or the manufacturer of the kit used for

the particular procedure. Polyacrylamide gel electrophoresis of proteins was performed as recommended in the technical literature supplied by Bio-Rad laboratories, Mississauga Ont. Restriction and DNA modification enzymes were purchased from New England Biolabs LTD., Mississauga Ont. Prep-A-Gene DNA purification matrix was purchased from Bio-Rad laboratories, Mississauga Ont. Sequenase, a DNA sequencing kit, was purchased from US Biochemicals, Cleveland Ohio. Oligonucleotide 3' end labelling was performed with a kit from Boehringer Mannheim Canada, Laval PQ. Protein concentration was determined from the molar extinction coefficient of the xylanase: 81,790 L.mol" ¹ .

A 2 ml culture of Escherichia coli RZ1032 (HfrKL16PO/45 [lγsA(61-62) ] , dutl, unσl. thil. relAl. Zbd- 279: :Tnl0.supE44) harbouring pBSX was grown at 37°C with vigourous shaking until the reached 0.5, at which time 10 μl of the helper phage, M13K07, (titre 1 x 10 ¹² /ml) , was added. After 1 h, 0.5 ml of the culture was subcultured into 20 ml of fresh media containing 50 μg kanamycin/ml, and 100 μg ampicillin/ml. This culture was shaken at 200 rpm for 16 h at 37°C. The supernatant containing the single stranded DNA containing particles (SSDNAP) was collected after centrifugation of the culture at 4°C for 20 min. at 7000 x g. The SSDNAP were precipitated by the addition of 1/4 volume of 15% PEG-8000/ 14.6% NaCl. After 30 min. at room temperature the precipitate was collected by centrifugation at 7000 x g for 20 min. The precipitate was resuspended in 0.5 ml TE buffer (lOmM Tris - HCl, 1 mM EDTA, pH 8) , and left on ice for 30 min. Any insoluble material was removed by a brief centrifugation in a microcentrifuge. The supernatant was made up to 1% acetic acid and the resultant precipitate was collected on a 1 cm glass fibre filter by suction filtration. Protein was removed from the precipitated SSDNAP'S, and the DNA bound to the filter by washing the filter under vacuum, with 2 ml of 4 M NaC10 ₄ in TE buffer. Excess NaCl0 ₄ was removed by

washing the filter with 2 ml of ice cold 70% ethanol. The filter was allowed to air dry for 5 min. at room temperature. The filter was placed into a 500 μl microcentrifuge tube with a hole in the bottom. This tube was placed inside a 1.5 ml microcentrifuge tube. The single stranded DNA was eluted by the addition of 50μl of 0.IX TE buffer to the filter and after 5 min centrifuging the two tubes to recover the liquid in the larger tube. The elution step was repeated. The single stranded DNA was quantified by analysis on agarose gel electrophoresis.

Oligonucleotides were synthesized using an Applied Biosystems model 380B DNA synthesizer. Synthetic oligonucleotides were purified by polyacrylamide gel electrophoresis (PAGE) in 15% gels containing 7 M urea. Oligonucleotides were detected by UV shadowing, cut out and eluted from the gel slices in 1 ml of 0.5 M ammonium acetate, 10 mM magnesium acetate. The oligonucleotides were desalted by passage through a Sep-Pak C18 reverse phase cartridge (Atkinson and Smith, 1984, In: Oligonucleotide synthesis a practical approach. Gait, N. J. , editor. IRL press, Washington) . The oligonucleotides were eluted with 20 % acetonitrile, and the concentration was determined by reading the A^o of the solution. For the production of S100C the following oligonucleotide was synthesized:

SEQ ID NO:4

Codons 96 97 98 99 100 101 102 103 104 5'- GGT ACT GTA AAA TGT GAT GGG GGT ACA - 3'

The oligonucleotide was phosphorylated for use in mutagenesis. 100 to 200 pmol of the oligonucleotide was mixed with 2μl of 10X kinase buffer (500 mM Tris-HCl, pH 7.6, 100 mM MgCl ₂ , 100 mM dithiothreitol (DTT) , 1.0 mM spermidine, 10 mM adenosine triphosphate (ATP)), and the volume was adjusted with water to a final volume of 20 μl.

The reaction was initiated by the addition of 10U of T4 polynucleotide kinase. The reaction was performed at 37°C for 1 h, after which the reaction was terminated by heating at 70°C for 10 min. Oligonucleotides prepared in this fashion were ready for use in the UDNA mutagenesis method.

The phosphorylated mutagenic oligonucleotide primer (20 p ol, 2μl) , was mixed with the ssUDNA (0.5 pmol, 7 μl) ; 1 μl of 10X DNA synthesis buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ , 500 mM NaCl, 10 mM DTT). The DNA'S were annealed by heating the mixture to 70°C, and then slowly cooled to room temperature. The mutagenesis reaction was then performed by the addition of 10 μl of the DNA synthesis mixture (1 μl 10X synthesis buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ , 100 mM DTT), 1 μl 5 mM deoxynucleotide triphosphates (dNTPs) , 1 μl 10 mM ATP, 6 μl H ₂ 0, 1 μl, 5U, T4 DNA ligase, 0.25 μl, 2.5U, T7 DNA polymerase) . The reaction mixture was kept on ice for 5 min, then at room temperature for 5 min. , and finally incubated at 37°C for 60 to 90 min. The reaction was terminated by the addition of 3 μl 50 mM EDTA. The reaction products were analyzed by standard agarose gel electrophoresis (Sambrook et al, 1989, Molecular cloning: A laboratory manual, 2nd edition) . A 5 μl portion of this mixture was used to transform the E. cόli strain MV1190

(deletion (lac-proAB) , thi, supE44. deletion (srl- recA)306::TnlO(tet ^r ) | ^" F':traD36. proAB. lacl"deletion M15]) or BHM 71-18 (deletion (lac-proAB) . thi. supE44. [mutS: :Tnl0(tet ^r ) ]) .

After transformation the bacteria were plated onto solid media (2YT) containing 150 μg ampicillin/ml. Individual colonies were picked and transferred to a fresh plate in a grid pattern of 100 colonies per plate. After overnight growth at 37°C, the colonies were transferred to Hybond-N nylon filter membranes (Amersha , Canada, Oakville Ont.), the colonies were lysed, and the DNA fixed to these

filters by well established procedures (Sambrook et al, 1989, Molecular cloning: a laboratory manual).

Oligonucleotide probes were synthesized using a 3' end labelling kit from Boehringer Mannheim. 100 pmol of oligonucleotide were dissolved in 4μl H ₂ 0. The reaction mixture was completed as follows: 4 μl of 5X tailing buffer (1M potassium cacodylate, 0.125 M Tris-HCl, bovine serum albumin (BSA) 1.25 mg/ml, pH 6.6), 4 μl 25 mM CoCl ₂ , 1 μl 1 mM digoxigenin-dideoxy-uracil triphosphate (DIG-ddUTP) , 6 μl H ₂ 0, 1 μl terminal transferase (50U) . The mixture was reacted at 37°C for 15 min., then 2 μl of glycogen was added (0.2 μg / μl in 0.2 M EDTA) . The labelled oligonucleotide was precipitated by the addition of 2.5 μl of 4 M LiCl, and 75 μl of ice cold, -20°C, 95% ethanol. After 2 h at - 20°C the precipitate was collected by centrifugation at 12,000 g for 30 min. The pellet was washed with 100 μl ice cold 70% ethanol, and then air dried in a fume hood.

The filters containing the colonies were washed free of cell debris in 2X SSC (SSC buffer is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.1% SDS, by gently rubbing the surface of the filter with a wet tissue. After removal of the cell debris, the filters were pre-hybridized in hybridization buffer (5X SSC, 0.1% SDS, 0.02% N- laurylsarkosine, 1% blocking reagent, a commercial product from Boehringer Mannheim) , for at least 5 h. After the pre-hybridization, the DIG-labelled oligonucleotide was dissolved.in 100 μl H ₂ 0, and added to 7 ml of hybridization buffer. The hybridization buffer-DIG labelled oligonucleotide mixture was poured onto the filter and the hybridization was then performed in plastic petri dishes at 37°C, with shaking overnight. Excess (unbound) probe was removed by washing the filters twice for 10 min, each time, with 2X SSC,0.1% SDS at room temperature. Non-specifically

bound probe was removed by washing the filters at 40°C with 0.5 X SSC, 0.1% SDS, again twice for 10 min each time.

The filters were then washed in 25 ml of M buffer (0.1 M maleic acid, 0.15 M NaCl, pH adjusted to 7.5 with concentrated NaOH) . The filter was then incubated in 20 ml M buffer containing 1% blocking reagent for 30 min. at room temperature. The solution was then discarded and then further incubated with 10 ml of fresh M buffer with 1% blocking reagent and 750 mU of anti-DIG-alkaline phosphatase-conjugate antibody. After 30 min. , the filter was washed with 3 x 20 ml washes of M buffer with 0.3% Tween 20. The filter was then equilibrated in alkaline phosphatase buffer (0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl ₂ pH 9.5) . The filter was then dipped in a solution of Lumi-phos, a chemiluminescent alkaline phosphatase substrate (Boehringer Mannheim) . The filter was sealed into a plastic bag, and incubated at 37°C for 30 min. The filter in the bag was then exposed to x-ray film for 5 to 60 min. Positive colonies were further characterized by DNA sequencing. The clone chosen for subsequent work contained plasmid pBSXSlOOC.

Removal of the mature coding sequence of the B. subtilis xylanase gene from its leader sequence was accomplished using PCR with specific primers. The primers were designed so that the resulting amplification product would contain a new Ndel restriction site to allow the precise fusion of the coding region to the transcriptional signals present in pCWori+. An Xbal site was introduced at the 3' end to facilitate cloning into pCWori-

The plasmid pBSXSlOOC was digested with EcoRI to linearize it, and then it was used as a template for PCR. The amplification reaction contained, 15 μl of template DNA

(50 ng) , 5 μl of 10X buffer (100 mM KC1, 100 mM ammonium sulfate, 200 mM Tris-HCl pH 8.8, 40 mM magnesium sulfate,

1% Triton X100, 100 μg/ml BSA) , 5 μl of 5 mM dNTPs, 2.5 μl 5' primer solution (25 pmol), 2.5 μl 3' primer solution (25 pmol) , 19 μl H ₂ 0, 1 μl (1U) Vent DNA poly erase (New England Biolabs). The 5' and 3' primers are shown below:

SEQ ID Nθ:5

Ndel site 5* BSYS GC CTG CAG CAT ATG GCT AGC ACA GAC TAC TGG CAA AAT TGG A SEQ ID Nθ:6 Xbal the

3' BSXY GC AAG CTT TCT AGA CTT TAA CCA TTA CTA ACG ATT TTA ATA ATC

The reaction was covered with 50 μl of paraffin oil to prevent evaporation. The reaction mixture was prewarmed to 94 ^β C without enzyme for 5 min., then the reaction mixture was cooled to 72°C, then the enzyme was added. The reaction was incubated in a temperature cycler for 30 cycles of 94°C 1 min., 55 ^β C 2 min., 72°C for 2 min. The yield of amplification product was approximately lμg of a 700 bp fragment. This fragment was purified from an agarose gel.

The resulting PCR product had an introduced Ndel site at the 5' end to provide a start codon for the xylanase gene when cloned into pCWori+. The xylanase gene was cloned into pCWori+ in two steps. First the 3' portion, codons 105 -185 (Figure 6) was inserted as a 330 bp Ndel - Xbal fragment. This fragment was generated by digesting 200 ng of the PCR product with Ndel and Xbal, and then was purified from an agarose gel. 50 ng of this fragment was ligated with 50 ng of Ndel - Xbal digested and gel purified pCWori+. The clone, pCW3', was identified by hybridization using a probe made by random priming labelling (Sambrook et al, 1989, Molecular cloning: a laboratory manual) , with DIG-dUTP, of 50 ng of the PCR product. The second step was to digest the PCR product with Ndel to generate a 318 bp fragment which encodes codons 1 - 104. After gel electrophoresis and

purification, 50 ng of this fragment was ligated to 50 ng of Ndel digested pCW3'. The clones containing a functional xylanase gene were identified by screening transformants on plates containing remazo-brilliant blue xylan (RBBX) . Those colonies which made halos, were expressing the gene for xylanase (Kluepfel, 1988, Methods in Enzymology, 154:367-382, which is incorporated herein by reference). This clone was called pCWBSX: :S100C.

A version of the B. circulans xylanase gene was synthesized to aid in subsequent mutagenesis studies, using the first 103 codons of the natural gene, and 82 codons derived from synthetic DNA fragments (pCWBCX3'SYN, Figure 7) . The synthetic gene portion is identical to the natural gene except codons were used, which reflect the frequency of usage for specific amino acid residues in the genes of E. coli. The 103 codon portion was replaced with the equivalent portion from pCWBSX: :S100C, by isolating the Nhel-Ndel restriction fragment of pCWBSX: :S100C and ligating it to Nhel-Ndel digested pCWBCX3'SYN. After ligation, the mixture was transformed into E. coli MV1190. Clones were analyzed by DNA sequencing to verify they carried the SIOOC mutation. The resultant plasmid was called pCWBCX: :S100C.

The plasmid pCWBCX: :S100C was purified and digested with the restriction enzymes EagI and Nsil to remove the portion encoding codons 136 to 153. The large plasmid fragment was isolated from an agarose gel as described above. A set of synthetic oligonucleotides: Xyl46C TOP (SEQ ID NO:7); Xyl47C BOTTOM (SEQ ID NO:8) and Xyl48C BOTTOM (SEQ ID NO:9) (as shown below) was synthesized to replace the EagI to Nsil fragment these oligonucleotides encoded the mutations at F146C, T147C, and N148C.

EagI Nsil

137138139140141 142143144145146147148149150151 152 GG CCG ACT GGT TCG AAC GCC ACC ATC ACT T∞ ACT AAC CAT GTC AAT GCA Xy146C TOP

C TGA CCA AGC TTG CGG TGG TAG TGA AAG ΛCA TTG GTA CAG TT Xy147C BOTTOM C TGA CCA AGC TTG CGG TGG TAG TGA AAG TGA ACG GTA CAG TT Xy148C BOTTOM

For the construction of restriction fragments for cassette mutagenesis, after kinase treatment, complementary oligonucleotides were mixed together (20 pmol of each oligonucleotide) , and the mixture was heated to 80°C for 10 min. The mixture was then allowed to slowly cool to room temperature. Portions from the annealed oligonucleotide mixture were then used directly in ligation reactions with the previously digested and purified pCWBCX: :S100C. After ligation and transformation, clones carrying the modified EagI-Nsil fragment were identified by hybridization with a DIG-ddUTP labelled oligonucleotide. Once hybridization positive colonies were identified, plasmid DNA was isolated and E. coli was re-transformed, to ensure the purity of the clone. DNA sequencing was performed to verify changes at the desired codons.

EXAMPLE 2

CONSTRUCTION OF THE MUTANT TS2

The plasmid pCWBCX3'SYN was used to transform E. coli RZ1032, and single stranded UDNA was prepared as previously described in Example 1. Two mutagenic oligonucleotides for making V98C, and A152C (see below) were used in an in vitro DNA synthesis reaction as described in Example 1.

SEQ ID NO:10

Codons 101 100 99 98 97 96 95 94 pCU V98C 5' C ATC ACT TTT <XA AGT ACC TTT ATA 3' SEQ ID NO:11

Codons 155154153152151150149 pCW A152C 5' GGA TTT CCA OA ATT CAC GTG 3'

After transformation, colonies were screened for hybridization to both mutagenic oligonucleotides. The xylanase gene from a hybridization positive clone, TS2, was completely sequenced to ensure only the desired mutations were present.

EXAMPLE 3

CONSTRUCTION OF THE MUTANT TS4

An additional thermostable mutant containing an inter-molecular disulfide bridge was constructed by a mutation at amino acid position 179 to change the amino acid from a serine to a cysteine (S179C) . This mutant was constructed using cassette mutagenesis to make TS4a using the oligonucleotides: S179C-1 (SEQ ID NO:12); S179C-2 (SEQ ID NO:13) and S179C-3 (SEQ ID NO:14) shown below:

Codons 178179180181182183184185 S179C-15' CT GGA JjE TCC AAT GTG ACA GTG TGG TAA AGA TCT TGA S179C-23' TCG AGA CCT ACG AGG TTA CAC S179C-33' TGT CAC ACC ATT TCT AGA ACT

The plasmid pCWBCX3'SYN was digested with Sad and Hindlll. The large fragment was purified from an agarose gel. The oligonucleotides for S179C were processed as described in Example 1, for cassette mutagenesis. This protein contained a single active thiol, however the protein dimerizes spontaneously. It forms a mixture based on the amount of free sulfhydryl from Ellmans reagent testing, and from purification of both the monomer and dimer from the mixture. This mutation was then combined

with TSl, by standard subcloning to produce a mutant called TS4. The mutant TS4M is the combined TSl + S179C monomeric species and the TS4D is the dimeric species of this mutant. The purity of the monomeric and dimeric species used in the thermostability assays is shown in Figure 8.

EXAMPLE 4

CONSTRUCTION OF MUTATIONS AT THE N-TERMINUS OF B. CIRCULANS

XYLANASE

Several mutant xylanases with mutations in the N- terminal region, have been constructed via the method of cassette mutagenesis. These mutants are either single, double or triple amino acid changes at positions 3, 4 or 8. All of the gene constructions involved ligations of 2 pairs of oligonucleotides which contained the mutations, into the Nhel/BspEI linearized plasmid pXYbc (Figure 9) , via the well established recombinant procedures of (i) phosphorylation of the oligonucleotides, (ii) their ligation with the linearized plasmid, (iii) transformation into the E. coli competent cells, (iv) identification of the mutant transformants via hybridization with the labelled oligonucleotide as probe, and (v) confirmation of the mutation through gene sequencing. These procedures have been fully described in the preceding Examples. The oligonucleotides used for the production of various N- terminus mutants are shown below.

(1) TS5a mutant, where the asparagine-8 (N-8) has been converted into tyrosine (Y) .

The oligonucleotides for the cassettemutagenesis are XY8Y-1 (SEQ ID NO:15), XY8Y-2 (SEQ ID NO:16), XY-11-2

(SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18), and are as sho below:

XY8Y-1 XY-11-2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2 N-ter A S T D Y W O Y U T D G G G I V N A V N G S 5'- CT AGT ACA GAT TAT TGG CAA TAT TGG ACA GAC GGT GGC GGT ATC GTT AAT GCC GTG AAC GGC T

A TGT CTA ATA ACC GTT ATA ACC TGT CTG CCA CCG CCA TAG CAA TTA CGG CAC TTG CCG AGG C

Nhel XY8Y-2 XY-16-1 BspEI

(2) TS6a mutant, where threonine-3 (T-3), aspartic acid- (D-4) and asparagine-8 (N-8) have been converted to glycin (G) , tyrosine (Y) and tyrosine (Y) , respectively.

The oligonucleotides for cassettemutagenesis ar XY3G4, 8Y-1; (SEQ ID NO:19) XY3G4, 8Y-2 (SEQ ID NO:20); XY 11-2 and XY-16-1 wherein the first two oligonucleotides ar shown below: XY3G4,8Y-1

1 2 3 4 5 6 7 8 9 10 11 12 13 N-ter Λ S G Y Y U Q Y V T D G G 5'- CT AGT GGA TAC TAT TGG CAA TAT TGG A CCT ATG ATA ACC GTT ATA ACC TGT CTG CCA CCG Nhel XY3G4.8Y-2

(3) TS3a mutant was prepared from a pair o oligonucleotides with mixed bases at codons-4 and 8. Th oligonucleotides for cassette mutagenesis are XY(l-8)Mu- (SEQ ID NO:21) , XY(l-8)Mu-2 (SEQ ID NO:22) , XY-11-2 and XY

16-1 wherein the first two oligonucleotides are shown below:

XY (l-8 ) Mu-l 1 2 3 4 5 6 7 8 9 10 11 12 13

N-ter A S T D/Y Y W Q W T D G G

5 ' - CT AGT ACA TAC TAC TGG CAA Tkl TGG A TGT CTG ATG ACC GTT Ami ACC TGT CTG CCA CCG

Nhel XY(l-8)Mu-2

k,l,m are mixed bases used in synthesis of oligonucleotide. k = T+A+G 1 = C+G m = T+A+C

4) TS3 mutant was constructed by combining the mutations from TSl with that from TS3a. The plasmid pXYbc was digested with Ssp I, Hind III and Avr II. Two fragments from this digest were isolated, a 2 KB Ssp I - Hind III (the vector portion of pXYbc) and a 245 bp Ssp I (a small portion of the vector and the first 78 codons of TS3a) . A second plasmid pCW::TSl was digested with Ssp I and Hind III and a 345 bp fragment was isolated (codons 79 - 185 of TSl) . These three fragments were mixed together and ligated. After transformation, clones were picked from RBBX-agar plates if they produced clearing zones (xylanase activity) . A clone was analyzed by DNA sequencing and was found to contain the desired mutations. This clone was designated TS3.

CHARACTERIZATION OF THE PROTEIN PRODUCTS Protein purification

Protein samples were prepared from cells by first making an extract of the cells by grinding 10 g of the cell paste with 25 g of alumina powder. After grinding to a smooth mixture, small amounts (5 ml) of ice cold buffer A

(lOmM sodium acetate, pH 5.5) were added and the mixture ground vigorously between additions. DNase, 10 μg/ml, was added to lower the viscosity of the extract. The alumina and cell debris were removed by centrifugation of the mixture at 8000 x g for 30 min. The supernatant (25 ml) was then dialyzed overnight at 4°C against 3 L buffer A, using dialysis tubing with a 3500 molecular weight cutoff. A slight precipitate formed in the dialysis bag, which is removed by centrifugation at 8000 x g for 15 min.

The cell extract was then pumped onto a 50 ml bed volume, S-sepharose fast flow, cation exchange column (Kabi-Pharmacia, Canada) , equilibrated in buffer A. The xylanase was eluted with a 300 ml linear gradient of 0 to 0.3 M NaCl in buffer A at a flow rate of 3 ml/min. The xylanase elutes at 100 to 150 ml of the gradient. The fraction are checked on SDS-PAGE, and those fractions having most of the xylanase were pooled, and concentrated by ultrafiltration using 3000 dalton molecular weight cutoff membranes (Amicon YM3) . The concentrated material, 5 ml, was then applied to a 1.5 cm x 85 cm TSK-HW50S gel filtration column, equilibrated in 50 mM ammonium acetate pH 6. The xylanase eluted at a volume of 90 to 100 ml. These fractions were analyzed by SDS-PAGE, and the peaks pooled as pure xylanase. The protein was quantified using the extinction co-efficient at 280 nm. e 0.1% solution = 4.08. Typical purified yield from 10 g of cells was 25 mg of xylanase.

Detection of free thiol groups

Two methods were used to indicate the presence of the SS bond. The first was shown in Figure 10. The electrophoretic mobility of the mutant proteins is faster under non-reducing conditions since the reduced protein is fully denatured and binds more SDS. This has also been observed for other disulfide bond containing proteins

(Mitchison and Wells, 1989, Biochemistry 28:4807-4815; Eder and Wilmanns, 1992, Biochemistry 31:4437-4444).

The second method checked for reactive sulfhydryl groups (SH groups) . Approximately 200 μg of protein (10 nmol) were used for each determination. The reaction was performed in cuvettes at room temperature. The reaction mixture contained 6M urea, 50 mM Tris-HCl, pH 8, 1 mM EDTA, and 9 - 13 nmol of xylanase, in a final volume of l ml. The reaction was initiated by the addition of 10 μl of 10 mM 5,5'-dithiobis(2-nitrobenzoic acid), Ellmans reagent. The A ₄₁₂ of the solution was monitored for 45 min. The amount of free thiol was calculated from the liberated 2- nitro-5-thiobenzoate anion, using a molar extinction coefficient of 13,700. A single thiol containing mutant (S100C) was the positive control.

Titration of these and other mutants with Ellman's reagent under denaturing conditions shows undetectable levels of SH groups in the SS bond mutants, but shows stoichiometric amounts in a control containing a single SH group (Table 3) . This shows that two of the mutants of the present invention (TSl and TS2) have their cysteine residues in SS bridges.

Table 3. Determination of free sulfhydryl groups in xylanase mutants

Measurement of enzymatic activity

The activity of the enzyme was measured two ways. The quantitative assay determined the number of reducing sugar ends generated from soluble xylan. The substrate for this assay was the fraction of birchwood xylan which dissolved in water from a 5% suspension of birchwood xylan (Sigma Chemical Co) . After removing the insoluble fraction, the supernatant was freeze dried and stored in a desiccator. The measurement of specific activity was performed as follows. Reaction mixtures containing 100 μl of 30 mg/ml xylan in assay buffer (50 mM sodium citrate pH 5.5), 150 μl assay buffer, 50 μl of enzyme diluted in 1 mg/ml BSA, in assay buffer. The substrate and buffer were mixed and prewarmed at 40 ^β C. The reaction was started by the addition of the enzyme. At various time intervals 50 μl portions were removed and the reaction stopped by diluting in 1 ml of 5 mM NaOH. The amount of reducing sugars was determined with the hydroxybenzoic acid hydrazide reagent (HBAH) (Lever, 1972, Analytical Biochem 47:273-279) . A unit of enzyme activity was defined as that

amount generating 1 μmol reducing sugar in 1 minute at 40°C. For the determination of kinetic parameters substrate concentrations from 0.4 mg/ml to 20 mg/ml were used. Kinetic parameters were calculated using the computer program Enzfitter (Leatherbarrow 1987, Enzfitter, a non-linear regression data analysis program for the IBM- PC. Elsevier Science Publishers BV. Amsterdam, The Netherlands. 1987) .

The second assay for activity was used for relative measurement, i.e. residual activity after heat treatment. This assay was performed with RBBX (Remazol Brilliant Blue Xylan) as the substrate (Biely et al, 1987, Methods in Enzymology 160: 536-541) . 100 μl of 10 mg/ml RBBX in H ₂ 0 was mixed with 100 μl of 100 mM ammonium acetate pH 6, and the mixture prewarmed to 40°C. The reaction was started by the addition of suitably diluted enzyme. After a fixed length of time, 5 or 10 min, the reaction was stopped by the addition of 0.5 ml 95% ethanol. The mixture was inverted to mix, and then allowed to stand at least 10 min. at room temperature. The mixture was then centrifuged in a microcentrifuge for 3 min. at 12,000 X g. The A ₅₉₅ of the supernatant was measured.

Thermostability assay

To determine the thermostability of the mutant and wild type enzyme, the following parameters were used. The proteins were diluted to between 100 to 150 μg/ml, in dilution buffer (50 mM ammonium acetate pH 6.0) . 400 μl of this solution were incubated in a 1.5 ml microcentrifuge tube, in a heating block containing glycerol. Portions of the solution were removed at specified times and immediately diluted 1 to 20 in dilution buffer and kept at room temperature until all samples had been taken. Portions of these diluted samples were then assayed for residual enzyme activity, using the RBBX assay. Results were expressed as percent residual activity compared to the

zero time point sample. Heating blocks were calibrated with a thermocouple and the measured temperature was that of the enzyme solution.

The data shown in Figures 11-16, clearly indicate the thermostabilizing effect of the introduction of non- native disulfide bridges into BCX. The mutant TSl is clearly stable at 61 ^β C for up to 3 h. The second SS bond mutant TS2 is not quite as stable, but maintains some activity after 1 h at 61°C (see Figure 12) . Figures 11 and 12 show curves for the mixed TS4 monomer/dimer preparation, which is more stable than wild type but less stable than TSl. It is however, more relevant to examine the individual components. The mutant which contains only the inter-molecular SS bond (TS4a) shows increased thermostability at 58°C (Figure 13) , and at this temperature is as stable as the intra-molecular SS bond mutant TSl (Figure 14) .

TS4 protein is produced as a mixture, but the monomer (TS4M) and the dimer (TS4D) are easily separated by rechromatographing the mixture on a cation exchange column. TS4M behaves very much like the TSl mutant, whereas TS4D results in additive thermostability such that TS4D is more stable than TSl. (Figures 14, 15 and 16) .

There has been another example of an artificial inter-molecular dimer using an SS bond, where the thermostability was increased, however this protein was not an enzyme, and the measurement of thermostability did not include a biological activity assay (Sauer et al, 1986, Biochemistry 25:5992-5998). The increase in thermostability seen in the intra-molecular SS bond mutants is similar to the effects seen in other proteins with an engineered intra-molecular SS bond; however, there is no significant decrease in specific activity, or change in kinetic parameters (Table 4) , as is sometimes the case with

SS bond mutants (Kanaya et al, 1991, Journal of Biological Chemistry 266(10) :6038-6044) .

Table 4 Kinetic Parameters of Thermostable Xylanase Mutants

These values were determined with a different batch of substrate, than the first 3 were, which accounts for the difference in K,,, and V,^

The decrease in the k^/K,,, for TS3 indicates that some effect on catalysis and/or substrate specificity has occurred (Table 4) . There is also a decrease in specific activity seen in TS4a and TS4D but this is not likely a severe limitation because decrease in the ability to hydrolyze soluble xylan may not be indicative of how well the protein performs in pre-bleaching applications on pulp (Table 5) . The activity of the thermostable mutants is higher at elevated temperatures (Figure 17) .

Table 5 Specific Activity of Thermostable Mutants of the

B. circulans Xylanase

'These activities were determined using the reducing sugar assay (HBAH) , and are the average of at least two determinations.

The N-terminal mutations, were found to confer thermostability, although less than that seen with the SS bond mutants. Thermostability of these mutants was determined in two ways: by a residual activity assay and by scanning calorimetry.

In the residual activity assay identical samples of a xylanase mutant were preheated in the assay buffer (50mM sodium citrate, pH 6.2) in Eppendorf tubes at different temperatures. The Eppendorf tubes with samples were heated in small water baths with lids. The heating temperatures were determined by a digital thermometer with a thermocouple sensor which measured to 0.1°C. After 30 min. , samples were cooled down to 20°C before being assayed at 40°C. The residual enzyme activity of the heated sample

was expressed as a percentage of the activity of an unheated sample. This residual activity was plotted against the heating temperature (Figure 18) . From the plot, the T _% the preheating temperature, at which the xylanase still retained 50% of its activity, was determined and is shown in Table 6.

TABLE 6 T _1/2 of pre-heating temperatures at which xylanase will retain 50% of activity

Samples of the xylanase were studied by scanning calorimetry to study the unfolding process of the xylanase structure as it relates to the rising temperature. Then T _m , the temperature at which half of the xylanase molecules have been denatured or melted, was determined. A higher T _m reflects a more thermostable molecule. It should be emphasized that calorimetry is concerned with the maintenance of the molecular structure, not enzymatic activity. However, the loss of enzyme activity through heating is often associated with the collapse of the molecular structure. The melting temperatures, T _m of wild type and mutant xylanases were determined from the calorimetric scans and are shown in Table 7.

TABLE 7 Melting temperature T _m of xylanase

Residue-8 appears to be essential to the thermostability of the BCX. The mechanism of the effect of this mutation may be explained by the 3-dimensional structure which reveals that a space existed between asparagine-8 and the other residues (Figure 19) . This space was occupied by two water molecules in the wild-type enzyme (BCX) . Molecular modelling indicated that substitution of asparagine-8 by phenylalanine or tyrosine could displace the water molecules (Figure 20) . Increased thermostability resulting from this substitution may be a result of increased hydrophobic interaction contributed by these larger hydrophobic side-chains. The aromatic ring may displace the buried water molecules thus gaining hydrophobic interaction. In addition, the phenolic hydroxy group may also form a hydrogen bond with the carbonyl of peptide linkage-16 on the main chain.

The mutations at the N-terminus, described above, in combination with one of the SS bond mutations, resulted in an additive thermostability (Figure 14, 15 and 16) . The combination of mutations in TS3 result in a protein with a thermostability of 64°C.

X-ray Cryatallographic Structure of the xylanases from Bacillus circulans and Trichoderma harzianum

Structure of the B. circulans xylanase

Crystals of the B. circulans xylanase were grown by the hanging drop vapour diffusion method. The reservoir buffer was 40mM Tris, pH 7.5, 22% saturated (NH ₄ ) ₂ S0 ₄ and lOOmM NaCl. Droplets were seeded after one day of equilibration. The space group of the crystals was P2 ₁ 2 ₁ 2 ₁ . The unit cell parameters for all structures are given in Table 8. The heavy-atom derivative was obtained by soaking crystals of the SerlOOCys mutant protein in lOmM HgCl ₂ for 6 days. All of the X-ray diffraction data sets for both the B. circulans and T. harzianum xylanase crystals were collected on a San Diego Multiwire Area Detector system on a Rigaku rotating anode generator.

All data reduction was performed using the San Diego software and the PHASES program package (Furey, W. & Swaminathan, S. (1990) "PHASES - A Program Package for the Processing and Analysis of Diffraction Data from Macromolecules", PA33, American Crystallographic Association Meeting Abstracts, Series 2, 18., pg 73). The initial electron density map for the B. circulans xylanase was calculated from phases based on the native (wild-type) data set and the positions of the mercury atoms found from the Patterson maps for the data collected on the HgCl ₂ derivative of the SerlOOCys mutant protein. The initial electron density map was "skeletonized" using BONES (Greer, J. (1985) Methods in Enzymology 115. 206) and the initial model was built with TOM/FRODO (Jones, T. (1978) J. Appl. Cryst. 11, 268) and with O (Jones, T.A. , Bergdoll, M. , & Kjeldgaard, M. (1990) O: A macromolecular modelling environment. In "Crystallographic and Modelling Methods in Molecular Design" (Bugg, CE. & Ealick, S.E., Editors).

Springer-Verlag, New York) . All refinements were carried out using the simulated annealing and minimization protocols of X-PLOR (Brunger, A.T. (1988) J. Mol. Biol. 203. 803) .

Structure of TSl mutant of the B. circulans xylanase

Crystals of the TSl mutant were grown as for the wild-type enzyme and were isomorphous with the crystals of the wild-type enzyme. A model for the cysteine side-chains was built into a difference electron density map that was calculated with data measured from the TSl crystals and with phases calculated from the refined model of the wild- type structure.

Structure of the T. harzianum xylanase

Crystals of the T. harzianum xylanase were grown by the hanging drop vapour diffusion method. The reservoir buffer was 16mM Tris, pH 7.5, 20% saturated (NH ₄ ) ₂ S0 ₄ . Droplets were seeded after one day of equilibration. The space group of the crystals was also B " 2{≥ 2. _* but with different unit cell dimensions and with a different crystal packing arrangement than for the B. circulans crystals.

The structure was solved by standard molecular replacement methods with the programs MERLOT (Fitzgerald,

P.M.D. (1988) J. Appl. Cryst. 21, 273-278) and BRUTE

(Fujinaga, M. and Read, R. (1987) J. Appl. Crystallogr. , 0, 517-521) using the B. circulans xylanase as the search model. Refinement statistics for all structures are given in Table 9 and plots of average main-chain B-factors for the three structures are shown in Figures 21, 22 and 23. Ramachandran diagrams indicating the stereochemical quality of the structures are shown in Figures 24 and 25.

Table 8 Data collection statistics

w ere.

<I> is the average intensity for a particular reflection and 1^, is one intensity measurement for that reflection.

'deriv and F^ _t are the amplitudes of the structure factors for the derivative (S100C + HgCl ₂ and TSl) and native (wild-type) data sets, respectively.

n/a = not applicable to this data set

Figure of merit is a measure of the quality of a derivative for calculating phases.

Table 9 Refinement statistics

w ere,

R-factor = ^Σ I ^« ^ ^" - ^F ^I

∑IF, obs I

F _cf c and F,*, are the amplitudes of the structure factors that are calculated from the refined model structure and from the measured reflections, respectively.

EXAMPLE 5

CONSTRUCTION OF OTHER MUTATIONS AT THE N-TERMINUS OF B.

CIRCULANS XYLANASE

Additional N-terminal mutants have been constructed and tabulated (Table 10) . Construction of mutants TS7a, TS8a, TS9a, TSlOa, TSlla, TS12a, TS13a, TS14a, TS15a, TS16a, TS17 and TS19a has been accomplished via the identical protocol of cassette mutagenesis which previously yielded TS3a, TS5a and TS6a as described in Example 4 of the application. This involved ligation of various oligonucleotides to the Nhel/BspEI linearized plasmid pXYbc (Figure 9) . Another mutant TSlδa was also prepared via the same protocol with the Nhel/BamHI linearized plasmid pXYbc. Different overlapping oligonucleotides encoding various mutation have been synthesized.

(a) TS7a mutant, where asparagine-8 has been converted to phenylalanine. The oligonucleotides for cassette mutagenesis are XY8F-1 (SEQ ID NO:23), XY8F-2 (SEQ ID

NO:24), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY8F-1 (SEQ ID N0:23) 1 2 3 4 5 6 7 8 9

A S T D Y U Q F U 5' -CT AGT ACA GAT TAT TGG CAA TTC TGG

A TGT CTA ATA ACC GTT AAG ACC TGT CTG CCA CCG XY8F-2 (SEQ ID N0:24)

(b) TS8a mutant, where asparagine-8 has been converted to tryptophan. The oligonucleotides for cassette mutagenesis are XY8W-1 (SEQ ID NO:25), XY8W-2 (SEQ ID NO:26), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY8W-1 (SEQ ID NO: 25)

1 2 3 4 5 6 7 8 9

A S T D Y W Q U W

5'-CT AGT ACA GAT TAT TGG CAA TGG TGG A TGT CTA ATA ACC GTT ACC ACC TGT CTG CCA CCG

XY8W-2 (SEQ ID NO:26)

(c) TS9a mutant, where aspartic acid-4 has been converted to tyrosine. The oligonucleotides for cassette mutagenesis are XY4Y-1 (SEQ ID NO:27), XY4Y-2 (SEQ ID NO:28), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY4Y-1 (SEQ ID NO:27) 1 2 3 4 5 6 7 8 9 A S T Y Y W Q N U

5'-CT AGT ACA TAC TAC TGG CAG AAC TGG

A TGT ATG ATG ACC GTC AAG ACC TGT CTG CCA CCG XY4Y-2 (SEQ ID NO:28)

(d) TSlOa mutant, where aspartic acid-4 and asparagine-8 have both been converted to tyrosine. The oligonucleotides for cassette mutagenesis are XY4,8Y-1 (SEQ ID NO:29), XY4,8Y-2 (SEQ ID NO:30), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18) .

XY4.8Y-1 (SEQ ID NO: 29) 1 2 3 4 5 6 7 8 9 A S T Y Y W Q Y W 5'-CT AGC ACA TAC TAT TGG CAA TAT TGG G TGT ATG ATA ACC GTT ATA ACC TGT CTG CCA CCG

XY4.8Y-2 (SEQ ID NO:30)

(e) TSlla mutant, where aspartic acid-4 and asparagine-8 have been converted to tyrosine and. tryptophan respectively. The oligonucleotides for cassette mutagenesis are XY4Y8W-1 (SEQ ID NO:31), XY4Y8W-2 (SEQ ID NO:32), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY4Y8W-1 (SEQ ID NO:31) 1 2 3 4 5 6 7 8 9 A S T Y Y W Q W W 5'-CT AGC ACA TAC TAT TGG CAA TGG TGG G TGT ATG ATA ACC GTT ACC ACC TGT CTG CCA CCG

XY4Y8W-2 (SEQ ID NO:32)

(f) TS12a mutant, where threonine-3 has been converted to glycine. The oligonucleotides for cassette mutagenesis are XY3G-1 (SEQ ID NO:33), XY3G-2 (SEQ ID NO:34), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY3G-1 (SEQ ID N0:33) 1 2 3 4 5 6 7 8 9 A S G D Y U Q N U

5'-CT AGC GGA GAC TAT TGG CAG AAT TGG

G CCT CTG ATA ACC GTT TTA ACC TGT CTG CCA CCG XY3G-2 (SEQ ID NO:34)

(g) TS13a mutant, where threonine-3 and aspartic acid-4 have been converted to glycine and tyrosine respectively. The oligonucleotides for cassette mutagenesis are XY3G4Y-1 (SEQ ID NO:35), XY3G4Y-2 (SEQ ID NO:36), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY3G4Y-1 (SEQ ID NO:35) 1 2 3 4 5 6 7 8 9 A S G Y Y W Q N U 5'-CT AGC GGA TAC TAT TGG CAG AAT TGG G CCT ATG ATA ACC GTT TTA ACC TGT CTG CCA CCG

XY3G4Y-2 (SEQ ID N0:36)

(h) TS14a mutant, where threonine-3, aspartic acid-4 and asparagine-8 have been converted to glycine, tyrosine and phenylalanine respectively. The oligonucleotides for cassette mutagenesis are XY3G4Y8F-2 (SEQ ID NO:37), XY3G4Y8F-3 (SEQ ID NO:38), XY-11-2 (SEQ ID NO:17) and XY-16-1 (SEQ ID NO:18).

XY3G4Y8F-2 (SEQ ID NO:37) 1 2 3 4 5 6 7 8 9 A S G Y Y U Q F U 5'-CT AGC GGA TAC TAT TGG CAA TTC TGG G CCT ATG ATA ACC GTT AAG ACC TGT CTG CCA CCG

XY3G4Y8F-3 (SEQ ID N0:38)

(i) TS15a mutant, where serine-22 has been converted to proline. The oligonucleotides for cassette mutagenesis are XY-11-3 (SEQ ID NO:39), XY-16-3 (SEQ ID NO:40), XY22P-1 (SEQ ID NO:41) and XY22P-2 (SEQ ID NO:42).

XY-11-3 (SEQ ID NO:39) 1 2 3 4 5 6 7 8 9 A S T D Y U Q N W

5'-CT AGC ACC GAT TAC TGG CAG AAC TGG

G TGG CTA ATG ACC GTC TTG ACC TGT CTG CCA CCG XY-16-3 (SEQ ID NO:40) XY22P-1 (SEQ ID NO:41)

10 11 12 13 14 15 16 17 18 19 20 21 22 23 T D G G G I V N A V N G P G 5' -ACA GAC GGT GGC GGT ATC GTT AAT GCC GTG AAC GGC C

CCA TAG CAA TTA CGG CAC TTG CCG GGG CC XY22P-2 (SEQ ID NO:42)

(j) TS16a mutant, where asparagine-8 and glycine-21 have been converted to phenylalanine and proline respectively.

The oligonucleotides for cassette mutagenesis are XY8F-1 (SEQ ID NO:23), XY8F-2 (SEQ ID NO:24), XY21P-1 (SEQ ID

NO:43) and XY21P-2 (SEQ ID NO:44) .

XY21P-1 (SEQ ID NO:43) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 T D G G G I V N A V N P S G

5' -ACA GAC GGT GGC GGT ATC GTT AAT GCC GTG AAC CCA T

CCA TAG CAA TTA CGG CAC TTG GGT AGG CC XY21P-2 (SEQ ID N0:44)

(k) TSl7a mutant, where asparagine-8 and serine-22 have been converted to phenylalanine and proline respectively. The oligonucleotides for cassette mutagenesis are XY8F-1 (SEQ ID NO:23), XY8F-2 (SEQ ID NO:24), XY22P-1 (SEQ ID NO:41) and XY22P-2 (SEQ ID NO:42) .

(1) TS19a mutant, where threonine-3, aspartic acid-4 asparagine-8 and serine-22 have been converted to glycine tyrosine, tyrosine and proline respectively. Th oligonucleotides for cassette mutagenesis are XY3G4,8Y- (SEQ ID NO:19), XY3G4,8Y-2 (SEQ ID NO:20), XY22P-1 (SEQ I NO:41) and XY22P-2 (SEQ ID NO:42) .

(m) TS18a is a mutant where asparagine-8 and glycine-2 have been converted to phenylalanine and proline. Th oligonucleotides for cassette mutagenesis in the Nhel/BamH linearized plasmid pXYbc are XY8F-1 (SEQ ID NO:23), XY8F- (SEQ ID NO:24), XY24P-1 (SEQ ID NO:45), XY23P-2 (SEQ I NO:46), XY-12a (SEQ ID NO:47) and XY-15a (SEQ ID NO:48) The plasmid obtained was subcloned to yield TSlδa.

XY24P-1 (SEQ ID NO:45)

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

T D G G G I V N A V N G S G P

5' -ACA GAC GGT GGC GGT ATC GTT AAT GCC GTG AAC GGC TCC GGA CCA CCA TAG CAA TTA CGG CAC TTG CCG AGG GGC CCT

XY23P-2 (SEQ ID N0:46) P G

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

N Y S V N U S N T G N F V V G -AAT TAT AGC GTC AAT TGG | TCT AAT ACT ^' GGG AAC TTC GTA GTC GGA

-TTA ATA TCG CAG TTA ACC AGA TTA TGA CCC|TTG AAG CAT CAG CCT

XY-12a (SEQ ID N0:47) 40 41 42 43 44 45

IC G T T G -AAA GGT TGG ACG ACA G -TTT CCA ACC TGC TGT CCT AG XY-15a (SEQ ID NO:48)

(n) TS20a mutant with extension from both the N-terminu (glycine-(-1)) and from the C-terminus (glycine-186 an cysteine-187) , has conversion of alanine-1 into cysteine. Its construction was accomplished via a 2-step protoco identical to that described in Example 1 of th application. Initially this involved a polymerase chai reaction (PCR) with the template plasmid pXYbc (Figure 9)

and primers XY(-1)G1C (SEQ ID NO:49) and XY186G187C (SEQ ID NO:50) . Subsequently the Nde/Hindlll linearized PCR product was ligated into the identically linearized plasmid pCWBCX3'SYN. XY(-1)G1C (SEQ ID N0:49)

Ndel -1 1 2 3 4 5 6 st G C S T D Y U

5'-CAT CGA TGC TTA GGA GGT CAT ATG GGA TGT AGC ACA GAT TAC TGG 7 8

Q N -CAA AAC-3'

XY186G187C (SEQ ID NO:50) 179 180 181 182 183 184 185 186 187

S S N V T V U G C ter Hindi 11

3'-GA AGG TTG CAA TGT CAC ACC CCA ACG ATT TCT AGA ACT TCG AAT AGC

-TAC TAT TCG ACA-5'

(o) TS21a mutant which has extension from both the N-terminus (glycine-(-1) ) and from the C-terminus

(glycine-186 and cysteine-187) , has conversion of alanine-1 and asparagine-8 into cysteine and phenylalanine respectively.. Its construction was accomplished with the protocol described for TS20a, with differences in the PCR template (plasmid of TS7a; primers XY(-1)G1C8F (SEQ ID

NO:51) and XY186G187C (SEQ ID NO:50)).

XY(-1)G1C8F (SEQ ID NO:51) Ndel -1 1 ^* 2 3 4 5 6 st G C S T D Y U 5'-CAT CGA TGC TTA GGA GGT CAT ATG GGA TGT AGT ACA GAT TAT TGG

7 8 Q F

-CAA TTC-3'

Table 10 Xylanase N-terminal lutants

Expression/ extraction and purification

The expression, extraction, purification and assay processes for the mutant xylanases were identical to the protocols described in Example 4 of the application.

The mutant xylanases have shown specific enzyme activity close (70% - 100%) to the wild type BCX.

Thermostability assay

Thermostability assay was identical to that described for the N-terminal mutant enzymes previously described in Example 4 of application, with a major difference in equipment used in the assay. The water bath for incubating enzymes in Example 4 has a fluctuation of ±1.5°C, while the circulating water bath (Haake type F 4391) used in the present examples fluctuated at ±0.1°C. The new water bath should therefore yield more accurate data in subsequent assay. Enzyme was initially incubated in buffer (50mM sodium citrate, pH 5.5) without substrate (xylan) at a set temperature for 30 min. The residual enzymic activity was then determined at a lower temperature of 40°C via hydrolysis of soluble birchwood xylan, with the deducing sugar estimated by the hydroxybenzoic hydrazide reagent (HBAH) . The thermostability of BCX and the earlier mutants TSl, TS2, TS3, TS3a, TS5a and TS6a has been reevaluated using the new equipment (Table 11) .

The thermostability temperature T at which various xylanase mutants retain 50% of activity was determined. The differences between the T _1/2 of mutants and wild type BCX have been tabulated (Table 11) . The discrepancies in the T _1/2 values of the earlier mutants in Table 6 (of the parent application) and Table 11 were most likely due to the differences in performance of the two water baths. The newly established T _1/2 of BCX is 55°C, as compared to an earlier value of 53.5°C in Table 6.

Various mutations N8F (in TS7a, TS3a, TS14a) , N8Y (in TS5a, TS6a, TSlOa) and S22P (in TS15a, TS17a, TS19a) , alone or in combination, caused a moderate increase in T _1/2 of 2-4°C, as compared to the greater gain of 5-8°C by the internal or N/C-terminal disulfide bond (in TSl, TS2, TS3, TS20a) .

Temperature optimal and pH optimum in soluble xylan

The temperature optimum assay was carried out substantially as for thermostability determinations except instead of incubating the enzymes in buffer without xylan for 30 min., xylanase is incubated in the same manner with xylan in the buffer. The modified process is designed to evaluate the hydrolysis of soluble xylan by the mutant enzyme at different temperatures for 30 min. The reducing sugar released was then determined by the HBAH reagent.

Highertemperature exerteddramaticallydifferent effects on the hydrolysis of soluble xylan by wild type BCX and mutants (Figure 26) . The temperature optimum T^ is defined as the highest temperature at which 150% relative enzymic activity was maintained as compared to performance at 40*. The temperature optimum T^ of BCX is 60*C. The change of temperature optimum of mutants versus wild type BCX was tabulated (Table 11) .

Mutations such as N8Y (in TS5a, TSlOa) , N8F (in TS7a, TS3a) , S22P (TS15a, TS17a) , with only minor gain of 2-3°C in the thermostability assay, caused an elevation of T^ by 5 ^β C (Table 11) . The positive effect of mutations N8Y and N8F indicates that the aromatic residues tyrosine and phenylalanine are advantageous to the T^,. However, the mutation to another aromatic residue tryptophan (N8W in mutants TS8a and TSlla) lowered the T^ by 4-5 ^β C. While the mutation S22P together with N8F elevated the T _op by 10°C in mutant TS17a, a similar conversion of the neighbouring residues to proline (G21P and G23P) dramatically lowered the T _op of TSlβa and TS18a by as much as 11°C.

In contrast, the internal disulfide (in TSl, TS2) , with a gain of 5-8°C in thermostability assay, demonstrated no gain in T^ (Table 11) . The N/C-terminal

disulfide bond (in TS20a) with good thermostability of 5°C has only a moderate elevation of 3°C in T^ (Table 11).

The mutations T3G (in TS12a) or D4Y (in TS9a) independently caused a decrease of 3-9 ^β C in T,, (Table 11) . However, the combination of this pair of seemingly damaging mutations T3G and D4Y, with N8Y generated mutant TS6a which demonstrated a ^ increase of 4°C as compared to TS5a (N8Y) . On the other hand, this conversion of mutant TS5a to TS6a resulted in a net loss of thermostability (l ^β C) . Such opposite effect on thermostability and temperature optimal was also repeated in the conversion of mutant TS7a to TS14a. With a further addition of mutation S22P in mutant TS19a, an elevation of T„ _p as much as 14°C has been achieved. However, despite this gain in the T^, the total gain of thermostability (T _1/2 ) remained not more than 4 ^β C.

There is no clear correlation between the thermostable temperature (T _1/2 ) determined from incubation without substrate and the temperature optimum (T,,) in the assay with soluble xylan (Table 11) . Therefore, a gain in thermostability does not necessarily correlate with a gain in temperature optimum to hydrolyse xylan or to bleach pulp.

The functional pH (pH optimum) of mutant enzymes was determined. Initially the enzyme was incubated in buffers of various pH without substrate at 50 ^β C for 20 hr. Then the enzymic activity of the incubated enzyme was determined via the 4 min. 40°C assay conducted in the incubation buffer, instead of the 50mM sodium citrate (pH 5.5) normally used for such assay (Example 4). The pH optimum (pH^) is defined as the highest pH at which the enzyme still maintains 50% of the maximal activity. Five mutants TS5a, TS7a, TS17a, TS19a and TS20a with elevated T^ were selected for this study. The enzymic activity of these mutants was compared to wild type BCX at different pH

(Figure 27) . BCX can only maintain maximal activity up to pH 5.5 while the selected mutants can endure up to pH 7. Therefore the pH optimum of these mutants have been increased by more than 1 pH unit (Table 11) .

Table 11

Change* of thermostability, temperature optimum and pH optimum of mutant xylanase* in soluble xylan assay.

In summary, the appropriate mutations (S22P,

N/C-terminal disulfide bond, N6F and NδY alone or in combination with T3G and D4Y) can permit the mutant xylanase to hydrolyse xylan at higher temperature and pH.

Biobleaching of Pulp by Mutant Xylanases

Although the 30 min. soluble xylan assay determines the effectiveness of mutant xylanase in direct hydrolysis of xylan at high temperature, the usefulness of the enzyme as a bleaching agent can only be established via the pulp bleaching test. The protocol for such small scale test has been published in detail (Tolan and Canovas, 1992; Nissen et al, 1992, both incorporated herein by reference) and well-known to researcher in the field. Pulp suspension at different temperature or pH was treated with the enzyme for 2 hr. The bleaching effect was measured in brightness gain or whiteness of the pulp after bleaching. In this process, the temperature optimum (T^) is defined as the highest temperature at which the enzyme still achieves a brightness gain of 0.4 in the bleached pulp. The pH optimum (pH^) is defined as the highest pH at which the enzyme still achieved a brightness gain of 0.4 in the bleached pulp.

Several mutants were chosen for the pulp test at pH δ. While the wild type BCX functioned well at pH 7 with a temperature optimum of 54 ^β C, it failed at pH δ (Figure 2δ) . In the same test at pH δ (Table 12), mutants TS20a, TS17a and TS19a showed increase of T,, by 3, 10 and 14°C, respectively over BCX (at pH 7) . Thus the mutant xylanase TS19a can efficiently bleach pulp at temperatures as high as 6δ°C (T _op ) . In addition to elevated temperature, the performance of these mutants at pH δ also indicated an pH

upshift of 1 unit over BCX. Such gains in ^ and pH^ in pulp bleaching (Table 12) are in general agreement to the gains already determined through the soluble xylan assay (Table 11) .

The mutant TSl, which has achieved greater thermostability (T _m ) than the three mutants above (Table 11) , was also tested for its effectiveness in pulp bleaching. However, its performance (temperature optimum of 54°C) was only equal to the wild type BCX (Table 12). This has already been predicted in the 30 min assay on soluble xylan (Table 11) . The general lack of agreement between the thermostability (T _1/2 ) and the temperature optimal T^ in pulp bleaching (Table 12) or soluble xylan assay (Table 11) confirmed that greater thermostability may not be reflected in a better performance in pulp bleaching at elevated temperature.

In conclusion, appropriate mutations (NδY, N8F, S22P and N/C- terminal disulfide bond; the combination of T3G, D4Y and N8Y or NδF) at the N-terminus region (1-25) region are beneficial in the performance of the enzyme in pulp bleaching or other applications at higher temperature and pH.

Sable 12

Change* in Thermostability, Temperature Optimum and pE

Optimum of mutant xylanases in soluble xylan and pulp bleaching.

The invention has been described with reference to particular embodiments, although it is understood that the specific details shown are merely illustrative, and the invention may be carried out in other ways without departing from the spirit and the scope of the following claims.