MODIFICATION OF XYLANASES TO INCREASE THERMOPHILICITY, THERMOSTABILITY AND

ALKALOPHILICITY

[0001] The present invention relates to the modification of xylanases. More specifically, the invention relates to modified xylanases that can perform at high temperature and pH.

BACKGROUND OF THE INVENTION

[0002] Xylanases are a group of enzymes with wide commercial utility. Major applications of xylanases include pulp biobleaching in the production of paper, clarifying agents in juices and wines, as a supplement to improve digestibility of poultry and swine feed and as a washing agent of precision devices and semiconductors (e.g. U.S. Patent No. 5,078,802).

[0003] In the manufacturing of pulp for the production of paper, fibrous material is subjected to high temperatures and pressures in the presence of chemicals. This treatment converts the fibers to pulp and is known as pulping. Following pulping, the pulp is bleached. Xylanase enzymes are used to enhance the bleaching of the pulp. The xylanase treatment allows subsequent bleaching chemicals such as chlorine, chlorine dioxide, hydrogen peroxide, or combinations of these chemicals, to bleach pulp more efficiently. Pretreatment of pulp with xylanase increases the whiteness and quality of the final paper product and reduces the amount of bleaching chemicals which must be used to bleach the pulp. This, in turn, decreases the amount of bleaching chemicals present in the effluent produced by such processes.

[0004] The most important chemical pulping process is the production of kraft pulp. For kraft pulp, following pulping, and prior to the treatment of pulp with xylanase, the pulp is exposed to a temperature of 55-70°C and a highly alkaline pH (e.g. Nissen et al., 1992). A drawback of many commercially available wild-type xylanases is that these enzymes exhibit an acidic pH optimum and a temperature optimum of about 55°C. Therefore, in order to utilize xylanases effectively for bleaching applications, the pulp must be acidified to a pH approximating the optimal pH for the specific xylanase used. In addition, the hot pulp must be cooled to a temperature close to the optimal temperature for enzymatic activity of the selected xylanase. Decreasing pulp temperatures for xylanase treatment decreases the efficiency of the subsequent chemical

bleaching. Acidification of pulp requires the use of large quantities of acids. Furthermore, the addition of acids leads to corrosion and lessens the lifetime of process equipment. Thus, xylanases optimally active at temperatures and pH conditions approximating the conditions of the pulp would be useful and beneficial in pulp manufacturing.

[0005] Xylanases which exhibit greater activity at higher temperatures could be used to treat pulp immediately following the pulping process, without the need to cool the pulp. Similarly, xylanases which exhibit greater activity at higher pH conditions would require less or no acid to neutralize the pulp. Xylanases with such properties would provide several advantages and substantial economic benefits within a variety of industrial processes.

[0006] Several approaches directed towards improving xylanase for use in pulp bleaching within the prior art include the isolation of thermostable xylanases from extreme thermophiles that grow at 80-100 ⁰ C, such as Caldocellum saccharolyticum, Thermatoga maritima and Thermatoga sp. Strain FJSS-B.l (Lϋthi et al., 1990; Winterhalter et al., 1995; and Simpson et al., 1991). However, these thermostable xylanase enzymes are large, with molecular masses ranging from 35-120 kDa (320-1100 residues), and have a reduced ability to penetrate the pulp mass compared with other smaller xylanases which exhibit better accessibility to pulp fibers. In addition, some of the extremely thermophilic xylanases, such as Caldocellum saccharolyticum xylanase A, exhibit both xylanase and cellulase activities (Lϋthi et al., 1990). This additional cellulolytic activity is undesirable for pulp bleaching due to its detrimental effect on cellulose, the bulk material in paper. Furthermore, hyper-thermostable xylanase enzymes, which function normally at extremely high temperatures, have low specific activities at temperatures in the range for optimal pulp bleaching (Simpson et al., 1991).

[0007] A number of xylanases have been modified by protein engineering to improve their properties for industrial applications. For instance, U.S. Patent No. 5,405,769 (Campbell et al.) discloses the modification of Bacillus circulans xylanase (BcX) using site-directed mutagenesis to improve the thermostability of the enzyme. The site specific mutations include replacing two amino acids with cysteine residues to create intramolecular disulfide bonds. The mutations to create disulfide bonds include S179C (i.e., serine at position 179 replaced with cysteine) for an intermolecular crosslink between two xylanase molecules, and S100C/N148C and V98C/A152C

for the creation of intramolecular crosslinks. These disulfide linkages contribute to the thermostability of the enzyme, and do not effect the thermophilicity or alkalophilicity of the enzyme. WO 00/29587 (Sung and Tolan) discloses the formation of the disulfide crosslinks, 110/154 and 108/158, in the fungal xylanase of Trichoderma reesei xylanase II (TrX or TrX II), corresponding to the 100/148 and 98/152 disulfide bonds of the BcX. As in the case of BcX, these crosslinks also increased the thermostability of TrX II, but do not have an effect on the thermophilicity or alkalophilicity of the enzyme.

[0008] U.S. Patent No. 5,405,769 (supra) also discloses the mutation of specific residues in the N-terminus of the xylanase and these mutations were found to further improve the thermostability of the enzyme. In in vitro assays, the disulfide mutants showed thermostability at 62°C, an improvement of 7°C over the native BcX xylanase enzyme. However, these thermostable disulfide mutants showed no gain in thermophilicity (Wakarchuck et al., 1994). Mutations T3G, (BcX xylanase amino acid numbering) D4Y(F) and N8Y(F), near the N- terminus of the BcX xylanase enzyme, provided thermostability to 57°C, an increase of 2°C over the native BcX (U.S. Patent No. 5,405,769). However, the use of these enzymes in industrial applications still requires cooling and acidification of pulp following pretreatment prior to enzyme addition. Therefore, further increases in thermostability, thermophilicity and pH optima are still required.

[0009] It is known in the art to modify Trichoderma reesei xylanase II (TrX II or TrX) to increase thermophilicity and alkalophilicity. For instance, U.S. Patent No. 5,759,840 (Sung et al.) and U.S. Patent No. 5,866,408 (Sung et al.) disclose mutations in the N-terminal region (residues 1-29) of TrX. Three mutations, at residues 10, 27 and 29 of TrX, were found to increase the enzymatic activity of the xylanase enzyme at elevated temperatures and alkaline pH conditions.

[0010] WO 01/92487 (Sung) discloses mutations S75A, L105R, N125A, I129E of TrX II, to produce a xylanase which maintains greater activity at higher temperature and pH. WO 03/046169 (Sung) also describes the application of multiple mutations to arginine residues (Y135R, H144R, Ql 61 R) in order to increase the pH optimum of the TrX II. The mutation, Yl 18C, allowed the xylanase to maintain its optimal activity at higher temperature.

[001 1] Turunen et al. (2002) describe the use of specific multiple arginines on the specific "Ser/Thr surface" of TrX II to increase the enzymatic activity at higher temperatures, but with decreased thermostability. It was also reported that another mutation, K58R, displayed slightly increased thermostability. However, this mutation in combination with other arginines showed a narrower range of effective pH.

[0012] Turunen et al. (2001) disclose mutations NI lD, N38E, Q162H of TrX II with a complement of similar disulfide bonds (S110C/N154C) to improve the thermostability of the xylanase. However, these mutations, including NI lD, also have an adverse effect on both the thermophilicity and the alkalophilicity of the xylanase, resulting in a decrease of enzymatic activity at higher temperatures and neutral-alkaline pH as compared to native TrX II.

[0013] There have been many attempts to stabilize proteins via the introduction of engineered disulfide bonds, with mixed results. Sowdhamini et al. (1989) describes a computational procedure called MODIP (Modeling of Disulfide bridges in Proteins) to aid in the design of proteins with disulfide bridges. By this method, a large number of sites for potential disulfide bond formation are usually predicted, with no way to foretell which are most likely to stabilize the protein. Dani et al. (2003) describe a refined version of this method to assist such selection. It predicted that a crucial requirement in any stabilizing disulfide bond is to enclose a loop of more than 25 amino acid residues between the two cysteines. A loop with less than 25 residues will offer little stabilization.

[0014] WO 00/29587 (Sung and Tolan) report the formation of two disulfide bonds in Trichoderma reesei xylanase II, one linking positions 1 10 and 154, and another linking positions 108 and 158 (both enclosed loops longer than 25 residues). Both disulfide bonds provide for enhanced thermostability of the enzyme, but do not enhance the thermophilicity.

[0015] Fenel et al. (2004) describe the formation of a disulfide bridge in TrX II through two mutations, T2C and T28C, which results in an increase in the temperature optimum and the thermostability of the enzyme without any change in the pH-dependent activity. The disulfide crosslink encloses a loop having a length of 26 amino acid residues between the two cysteine residues.

[0016] While the prior art discloses the modification of xylanases to alter various characteristics, the needs of current industrial processes require enzymes with increasingly robust activity. There is a need in the art for novel xylanases which exhibit increased enzymatic activity at elevated temperatures and pH conditions. Such enzymes would be adaptable to uses in various fields, for example the production of paper pulp and the washing of precision devices and semiconductors.

SUMMARY OF THE INVENTION

[0017] The present invention relates to modified xylanases. More specifically, the invention relates to modified xylanases with improved performance at conditions of high temperature and/or pH.

[0018] This invention relates to a modified xylanase comprising cysteine residues at positions 99 and 118 to form an intramolecular disulfide bond, the xylanase produced by substitution of an amino acid at positions 99, 118 or both positions 99 and 118 with a cysteine. The positions of the amino acid substitution(s) are determined from sequence alignment of the modified xylanase with a Trichoderma reesei xylanase II amino acid sequence as defined in SEQ ID NO: 16. The modified xylanase exhibits thermophilicity, alkalophilicity, thermostability or a combination thereof.

[0019] The modified xylanase may be derived from a Family 11 xylanase, including, but not limited to, a Trichoderma reesei xylanase. The modified xylanase is preferably not a native Aspergillus xylanase.

[0020] According to the present invention, there is also provided a modified xylanase, as described above, further comprising a substituted amino acid residue at position 40. The substituted amino acid at position 40 may be selected from the group consisting of His, Cys, Phe, Lys, Tyr and Arg. In a specific example, the substituted amino acid at position 40 is a basic amino acid, including, but not limited to, His.

[0021] The present invention also pertains to the modified xylanase comprising cysteine residues at positions 99 and 1 18 and further comprising a substituted amino acid at position 58, including, but not limited to, a basic amino acid, such as Arg. In addition, the modified xylanase just described may further comprise a basic substituted amino acid at position 10, a hydrophobic substituted amino acid at position 27 and a hydrophobic substituted amino acid at position 29. The basic substituted amino acid at position 10 may be His, the hydrophobic substituted amino acid at position 27 is a Met and the hydrophobic substituted amino acid at position 29 is a Leu (HML). In addition to these mutations, the modified xylanase may comprise a non-polar substituted amino acid at position 75, a basic substituted amino acid at position 105, a non-polar substituted amino acid at position 125 and an acidic amino acid at position 129. The non-polar amino acid at position 75 may be an Ala, the basic amino acid at position 105 may be a His, the non-polar amino acid at position 125 may be an Ala and the acidic amino acid at position 129 may be a GIu. The modified xylanase may further comprise an acidic amino acid at position 11 , such as an Asp. In addition to a mutation at position 11, the modified xylanase may further comprise a mutation at position 131 to an Asn.

[0022] This invention also includes a modified xylanase comprising cysteine residues at positions 99 and 1 18 and further comprising basic amino acids at positions 40 and 58. The modified xylanase may further comprise a basic substituted amino acid at position 10, a hydrophobic substituted amino acid at position 27, and a hydrophobic substituted amino acid at position 29. The basic substituted amino acid at position 10 may be His, the hydrophobic substituted amino acid at position 27 may be Met and the hydrophobic substituted amino acid at position 29 may be Leu (HML). In addition to these mutations, the modified xylanase just described may further comprise a non-polar substituted amino acid at position 75, including, but not limited to, Ala; a basic substituted amino acid at position 105, including, but not limited to, His; a non-polar substituted amino acid at position 125, including, but not limited to, Ala; and an acidic amino acid at position 129, including, but not limited to, GIu. The modified xylanase may further comprise an acidic substituted amino acid at position 11 , including, but not limited to, Asp; and, optionally, an Asn at position 131. The modified xylanase as just described may further comprise a substituted amino acid at position 52, including, but not limited to, Cys. In addition, the modified xylanase may further comprise basic substituted amino acids at positions 144 and 161, including, but not limited to, Arg residues.

[0023] The present invention also relates to a modified xylanase comprising substituted amino acid residues at positions 99 and 118 and having a maximum effective temperature (MET) between about 65°C and about 85°C or having a maximum effective pH (MEP) between about pH 6.5 and about pH 8.0.

[0024] The present invention also relates to a modified xylanase selected from the group consisting of:

MUTANT NAME SEQUENCE

TrX-99C-118C TrX-CC SEQ ID NO:66

TrX-40H-99C-118C TrX-H CC SEQ ID NO:67

TrX-58R-99C-118C TrX-R CC SEQ ID NO:68

TrX-40H-58R-99C-l 18C TrX-HR CC SEQ ID NO:69

TrX- 10H-27M-29L-40R-58R-99C- 118C TrX-HML RR CC SEQ ID NO:70

TrX-10H-27M-29L-40R-58R-75A-99C-118C TrX-HML RRA CC SEQ ID NO:71

TrX- 10H-27M-29L-75 A-99C- 105H- 118C- 125 A- 129E TrX-HML A CHC AE SEQ ID NO:72

TrX- 10H-27M-29L-58R-75 A-99C- 105H- 118C- 125 A-

TrX-HML RA CHC AE SEQ ID NO:73

129E

TrX-I OH-11D-27M-29L-58R-75A-99C-105H-118C -

125A-129E TrX-HDML RA CHC AE SEQ ID NO:74

TrX-I OH-11D-27M-29L-40X-58R-75A-99C-105H-118C ■

125A-129E, wherein X is:

X is C C- SEQ ID NO:75

X is F TrX-HDML XRA CHC AE F- SEQ ID NO:76

X is H H- SEQ ID NO:77

X is Y Y- SEQ ID NO:78

X is R R- SEQ ID NO: 79

TrX- 1 OH- 11 D-27M-29L-40H-52C-58R-75 A-99C- 105H-

118C-125A-129E TrX-HDML HCRA CHC AE SEQ ID NO:80

TrX-lOH-11D-27M-29L-40R-58R-75A-99C-105H-118C- TrX-HDML RRA CHC

SEQ ID NO: 81

125A-129E-144R-161R AERR

TrX-I OH-11D-27M-29L-58R-75A-99C-105H-118C-

125A-129E-131N TrX-HDML RA CHC AEN SEQ ID NO:82

[0025] Xylanases of the present invention comprising cysteine residues at positions 99 and 118 display improved thermophilicity, alkalophilicity or thermostability relative to wild-type xylanases. Such xylanases find use in a variety of applications in industry that require enzyme activities at temperatures and/or pH values above that of the native enzyme. For example, modified xylanases, as described herein, may be used for the purposes of bleaching pulp, improving the digestibility of poultry and swine feed, or the processing of precision devices.

[0026] The present invention also pertains to a modified xylanase comprising a substituted amino acid at position 40, the position determined from sequence alignment of the modified xylanase with a Trichoderma reesei xylanase II amino acid sequence as defined in SEQ ID NO: 16. The modified xylanase as just defined may further comprise an intramolecular disulfide bond having a loop of between 10 and 24 amino acids. The intramolecular disulfide bond may be produced by substitution of an amino acid at position 99, 118 or both positions 99 and 118 with a cysteine. The amino acid substitution at position 40 is preferably a basic amino acid, including, but not limited to, His.

[0027] This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0029] FIGURE 1 shows an amino acid sequence alignment among Family 11 xylanases. The amino acid numbering is relative to Trichoderma reesei xylanase II (Tr2, also refered to herein as TrX II) as indicated at the top of the sequences. The residues at position 99 and 118 (relative to Tr2) are in italics and indicated with an asterisk. The amino acids common to at least 75% of the listed Family 11 xylanases are indicated in bold. The residues common to all Family 11 xylanases are underlined. For xylanases with a cellulose-binding domain, only the catalytic core sequences are presented.

[0030] FIGURE 2 shows the nucleotide sequence of TrX xylanase (SEQ ID NO:40), and the synthetic oligonucleotides TrX(I -91) and TrX (92-190) (SEQ ID NOs:61 to 64) used to construct the sequence encoding the Trichoderma reesei xylanase II enzyme (TrX) in the plasmid pTrX.

[0031] FIGURE 3 shows the effect of temperature on the enzymatic activity of modified xylanases TrX-99C, TrX-58R, TrX-40H, TrX-118C, TrX-99C-118C, TrX-58-R99C-118C, TrX-

40H-99C-1 18C, TrX-40H-58R-99C-118C compared with TrX, at pH 5.0 during 30-minute incubations. The data are normalized to the activity observed at 4O ⁰ C.

[0032] FIGURE 4 shows the effect of temperature on the enzymatic activity of modified xylanases TrX-99C-118C, TrX-58R-99C-118C, TrX-40H-99C-118C, and TrX-40H-58R-99C- 118C, compared with TrX, during 30-min incubations at pH 5.0. The data are based on those of Figure 3, but normalized to the activity observed at the temperature optimum.

[0033] FIGURE 5 shows the effect of temperature on the enzymatic activity of modified xylanases TrX-118C, TrX-99C-118C, TrX-40H-58R-99C-118C and TrX-10H-27M-29L-40R- 58R-99C-1 18C compared with the known xylanases, TrX, TrX-10H-27M-29L and TrX-IOH- 27M-29L-75A-105H-125A-129E during 30-minute incubations at pH 5.0 unless otherwise indicated. The data are normalized to the activity observed at the temperature optimum.

[0034] FIGURE 6 shows the effect of temperature on the enzymatic activity of modified xylanases TrX-10H-27M-29L-40R-58R-99C-l 18C, TrX-10H-27M-29L-40R-58R-75A-99C- 1 18C, TrX- 10H-27M-29L-75 A-99C- 105H- 118C- 125A- 129E, TrX- 10H-27M-29L-58R-75A- 99C- 105H- 118C- 125 A- 129E, TrX- 1 OH- 11 D-27M-29L-58R-75 A-99C- 105H- 118C- 125 A- 129E, TrX- 1 OH- 11 D-27M-29L-40H-58R-75 A-99C- 105H- 118C- 125 A- 129E and TrX- 1 OH- 11 D-27M- 29L-40H-52C-58R-75A-99C-105H-118C-125A-129E, compared with the known xylanase, TrX- 10H-27M-29L-75A-105H-125A-129E, at pH 5.5 during 30-minute incubations. The data are normalized to the activity observed at the temperature optimum.

[0035] FIGURE 7 shows the effect of temperature on the enzymatic activity of modified xylanases TrX-10H-l lD-27M-29L-58R-75A-99C-105H-118C-125A-129E and TrX- 1 OH- 1 ID- 27M-29L-40X-58R-75A-99C-105H-118C-125A-129E (where X is T, C, F, Y, R and H) at pH 5.5 during 30-minute incubations. The data are normalized to the activity observed at the temperature optimum.

[0036] FIGURE 8 shows the effect of temperature on the enzymatic activity of modified xylanases TrX-10H-27M-29L-75A-99C-105H-l 18C-125A-129E, TrX-I OH-11D-27M-29L-58R- 75 A-99C- 105H- 118C- 125A- 129E, TrX- 1 OH- 11 D-27M-29L-40H-58R-75A-99C-105H- 118C- 125 A- 129E and TrX- 1 OH- 1 1 D-27M-29L-40H-52C-58R-75 A-99C- 105H- 118C- 125 A- 129E,

compared with the known xylanases, TrX, TrX-10H-27M-29L and TrX-10H-27M-29L-75A- 105H-125A-129E (pH 5.5), at pH 5.5 during 30-minute incubations. The data are normalized to the activity observed at 40 ⁰ C.

[0037] FIGURE 9 shows the effect of temperature on the enzymatic activity of modified xylanases TrX- 1 OH- 11 D-27M-29L-40R-58R-75 A-99C- 105H- 118C- 125 A- 129E- 144R- 161 R (pH 6.5), compared to the known xylanase, TrX-10H-l lD-27M-29L-75A-105H-118C-125A-129E- 144R- 161 R (pH 6), during 30-minute incubations. The data are normalized to the activity observed at the temperature optimum.

[0038] FIGURE 10 shows the effect of pH on the enzymatic activity of modified xylanases TrX-99C and TrX-99C-l 18C, compared with native TrX and the known xylanase, TrX-118C, at pH 4.5 - 7.5, at 55°C during a 30-minute incubation. The data are normalized to the activity observed at the pH optimum for each enzyme.

[0039] FIGURE 11 shows the effect of pH on the enzymatic activity of modified xylanases TrX- 10H-27M-29L-75A-99C- 105H- 1 18C- 125A- 129E, TrX- 10H-27M-29L-58R-75A-99C-

105H-118C-125A-129E, TrX-lOH-11D-27M-29L-58R-75A-99C-105H-118C-125A-129E and TrX-10H-l lD-27M-29L-40H-58R-75A-99C-105H-118C-125A-129E, compared with the known xylanase, TrX-10H-27M-29L-75A-105H-125A-129E, at pH 5.0-8.0, at 65°C during a 30-minute incubation. The data are normalized to the activity observed at the pH optimum for each enzyme.

[0040] FIGURE 12 shows the effect of pH on the enzymatic activity of modified xylanase TrX- lOH-11D-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E-144-R161 R, compared with the known xylanases, TrX-10H-27M-29L-75A-105H-125A-129E, TrX-10H-27M-29L-75A-105H- 125 A- 129E- 144R- 161 R and TrX- 1 OH- 11 D-27M-29L-75 A- 105H- 116G- 118C- 125 A- 129E- 144R-161R, at pH 5.0-8.0, at 65°C during a 30-minute incubation. The data are normalized to the activity observed at the pH optimum for each enzyme.

[0041] FIGURE 13 shows the effect of temperature on the residual enzymatic activity of modified xylanase, TrX-99C-118C, compared with the natural xylanase, TrX, at 48 ⁰ C, 52°C,

56 ⁰ C, and 60 ⁰ C during 30-minute incubations without any soluble xylan substrate. The data are normalized to the activity observed at room temperature after a preincubation at 48 ⁰ C.

[0042] FIGURE 14 shows the effect of temperature on the percentage of maximum xylose released for the modified xylanases TrX-lOH-l lD^M^L^OR-SSR^A^C-lOSH-mC- 125A-129E-131N and TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E on 1 % wheat arabinoxylan substrate at pH 7 for 60 minutes.

[0043] FIGURE 15 shows the effect of pH on the percentage of maximum xylose released for the modified xylanases TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E- 13 IN and TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E on hardwood pulp (10% consistency) at 70 ⁰ C for 60 minutes.

[0044] FIGURE 16 shows the effect of pH on the percentage of maximum xylose released for the modified xylanases TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E- 131N and TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E on softwood pulp (10% consistency) at 70 ⁰ C for 60 minutes.

[0045] FIGURE 17 shows the effect of pre-incubation temperature on the relative residual activity (%) of the modified xylanases TrX-I OH-1 1D-27M-29L-40R-58R-75A-99C-105H-1 18C- 125A-129E, TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125E-131N and TrX- 10H-27M-29L after 30-minute incubation at 50 ⁰ C, 6O ⁰ C, 70 ⁰ C, and 80 ⁰ C.

DESCRIPTION OF PREFERRED EMBODIMENT

[0046] The present invention relates to modified xylanases. More specifically, the invention relates to modified xylanases with improved performance at conditions of high temperature and pH and improved stability at high temperature.

[0047] The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0048] The mechanism by which xylanases facilitate bleaching of pulp is not fully understood. Without wishing to be bound by theory, it has been postulated that the coloured lignin is

connected to crystalline cellulose through xylan and xylanase enzymes facilitate bleaching of pulp by hydrolysing xylan, releasing coloured lignin in the pulp. Modified xylanases as outlined herein, may be used for the purposes of bleaching pulp or other applications requiring activities at temperatures and pH values above that of the wild-type enzyme. For the bio-bleaching of pulp, the preferred xylanase is derived from a xylanase classified in Family 11 (see Table 1).

[0049] Family 1 1 xylanase enzymes are a group of small enzymes of relatively low molecular mass (approximately 20 kDa and about 200 amino acid residues). The small size associated with Family 11 xylanases permits ready penetration of the pulp mass. Another advantage of Family 11 xylanases is that they are free of cellulase activity.

[0050] Family 11 xylanases share extensive amino acid sequence similarity (Figure 1). Structural studies of several Family 1 1 xylanases indicate that Family 11 xylanases from bacterial and fungal origins share the same general molecular structure (U.S. Patent No. 5,405,769; Arase et al., 1993). In addition, most Family 11 xylanases identified so far exhibit three types of secondary structure including beta-sheets, turns and a single alpha helix. The helix of the Trichoderma reesei xylanase II enzyme encompasses the region from amino acid 151 to amino acid 162 (Torronen et al., 1995, Biochem. 34:847-856.).

[0051] A xylanase is classified as a Family 11 xylanase if it comprises amino acids common to other Family 11 xylanases, including two glutamic acid (E) residues which may serve as catalytic residues. The glutamic acid residues are found at positions 86 and 177 (see Figure 1; based on Trichoderma reesei xylanase II (TrX II, or Tr2) amino acid numbering). Most of the Family 11 xylanases identified thus far are mesophilic and have low molecular masses (20 kDa). However, this family also includes at least three thermostable xylanases of higher molecular mass, Thermomonospora fusca xylanase A (TfX-A) of 296 amino acids and a molecular mass of approximately 32 kDa (Irwin et al., 1994; WO 95/12668), Thermomyces lanuginosus xylanase (Tin) of 194 amino acids and a molecular mass of approximately 22 kDa (Gruber et al., 1998), and Clostridium stercorarium xylanase A of 511 amino acids and a molecular mass of approximately 56 kDa. The Clostridium stercorarium xylanase A enzyme exhibits maximum activity at a temperature of 70 ⁰ C (Sakka et al., 1993).

[0052] Some large thermostable Family 11 xylanases differ from the small mesophilic enzymes by the possession of a hydrophobic cellulose-binding domain (CBD) in the extended C-terminus of the enzyme. The TfX-A enzyme is composed of a catalytic core sequence of 189 residues common to all Family 1 1 xylanases, and a cellulose-binding domain of 107 residues. The larger C. stercorarium xylanase A has two copies of the cellulose-binding domain.

[0053] Non-limiting examples of Family 11 xylanases that may be modified following the general approach and methodology as outlined herein are described in Table 1 below.

[0054] TABLE 1: Family 11 xylanase enzymes

[0055] Examples of preferred Family 11 xylanases, which are not meant to be limiting, include Trichoderma reesei xylanase II, Trichoderma reesei xylanase I, Trichoderma viride xylanase, Streptomyces lividans xylanase B and Streptomyces lividans xylanase C. For example, the mutant xylanase of the present invention may comprise a mutant Trichoderma reesei xylanase II enzyme.

[0056] By "modified xylanase", it is meant a xylanase comprising a mutation or alteration of the natural xylanase sequence. The mutation or alteration is not found in the corresponding native xylanase. A xylanase molecule may be modified using techniques that are known to one of skill in the art. These techniques include, but are not limited to, site directed mutagenesis, cassette mutagenesis, random mutagenesis, synthetic oligonucleotide construction, cloning and other genetic engineering techniques. An example of a suitable technique to produce mutations in xylanases which render the enzyme more thermophilic and/or alkalophilic compared to the native enzyme is site-directed mutagenesis. However, it is also considered within the scope of the invention to use other techniques to introduce mutations that are known to those of skill in the art.

[0057] By the term "optimal activity", it is meant the activity of the particular enzyme at a pH where maximaum activity is obersved (i.e. optimal pH) and a temperature where maximal activity is observed (i.e.optimal temperature) over a given length of time.

[0058] A xylanase is "thermophilic," as used herein, if the xylanase exhibits a maximum effective temperature of between about 60 ⁰ C and about 9O ⁰ C. By "maximum effective

temperature" or "MET", it is meant the highest temperature at which a xylanase exhibits at least 80% of its optimal activity. For the purposes of this specification, the MET of a xylanase is determined by measuring the temperature profile of a xylanase using the standard assay for measurement of xylanase activity as detailed in Example 2.3 and modified as detailed in Example 3. The activity of the xylanase is measured at its pH optimum. The temperatures at which the modified xylanase exhibits at least about 80% of its optimal (maximum) activity are determined and the highest temperature is the MET.

[0059] The modified xylanase may have a MET of about 6O ⁰ C, 62 ⁰ C, 64°C, 65°C, 66 ⁰ C, 67 ⁰ C, 68°C, 69°C, 7O ⁰ C, 71°C, 72 ⁰ C, 73 ⁰ C, 74 ⁰ C, 75°C, 76°C, 77 ⁰ C, 78 ⁰ C, 79°C, 8O ⁰ C, 81°C, 82°C, 83°C, 84°C, 86°C, 88°C, or 90 ⁰ C, or any temperature therebetween. In a non-limiting example, the modified xylanase may have a MET between about 62° and about 85 ⁰ C or any range therebetween; between about 65°C and about 85°C or any range therebetween; between about 68°C and about 85 ⁰ C or any range therebetween; or between about 70 ⁰ C and about 85°C or any range therebetween.

[0060] A xylanase is "thermostable," as used herein, if it has a T ₅₀ of between about 55°C and about 85°C. The "T ₅₀ " is the incubation temperature at which the modified or the natural enzyme retains 50% of its residual activity, after an incubation time of 30 minutes. The T ₅₀ of a xylanase may be determined by the assay detailed in Example 5. As set forth in Example 5, the residual activity at 48°C is normalized to 100%.

[0061] The modified xylanase may have a T ₅₀ of about 55°C, 56 ⁰ C, 57°C, 58°C, 59 ⁰ C, 6O ⁰ C, 64°C, 68 ⁰ C, 72°C, 76°C, 8O ⁰ C or 85°C, or any temperature therebetween. In a non-limiting example, the modified xylanase may have a T ₅₀ between about 54°C and about 8O ⁰ C or any range therebetween; between about 56°C and about 8O ⁰ C or any range therebetween; or between about 58°C and about 8O ⁰ C or any range therebetween.

[0062] The use of the terms thermophilicity and thermostability has, in the past, been confused in the literature, as they have been used interchangeably. However, the use of the terms as defined herein is consistent with the usage of the terms in the art (Mathrani and Ahring, 1992).

[0063] A xylanase is alkalophilic, as used herein, if the xylanase has a maximum effective pH (MEP) of between about pH 6.0 and about pH 8.5. By "maximum effective pH" or "MEP", it is meant the highest pH at which a xylanase exhibits at least 80% of its optimal activity. The MEP may be determined by measuring the pH profile of a xylanase as set out in Example 4. The pH for which at least 80% of the optimal (maximum) activity is determined and the highest pH is the MEP.

[0064] The modified xylanase may have a MEP of pH 6.2, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0, or 8.5, or any pH therebetween. In a non-limiting example, the MEP may be between about pH 6.5 and about 8.5 or any range therebetween; or between about pH 6.8 and 8.0 or any range therebetween; or between about pH 7.0 and about 8.0 or any range therebetween.

[0065] By "TrX numbering", it is meant the numbering corresponding to the position of amino acids based on the amino acid sequence of TrX (Xyn II - Table 1 ; Tr2 - Figure 1 ; SEQ ID NO: 16). As disclosed below, and as is evident by Figure 1, Family 11 xylanases exhibit a substantial degree of sequence similarity. Therefore, by aligning the amino acids to optimize the sequence similarity between xylanase enzymes, and by using the amino acid numbering of TrX as the basis for numbering, the positions of amino acids within other xylanase enzymes can be determined relative to TrX. Standard methods known to one of skill in the art may be used to to align these seuqneces.

[0066] As described in more detail herein, several mutant xylanases have been prepared that exhibit enhanced thermophilicity, alkalophilicity and/or thermostability. A list of several mutants, which is not to be considered limiting in any manner, is presented in Table 2.

[0067] TABLE 2: Modified xylanases

SEQ ID Mutant TrX Mutations involved

NO:

TrX-R TrX: K58R

TrX-H TrX: S40H 65

TrX-C TrX: S99C

TrX-CC TrX: S99C and Yl 18C 66

TrX-R CC TrX: K58R, S99C and Yl 18C 68 TrX-H CC TrX: S40H, S99C and Yl 18C 67 TrX-HR CC TrX: S40H, K58R, S99C and Yl 18C 69 TrX-HML RR CC TrX: NlOH, Y27M, N29L, S40R, K58R, S99C and Y118C 70

TrX: NlOH, Y27M, N29L, S40R, K58R, S75A, S99C and

TrX-HML RRA CC 71

Y118C

TrX: NlOH, Y27M, N29L, S75A, S99C, L105H, Y118C, TrX-HML A CHC AE 72

Q125A and I129E

TrX: NlOH, Y27M, N29L, K58R, S75A, S99C, L105H, TrX-HML RA CHC AE 73

Y118C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, K58R, S75A, S99C, TrX-HDML RA CHC AE 74

L105H, Yl 18C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40H, K58R, S75A, TrX-HDML HRA CHC AE 77

S99C, L105H, Yl 18C, Ql 25 A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40C, K58R, S75A, TrX-HDML CR ACHC AE 75

S99C, L105H, Yl 18C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40T, K58R, S75A, TrX-HDML TRA CHC AE

S99C, L105H, Y118C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40Y, K58R, S75A, TrX-HDML YRA CHC AE 78

S99C, L105H, Yl 18C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40F, K58R, S75A, TrX-HDML FRA CHC AE 76

S99C, L105H, Yl 18C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40R, K58R, S75A, TrX-HDML RRA CHC AE 79

S99C, L105H, Yl 18C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40A, K58R, S75A, TrX-HDML ARA CHC AE

S99C, L105H, Yl 18C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40H, Q52C, K58R, TrX-HDML HCRA CHC AE 80

S75A, S99C, L105H, Y118C, Q125A and I129E

TrX: NlOH, Nl ID, Y27M, N29L, S40R, K58R, S75A, TrX-HDML RRA CHC AERR 81

S99C, L105H, Yl 18C, Q125A, I129E, H144R and Q161R

TrX: NlOH, Nl ID, Y27M, N29L, 58R, 75A, 99C, 105H, TrX-HDML RA CHC AEN 82

118C, 125A, 129E and l31N

[0068] Mutant xylanases described in WO 03/046169, U.S. Patent No. 5,759,840, WO 01/92487 and WO 2005/093072 (the contents of which are incorporated herein by reference) may be further modified to introduce cysteine residues at positions 99 and 118. Non-limiting examples of mutant xylanases that may be modified in accordance with the present invention are listed in Table 3.

[0069] TABLE 3: Modified xylanases described in WO 03/046169, U.S. Patent No. 5,759,840 and WO 01/92487

Mutant TrX Mutation

^" TrX-C ^{a ~~} TrX: YI l 8C ^{~ ~ ~~ ~}

TrX-HML ^b TrX: NlOH, Y27M and N29L

TrX-HML-AHAE ^c TrX: NlOH, Y27M, N29L, S75A, L105H, Q125A and I129E

TrX-HDML AH CAERR ³ TrX: NlOH, NI lD, Y27M, N29L, S75A, L105H, Y118C, Q125A,

I129E, H144R and Q161R TrX-HDML AHGC AERR ³ TrX: NlOH, NI lD, Y27M, N29L, S75A, L105H, D116G, Y118C,

Q125A, I129E, H144R and Q161R TrX-HML AHAE RR ^c TrX: NlOH, Y27M, N29L, S75A, L105H, Q125A, I129E, H144R and

Q161R ^a WO 03/046169 (Sung) ^b U.S. Patent No. 5,759,840 (Sung et al.) ⁰ WO 01/92487 (Sung)

Increasing the Thermophilicity of Xylanase

[0070] The effect of temperature on the hydrolysis of xylan by Trichoderma reesei xylanase TrX with the single mutations S40H (TrX-40H), K58R (TrX-58R), S99C (TrX-99C), or Y118C (TrX-118C) is shown in Figure 3.

[0071] The increase in thermophilicity of a Family 11 xylanase by the single mutation, Yl 18C, as in the modified xylanase, TrX-118C, has been described in WO 03/046169 (Sung). However, the possibility of producing a disulfide bond based on a cysteine at position 118 and another cysteine to increase thermostability, thermophilicity or alkalophilicity of a xylanase has never been reported. The present invention involves the construction of a 99C/118C-disulfide linkage

for such purpose, based on either a naturally occurring or generated Cys-118 with a second cysteine at residue-99 which is either naturally occurring or created via a mutation S99C.

[0072] To verify that any improved activity at higher temperatures is the result of the formation of a disulfide bond, the single mutation S99C was tested (TrX-99C; Figure 3). This mutant xylanase, TrX-99C, showed no improvement of enzymatic activity at higher temperature, as compared to natural TrX. Therefore, the S99C single mutation alone had no effect on the temperature/activity profile of TrX.

[0073] However, when mutations S99C and Y118C were incorporated in the form of a double mutant xylanase, TrX-99C-l 18C, there was a dramatic enhancement of thermophilicity (Figures 3 and 4), even when compared to the single mutant TrX-118C. The improvement of the temperature optima of the double mutant TrX-99C-118C over the natural xylanase (TrX) and TrX-118C is about 7°C and 5°C, respectively (Figures 3 and 4). In addition to a higher temperature optimum, TrX-99C- 118C also exhibited higher optimal activity than TrX at their respective temperature optima (Figure 3).

[0074] The single mutation, S40H, in xylanase TrX-40H showed an improved enzymatic activity at higher temperature (Figure 3), as compared to the wild type TrX. The positive effect on thermophilicity due to this mutation was confirmed in another mutant xylanase, TrX-40H-99C- 118C, (Figures 3 and 4) when compared to TrX-99C-l 18C.

[0075] In the case of the modified xylanase TrX-58R, the mutation K58R by itself could not improve the activity of the natural TrX (Figure 3), as previously reported by Turunen et al. (2002). However, in the mutant xylanases containing the mutations S99C and YI l 8C, the mutation K58R increased enzymatic activity at higher temperatures. This was confirmed by comparing the thermophilicity of the mutant xylanases TrX-58R-99C-118C and TrX-40H-58R- 99C-118C (Figures 3 and 4) to that of the xylanases TrX-99C-118C and TrX-40H-99C-118C, respectively. Although the mutation, K58R, by itself failed to improve the activity of xylanase at higher temperature, it has a positive effect on thermophilicity in combination with the other mutations S40H and S99C/Y118C.

[0076] The mutations above are compatible with other advantageous xylanase mutations previously described in the art. The additive effect of these mutations in combination with previously disclosed mutations was demonstrated in the construction of the combined variant xylanases possessing a higher temperature optima and optimal activity, as described below.

[0077] The mutations NlOH, Y27M and N29L have been shown to increase the thermophilicity of TrX in the form of the mutant TrX-10H-27M-29L (TrX-HML; see U.S. Patent No. 5,759,840). Incorporation of mutations S40H, K58R and S99C/Y118C in TrX-10H-27M-29L created the variant xylanase TrX-10H-27M-29L-40R-58R-99C-l 18C, with further improvement in enzymatic activity at higher temperatures (Figure 5).

[0078] The mutations NlOH, Y27M, N29L, S75A, L105H, Q125A and I129E have also been shown to increase the thermophilicity of TrX (mutant TrX-10H-27M-29L-75A-105H-125A- 129E; see WO 01/92487). Incorporation of mutations S40H, K58R and S99C/Y1 18C to TrX- 10H-27M-29L-75A-105H-125A-129E created the variant xylanases TrX-10H-27M-29L-40R- 58R-75A-99C-118C, TrX-10H-27M-29L-75A-99C-105H-118C-125A-129E, and TrX-IOH- 27M-29L-58R-75A-99C-105H-118C-125A-129E, showing enhanced enzymatic activity at higher temperatures (Figures 6 and 8).

[0079] Addition of an Nl ID mutation created modified xylanase TrX- 1 OH- 11D-27M-29L-58R- 75A-99C-105H-118C-125A-129E (Figures 6, 7 and 8). A series of mutants based on this xylanase bearing another mutation at position 40 were constructed to determine those amino acid residues that enhance the thermophilicity of the enzyme. Different mutations at position 40 (S40C, F, R, Y, A or T) were introduced to create seven new mutant xylanases: TrX- 1 OH- 1 ID- 27M-29L-40X-58R-75A-99C-105H-118C-125A-129E (where X is A, C, F, H, R, Y or T). Similar to xylanases TrX-40H and TrX-40H-99C-118C, with fewer mutations, the introduction of mutations S40H or S40R moderately improved the relative activity of the resultant variant xylanases at a higher temperature as compared to the parent enzyme (Figures 6, 7 and 8). Other mutations like S40C, S40F and S40Y also exhibited the same enhancing effect (Figure 7), while S40T and S40A showed no such enhancing effect on the temperature/activity profile (Figure 7).

[0080] The positive effect on the thermophilicity of Family 11 xylanases via the mutation of Ser 40 into Cys, Phe, Tyr, His or Arg has not been described previously. No known Family 11

xylanases possess the residue Cys, Phe, Tyr or His at position 40. The Arg residue, though present in the thermophilic Thermomyces lanuginosus Xyn (Figure 1), also exists in the mesophilic Steptomyces lividan XIn B.

[0081] Another mutation, Q52C, was introduced into TrX-10H-l lD-27M-29L-40H-58R-75A- 99C-105H-118C-125A-129E. The resulting mutant xylanase TrX- 1 OH- 11D-27M-29L-40H- 52C-58R-75A-99C-105H-1 18C-125A-129E was able to retain significantly higher relative activity at 80 and 85°C, in comparison to the parent enzyme (Figures 6, 7 and 8). No Family 11 xylanase possesses the residue Cys at position 52.

[0082] The mutations H144R and Q161R have previously been shown to increase the pH optimum of xylanase TrX-I OH- 1 1D-27M-29L-75 A-105H- 118C-125A-129E-144R-161R (TrX- HDML-AH-118C- AE-RR; WO 03/046169). Addition of mutations S40R, K58R and S99C resulted in the mutant xylanase TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A- 129E-144R-161R, which retains greater activity at higher temperatures of 80 and 85°C (Figure 9).

[0083] The above results demonstrate that the enhancing effect of the mutations S40X (where X is C, F, H, R or Y), Q52C, K58R and S99C/Y118C on the thermophilicity of the mutant xylanase are not only complementary or additive to each other, but also to other mutations described in U.S. Patent No. 5,759,840, WO 01/92487 and WO 03/046169.

[0084] The mutation 13 IN was introduced into the modified xylanase TrX-I OH-1 1D-27M-29L- 40R-58R-75A-99C-105H-118C-125A-129E. The resulting mutant xylanase TrX-I OH- HD- 27M-29L-40R-58R-75A-99C-105H-118C-125A-129E-131N showed a slightly higher temperature optimum than TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E (Figure 14). These results demonstrate that the mutation 13 IN is compatible with the 99C/1 18C disulfide mutation and other mutations which increase thermophilicity.

Increasing the Alkalophilicitv of Xylanase

[0085] The effect of the disulfide mutation S99C/Y118C on the pH/activity profile of xylanase is shown in Figure 10. The mutant xylanase TrX-99C-118C maintained greater activity at the higher pH values of 6.5-7.5 as compared to the natural xylanase TrX. The pH range for xylanase

TrX-99C-118C to maintain 80% optimal activity is 4.8-7.0, which is broader than the range of 4.8-6.0 for the corresponding natural or native xylanase TrX. These results demonstrate the positive contribution of the 99C/118C mutations on the alkalophilicity of the xylanase TrX.

[0086] In order to identify the direct cause of higher activity at higher pH, xylanases with only one of the S99C/Y118C mutations were compared to both TrX-99C-118C and the natural xylanase TrX. These two xylanases with a single mutation, TrX-99C or TrX-118C, showed similar pH/activity profiles as TrX (Figure 10). This confirmed that the improvement of activity at higher pH is a result of the disulfide bond formed via a combination of mutations S99C and Yl 18C, and not the single Cys mutations.

[0087] The effect of the mutations S40X (where X is H or R), K58R and S99C/Y118C on the pH/activity profile of xylanase was also studied in two groups of mutants constructed as described above.

[0088] The first group was derived from the mutant TrX-10H-27M-29L-75A-105H-125A-129E (see WO 01/92487). The disulfide mutant xylanase TrX-10H-27M-29L-75A-99C-105H-118C- 125A-129E showed enhanced activity of 75, 60 and 50% at respective pH values of 7.0, 7.5 and 8.0 (Figure 11) versus the parent xylanase, TrX-10H-27M-29L-75A-105H-125A-129E, which only showed 60, 40 and 22% enhanced activity at these pH values, respectively. This confirms the contribution of the 99C/1 18C mutations on the alkalophilicity of xylanases, with compatibility to the alkalophic and thermophilic mutations previously disclosed in the art.

[0089] Other members of this group were constructed with additional mutations at positions 40 and 58, which also contained the 99C/118C disulfide mutation. This includes TrX-10H-27M- 29L-58R-75 A-99C- 105H- 118C-125A- 129E, TrX- 1 OH- 11 D-27M-29L-58R-75 A-99C- 105H- 118C-125A-129E and TrX-I OH- 11D-27M-29L-40H-58R-75A-99C-105H- 1 18C-125A-129E. However, these mutants did not show any improved activity at the higher pH (Figure 11) as compared to the parent TrX-10H-27M-29L-75A-99C-105H-118C-125A-129E xylanase. Although these thermophilic mutations at positions 40 and 58 could not improve the alkalophilicity of the xylanase, they have no adverse effect, thus demonstrating that they are compatible in the construction of a thermophilic and alkalophilic xylanase with other advantageous mutations.

[0090] The enhancing effect of the S99C/Y 118C mutations was further demonstrated in a second group based on TrX-10H-27M-29L-75A-105H-125A-129E-144R-161R, a xylanase containing two mutations, H144R and Ql 6 IR, which have successfully increased the pH optimum of a xylanase in WO 01/92487. With the 99C/118C mutations, the mutant xylanase TrX-I OH-I ID- 27M-29L-40R-58R-75A-99C-105H-118C-125A129E-144R-161R exhibited greater activity at higher pH than its parent TrX-10H-27M-29L-75A-105H-125A-129E-144R-161R (Figure 12). It also outperformed another xylanase, TrX-10H-l lD-27M-29L-75A-105H-116G-118C-125A- 129E-144R-161R (Figure 12), a mutant xylanase which showed the most improved pH/activity profile among mutant xylanases in WO 03/046169. This is another non-limiting example demonstrating the compatibility of the 99C/118C mutations with other alkalophilic mutations.

[0091] The effect of the 13 IN mutation on the pH/activity profile of the mutant xylanase TrX- 10H-11D-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E was also investigated. As shown in Figures 15 and 16, the modified xylanase TrX-I OH-11D-27M-29L-40R-58R-75A-99C- 105H-118C-125A-129E-131N has a slightly broader pH optimum on both hardwood (Figure 15) and softwood (Figure 16) than TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A- 129E. This is yet another non-limiting example demonstrating the compatibility of the 99C/118C mutations with other mutations which increase alkalophilicity.

Increasing the Thermostability of Xylanase

[0092] The thermostability of the mutant xylanase was compared via incubation in the absence of substrate at different temperatures. After 30 minutes, the residual activity of the xylanase was determined via a standard assay with soluble xylan as a substrate.

[0093] The effect of the 99C/118C mutations on the thermostability of xylanase was determined via comparative study of the TrX-99C-118C mutant and the natural TrX. After incubation at higher temperatures for 30 minutes, the former retained greater residual activity than the latter (Figure 13).

[0094] The T ₅₀ was determined. For the disulfide xylanase TrX-99C-l 18C, the T ₅₀ was 58 ⁰ C, as compared to 51°C for the natural xylanase TrX. This represented an increase in the

thermostability, as measured by the "T ₅₀ ", by about 7°C through the introduction of the 99C/118C mutations.

[0095] The thermostability of modified xylanases containing the 99C/118C mutations in combination with additional mutations was also tested and compared with TrX-10H-27M-29L. As shown in Figure 17, both TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A- 129E-131N and TrX-lOH-1 1D-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E exhibit superior thermostability relative to TrX-10H-27M-29L.

[0096] In summary, improved thermophilic, alkalophilic and/or thermostable mutant xylanases of the invention comprise cysteine residues at positions 99 and 118. The modified xylanase may further comprise one or more than one of the following amino acid substitutions:

(i) a substituted amino acid at position 58 such as a basic amino acid, including, but not limited to, Arg;

(ii) a substituted amino acid at position 40, including, but not limited to, an amino acid selected from Arg, Cys, Phe, His and Tyr;

(iii) amino acid substitutions at positions 10, 27 and 29, such as a basic substituted amino acid at position 10, including, but not limited to, His; a hydrophobic substituted amino acid at position 27, including, but not limited to, Met; and a hydrophobic substituted amino acid at position 29, including, but not limited to, Leu; and

(iv) any combination of the mutations set out in (i) to (iii).

[0097] In addition, the modified xylanase described above may further comprise one or more than one of the following amino acid substitutions:

(v) substitutions at positions 75 and 125 such as non-polar substituted amino acids, including, but not limited to, Ala or GIy; an amino acid substitution at position 105 such as a substituted basic amino acid, including, but not limited to, His, Arg or Lys;

and/or an amino acid substitution at position 129 such as a substituted acidic amino acid including, but not limited to, Asp or GIu;

(vi) an amino acid substitution at position 52, including, but not limited to, Cys;

(vii) an amino acid substitution at position 11 , such as an acidic amino acid, including, but not limited to, Asp;

(viii) an amino acid substitution at position 144 and/or 161, including, but not limited to, a basic amino acid such as Arg;

(ix) an amino acid substitution at position 131 to an Asn; and

(x) any combination of the mutations described in (v) to (viii).

[0098] Non-limiting examples of xylanase mutants comprising a 99C/1 18C disulfide bond in combination with the amino acid substitutions listed above are given in Table 2.

[0099] It is also within the scope of the invention to introduce one or more than one of the amino acid substitutions of (v) to (x) into a modified xylanase comprising a 99C/118C mutation and which does not contain the mutations set out in (i) to (iv). Furthermore, the modified xylanase may comprise amino acid substitutions not listed above in combination with the 99C/118C mutations. In addition, the 99C/118C mutations may also be introduced into any of the xylanase mutants described in U.S. Patent No. 5,759,840, WO 03/046169, WO 01/92487 or WO 2005/093072, which are incorporated herein by reference.

[00100] It will also be appreciated that if one of the two positions, 99 or 118, already has a

Cys residue, the creation of a 99C/118C disulfide bond could also be produced by a substitution of an amino acid at only one of positions 99 or 118 to Cys. Thus, the present invention relates to a modified xylanase comprising cysteine residues at positions 99 and 118 to form a 99C/1 18C disulfide bond, the xylanase produced by substitution of an amino acid at position 99, 118 or both positions 99 and 118 with a cysteine.

[00101] There are natural examples of Aspergillus xylanases with cysteine residues at positions that correspond to positions 99 and 118 of Trichoderma reesei xylanase II, for example A. niger,

var. awamori; A. kawachii XynC; A. tubigensis (Figure 1). However, like the natural TrX, the Aspergillus xylanases can only function at low temperature (Fushinobu et al., 1998) and acidic pH (Krengel and Dijkstra, 1996), and are only stable up to 40°C (Ito et al., 1992, Biosci. Biotechnol. Biochem. 56:906-912). Therefore, the existence of cysteine residues at positions 99 and 118 in these mesophilic Aspergillus xylanases does not suggest that the creation of a disulfide bond in a xylanase will enhance its activity at high temperatures. Furthermore, these Aspergillus xylanases can only function at acidic pH values, with an acidic optimum pH of around 2-3 (Krengel and Dijkstra, 1996; Fushinobu et al., 1998, Esteves et al., 2004; Hakulinen et al., 2003). It therefore does not suggest that the creation of a similar disulfide bond in another acidophilic xylanase, including, but not limited to, TrX will enhance its activity at higher pH. Moreover, disulfide mutations are seldom observed to contribute to the alkalophilicity of an enzyme.

[00102] Therefore, the modified xylanase of the present invention may be dereived from a Family 1 1 xylanase, including but not limited to a Trichoderma reesei xylanase. The modified xylanase preferably is not native Aspergillus xylanase. However, the Aspergillus xylanase, comprising naturally occurring cysteine residues at postions 99 and 118 (TrXII numbering) may be used to derive a modified xylanase comprising additional mutations as described herein in order to enhance the properties of thermophilic! ty and alkalophilicity of the Aspergillus xylanase.

[00103] Furthermore, a computational procedure called MODIP (Sowdhamini et al., 1989; Dani et al., 2003), which was established to aid in the design of a disulfide bridge to stabilise protein, has predicted that any stabilizing disulfide bond must enclose a loop, i.e., the number of residues between the two cysteines, of 25 amino acid residues or more. Another related study has also concluded that there is little stabilization if the loop length is smaller than 25 residues. The 99C/118C disulfide bond of the present invention, with a loop of 19 residues, is considerably smaller than the predicted minimum of 25 residues for a stabilising disulfide bond. The smallest loop of any stabilizing disulfide bond reported in a Family 11 xylanase is a 2/28 disulfide bond with a loop length of 26 resides, created at the N-terminus of TrX (Fenel et al., 2004). This 2/28 disulfide bond did not enhance xylanase activity at higher pH range.

[00104] The above description is not intended to limit the claimed invention in any manner. Furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.

[00105] The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

EXAMPLES

[00106] The construction of variant xylanases in the examples required the use of a precursor plasmid which contained only a partial xylanase gene (Table 4). The precursor plasmid is incapable of expressing a xylanase. Synthesis of the precursor plasmid has been described previously.

TABLE 4: Plasmid containing a partial xylanase gene for the construction of new mutant xylanases

³ WO 01/92487 (Sung)

[00107] The new variant xylanases constructed in the following examples were also compared to various selected known mutant xylanases. The plasmids which expressed these previously described mutant xylanases are described in Table 5.

TABLE 5: Xylanase-expressing plasmids reported in the art

³ WO 03/046169 (Sung) ^b U.S. Patent No. 5,759,840 (Sung et al.)

^C WO 01/92487 (Sung)

EXAMPLE 1: Construction of Trichoderma reesei mutant xylanases

[00108] Basic recombinant DNA methods like plasmid preparation, restriction enzyme digestion, polymerase chain reaction, oligonucleotide phosphorylation, ligation, transformation and DNA hybridization were performed according to well-established protocols familiar to those skilled in the art (e.g. Sung et al., 1986), or as recommended by the manufacturer of the enzymes or kit. The buffers for many enzymes were supplied as part of a kit or made according to the manufacturer's instructions. Restriction enzymes, T4 polynucleotide kinase and T4 DNA ligase were purchased from New England BioLabs Ltd, Mississauga, Ontario. The GeneAmp PCR reagent kit was purchased from Perkin-Elmer. A precursor plasmid, pXYbc, which is a pUC type plasmid with a Bacillus circulans xylanase gene inserted, has previously been prepared and published (Sung et al., 1993; Campbell et al., U.S. Patent No. 5,405,769). A commonly used E. coli strain, HBlOl (Clonetech Lab, Palo Alto, CA), was used as a transformation and expression host for all gene constructs. Birchwood xylan and Remazol Brilliant Blue R-D-Xylan were purchased from Sigma (St. Louis, Mo). Hydroxybenzoic acid hydrazide (HBAH) was purchased from Aldrich. Oligonucleotides were prepared with an APPLIED BIOSYSTEM DNA synthesizer (model 380B). All xylanase enzymatic assays were performed in a covered circulating water bath (Haake type F 4391) and maintained within a temperature range of + 0.1°C.

1.1 Construction of precursor plasmid pTrX harbouring synthetic TrX (SEQ ID NO:40)

[00109] The precursor plasmid pTrX for mutations disclosed below has been described (Sung et al., 1995). This plasmid was derived from a pUC119 plasmid with a synthetic nucleotide

sequence encoding a Trichoderma reesei xylanase (TrX; Figure 2). Expression of this xylanase and other mutant xylanases subsequently described are under the control of the lac Z promoter of the pUC plasmid. The total assembly of the Trichoderma xylanase gene required two stages, initially involving ligation of the 92-190 region, then followed by the 1-92 region (TrX numbering). The protocol for the construction of this gene is routine and identical to the standard published procedure for many other genes. The protocol requires enzymatic phosphorylation of overlapping synthetic oligonucleotides which encodes a xylanase. This is followed by their ligation into an appropriately cut plasmid.

[00110] For the construction of TrX (92-190), the following ten overlapping oligonucleotides (see Figure 2) were designed:

XyTv-101, SEQ ID NO:30;

XyTv- 102, SEQ ID NO:31;

TrX-103, SEQ ID NO:32;

XyTv-104, SEQ ID NO:33;

XyTv-105, SEQ ID NO.34;

XyTv- 106, SEQ ID NO:39;

XyTv-107, SEQ ID NO:38;

TrX-108, SEQ ID NO:37;

XyTv-109, SEQ ID NO:36; and

XyTv-110, SEQ ID NO.35.

[001 11] These mutants were designed with a codon usage frequency imitating that of E. coli. The Sail and BgIU cohesive ends of two terminal oligonucleotides enabled the enzymatic ligation of the ten fragments into the linearized plasmid pXYbc. The ten oligonucleotides (50 pmol, 1 μL for each) encoding the TrX(92-190) region of Trichoderma xylanase were phosphorylated in a mixture containing 1OX standard kinase buffer (0.4 μL), 1 mM ATP (4 μL), T4 DNA kinase (5 units) and water (3 μL). Phosphorylation reactions were carried out for 1 hour at 37°C. The solutions were then combined and heated to 70 ⁰ C for 10 minutes. After being cooled slowly to room temperature, the combined solutions were added to a mixture of 4 mM ATP (3.5 μL), SaWBgHl linearized plasmid pXYbc (0.1 pmol), and T4 DNA ligase (3.5 μL) and

incubated at 12 ⁰ C for 20 h. Aliquots of the ligation mixture were used to transform E. coli HBlOl on YT plates (8 g yeast extract, 5 g bacto-tryptone, 5 g NaCl, 15 g of agar in 1 L of water) containing ampicillin (100 mg/L).

[00112] For the preparation of a hybridization probe, one of the oligonucleotides, for example, XyTv-I lO (10 pmol, 1 μL) was phosphorylated with ³² P-ATP (10 pmol, 3 μL) using T4 DNA kinase (1 μL), 1OX kinase buffer (1 μL), and water (4 μL) at 37°C for 1 h.

[00113] Transformants were selected randomly for hybridization analysis. Colonies were grown on YT plates with ampicillin overnight and transferred onto nylon filters. They were then denatured with 0.5 N NaOH - 1.5 M NaCl (10 minutes) and neutralized with 0.5 N Tris-HCl (pH 7.0) - 1.5 M NaCl (10 minutes). After ultraviolet irradiation at 254 nm for 8 minutes, the filters were washed with 6X SSC - 0.05% Triton X-100 for 30 minutes. Cell debris was scraped off completely. After another 30 minutes in fresh solution, duplicate filters were transferred individually into separate mixtures of 6X SSC - 1% dextran sulphate - 0.05% TritonX-100 - IX Denhardt's hybridization fluid. The ³² P-labelled probe was added to the filter. After 16 h at 45°C, the filter was washed twice with 6X SSC - 0.05% TritonX-100 at room temperature for 5 minutes and then at 65°C for 30 minutes. Positively hybridized clones with the intermediate plasmid pBcX-TrX were identified by autoradiographic analysis.

[00114] The above protocol, involving enzymatic phosphorylation of synthetic overlapping oligonucleotides and ligation into a linearized plasmid, was employed in the assembly of the TrX(I -92) region and in the cassette mutagenesis for the subsequent generation of other mutant xylanases described in this invention.

[00115] For the assembly of the TrX(l-92; TrX numbering) region to complete the full-length Trichoderma reesei xylanase II gene (TrX), the intermediate plasmid pBcX-TrX was linearized by MeI and Kpnl endonucleases to release the DNA insert for BcX(l-83). With MeI and Kpnl cohesive ends, eight overlapping oligonucleotides:

TrX-I, SEQ ID NO:22;

XyTv-2, SEQ ID NO:23;

TrX-3, SEQ ID NO:24;

XyTv-4, SEQ ID NO:25;

XyTv-5, SEQ ID NO:29;

TrX-6, SEQ ID NO:28;

XyTv-7, SEQ ID NO:27; and

TrX-8, SEQ ID NO:26

encoding the TrX(I -91) sequence were ligated into the linearized plasmid pBcX-TrX (Figure 2) via the protocol described above. The new plasmid pTrX, therefore, harbored a synthetic TrX gene (SEQ ID NO:40).

[00116] All mutant xylanase genes described below were constructed via the method of cassette mutagenesis. The protocol for cassette mutagenesis was identical to that described for gene assembly described above. Generally, cassette mutagenesis involved (i) enzymatic phosphorylation of overlapping synthetic oligonucleotides, (ii) ligation of synthetic oligonucleotides with a linearized plasmid, (iii) transformation of the plasmid into E. coli HBlOl competent cells, (iv) identification of mutant transformants via hybridization with the labelled oligonucleotide, and (v) confirmation of the mutation through dideoxy nucleotide sequencing.

1.2 Construction of plasmid pOmp-TrX harbouring the secretion leader sequence of the outer membrance protein A (SEQ ID NOs:41 and 42)

[00117] Following the experimental protocol of 1.1, the oligonucleotides Omp-TX-1, -2, -3 and -4, which encode the secretion leader sequence of the E. coli outer membrance protein A and the reconstructed TrX(I -7) region, were ligated to the NheV Pin Al-cut plasmid pTrX. The resulting plasmid pOmp-TrX can produce the functional xylanase via expression and secretion.

Omp-TX-1

[OmpA 2 3 4 5 6 7 8 9 10 11 12 13 14

K K T A I A I A V A L A G

5'-CT AGC AAG AAG ACA GCA ATA GCA ATC GCT GTG GCA TTA G | CC GGC G TTC TTC TGT CGT TAT CGT TAG I CGA CAC CGT AAT C GG CCG

Nhel Omp-TX-4 Omp-TX-3

Oxαp - TX - 2 TrX sequence

15 16 17 18 19 20 21] [ 1 2 3 4 5 6 7

F A T V A Q A Q T I Q P G T TTT GCG ACC GTT GCT CAG GCC CAG ACC ATA CAA CCA GGA A (SEQ ID NO -.41 AAA CGC TGG CAA CGA GTC CGG GTC TGG TAT GTT GGT CCT TGG CC (SEQ ID NO : 42) PinAI

1.3 Construction of the precursor plasmid pOmp-TrX(l-l 13)

[00118] Plasmid pOmp-TrX-(l-113) comprises the amino acid sequence 1-113 of TrX and cannot express an active xylanase. Such transformants were confirmed by the absence of a clearing zone or halo around the transformant colonies on blue xylan plates.

[00119] The plasmid was constructed via (i) the removal of the TrX(1 14-190) coding sequence of plasmid pOmp-TrX through cutting with restriction enzymes BamHl and Bglll, (ii) ligation of the identical cohesive ends of the linearized plasmid, (iii) transformation into the E. coli HBlOl competent cells followed by plating on YT plate (containing 5 g yeast extract, 3 g bacto- tryptone, 5 g NaCl, 15 g of agar in 1 L of water, 1 g Remazol Brilliant Blue R-D-xylan) and ampicillin (100 mg/L), (iv) identification of the mutant transformants through the loss of xylanase activity (absence of a clearing zone or halo around the colonies on the blue xylan plate overnight at 40°C), and (v) confirmation of the mutation through dideoxy nucleotide sequencing. The protocol for each of these steps was similar to that for gene assembly described above.

1.4 Construction of the precursor plasmid pTrX-HML

[00120] The construction of this precursor plasmid pTrX-HML has been described in detail in U.S. Patent No. 5,759,840 (see Example IN, herein incorporated by reference; plasmid termed pNI-TX13). TrX-HML comprises the native TrX xylanase, along with three mutations at NlOH (Asn at position 10 is replaced with His), Y27M and N29L. The first thirty amino acids of the sequence comprising NlOH, Y27M and N29L are shown below.

TrX 1 2 3 4 5 6 7 8 amino acid Q T I Q P G T G

5'-CT AGC TAA GGA GG CTG CAG ATG CAA ACA ATA CAA CCA GGA ACC GGT

3 ' -G ATT CCT CC GAC GTC TAC GTT TGT TAT GTT GGT CCT TGG CCA

Nhel PinAI

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Y H N G Y F Y S Y W N D G H G G

TAC CAC AAC GGT TAC TTT TAC AGC TAT TGG AAC GAT GGC CAT GGA GGC

ATG GTG TTG CCA ATG AAA ATG TCG ATA ACC TTG CTA CCG GTA CCT CCG

25 26 27 28 29 30

V T M T L G

GTC ACA ATG ACT CTG GGG (SEQ ID NO: 43) CAG TGT TAC TGA GAC CCC (SEQ ID NO: 44)

/.5 Construction of the precursor plasmids pOmp-TrX-HML(l-113) and pOmp-TrX- HDML(I-IU)

[00121] Plasmids pOmp-TrX-HML(l-113) and pOmp-TrX-HDML(l-113) comprise the amino acid sequence 1-113 of TrX and cannot express an active xylanase. Such transformants are confirmed by the absence of a clearing zone or halo around the transformant colonies on blue xylan plates.

[00122] In the construction of plasmids pOmp-TrX-HML(l-113), PCR was used to generate a DNA fragment encoding (8-C terminus) region with the PCR primers TX-I OH-I (SEQ NO:45) and TX-Cl, and template pTrX-HML (Table 5). Cutting of the PCR product with restriction enzymes PmAI and BamHl yielded the (8-113) sequence.

[00123] In the construction of the plasmid pOmp-TrX-HDML(l-113), PCR was repeated with the primer TX-10H1 ID-I (SEQ NO:46, which has been described in WO 03/046169), replacing the TX-I OH-I, to generate the (8-C terminus) sequence. Cutting of the PCR product with the restriction enzymes, PmAI and BamHl, yielded the (8-113) sequence.

TX-lOH-1 (SEQ ID NO:45)

6 7 8 9 10 11 12 13 14 15 16 17 18 G T G Y H N G Y F Y S Y W 5'-GGA ACC GGT TAC CAC AAC GGT TAC TTT TAC AGC TAT TGG PinAI

TX-lOHllD-1 (SEQ ID NO:46)

6 7 8 9 10 11 12 13 14 15 16 17 18

G T G Y H D G Y F Y S Y W

5'-GGA ACC GGT TAC CAC GAC GGT TAC TTT TAC AGC TAT TGG

PinAI Reverse PCR primer TX-Cl comprised :

TX-Cl (SEQ ID NO : 47) 183 184 185 186 187 188 189 190 ter

G S A S I T V S

CCA AGG CGA TCA TAA TGT CAC TCG ATT TCT AGA ACT TCG AAC CC-5'

BgII HindIII

[00124] The appropriate PCR template, pTRX-HML (Table 5), primers and the restriction enzymes to cut the end of the PCR products are listed below (Table 6).

TABLE 6

PCR PCR upstream PCR reverse PCR Restriction product primer primer template enzymes for PCR product

(a) TX-I OH-I TX-Cl pTrX-HML PinAVBamHl

(b) TX-IOHl ID-I TX-Cl pTrX-HML PinkVBamαl

[00125] The cut PCR products (a) and (b) (Table 6) were ligated into a PmAIASg/II-linearized plasmid pOmp-TrX (described in 1.2) to generate plasmids pOmp-TrX-HML(l-113) and pOmp- TrX-HDML(I -1 13), respectively.

[00126] Subsequent steps involved (i) transformation into the E. coli HBlOl competent cells, followed by plating on a YT plate (containing 5 g yeast extract, 3 g bacto-tryptone, 5 g NaCl, 15 g of agar in 1 L of water, 1 g Remazol Brilliant Blue R-D-xylan) and ampicillin (100 mg/L), (ii) identification of the mutant transformants through the loss of xylanase activity (absence of a clearing zone or halo around the colonies on the blue xylan plate overnight at 40°C), and (iii) confirmation of the mutation through dideoxy nucleotide sequencing. The protocol for each of these steps was similar to that for the construction of plasmid pOmp-TrX(l-113) described in 1.3.

1.6 Construction of plasmid pTrX-58R

[00127] The pTrX-58R plasmid , with the additional mutation K58R compared to the precursor plasmid pTrX, were prepared.

[00128] PCR was used to generate a DNA fragment encoding the (54-190) region with the K58R mutation. The PCR primer with the K58R mutation (in bold type) is shown below.

TX-58R-1 (SEQ ID NO:48) 53 54 55 56 57 58 59 60 61 P G T K N R V I N 5 ' - CAA CCC GGG ACC AAA AAT AGG GTG ATC AAC Xmal

[00129] With the appropriate PCR template pTrX (Table 5), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 7).

TABLE 7

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

__ XX.58R.! TX-Cl XmallHindlll

[00130] The cut PCR product (c) (Table 7) was ligated into a Xmαl/HzWIII-linearized plasmid pTrX(l-l 13) to generate the plasmid pTrX-58R.

1.7 Construction of plasmids pTrX-40H and pTrX-40R

[00131] Plasmids pTrX-40H and pTrX-40R, with the additional mutations S40H and S40R compared to the precursor plasmid pTrX, were prepared.

[00132] PCR was used to generate a DNA fragment encoding (39-190) region with the S40H and S40R mutation. The PCR primers with the 4OH and 4OR mutations (in bold type) are shown below.

TX- 40H - 1 ( SEQ ID NO : 49 )

39 40 41 42 43 44 45 46 47 48 W H N S G N F V G G

5 ' - GTC AAT TGG CAT AAC TCC GGA AAC TTC GTA GGT GGA Mun I

TX-40R-1 (SEQ ID NO:50)

39 40 41 42 43 44 45 46 47 48 W R N S G N F V G G

5 ' - GTC AAT TGG CGT AAC TCC GGA AAC TTC GTA GGT GGA Mun I

[00133] With the appropriate PCR template pTrX, the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 8).

TABLE 8

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(d) TX-40H-1 TX-Cl MunVHindlll

(e) TX-40R-1 TX-Cl Munl/HindlU

[00134] The cut PCR products (d) and (e) (Table 8) were ligated into a plasmid pTrX(l-l 13) to generate the plasmids pTrX-40η and pTrX-40R.

1.8 Construction of plasmid pTrX-99C

[00135] Plasmid pTrX-99C, with the additional mutation S99C not present in the precursor plasmid pTrX, was prepared.

[00136] PCR was used to generate a DNA fragment encoding (95-190) region with the S99C mutation.

[00137] The PCR primers with mutation (in bold type) is shown below.

TX-99C-1 (SEQ ID NO: 51) 95 96 97 98 99 100 101

T Y N P C T G

5 ' - TTC GGT ACC TAC AAT CCG TGT ACC GGC Kpn I

[00138] With the appropriate PCR template, pTrX, the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 9).

TABLE 9

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(t) TX-99C-1 TX-Cl Kpnl/HindlU

[00139] The cut PCR product (f) (Table 9) was ligated into a A>«I/HzWIII-linearized plasmid pTrX(l-l 13) (Table 4) to generate the plasmid pTrX-99C.

1.9 Construction of plasmid pOmpTrX-99C-U8C

[00140] The pOmpTrX-99C-l 18C plasmid, with the additional mutations S99C and Yl 18C and a secretion leading signal sequence not present in the precursor plasmid pTrX, was prepared.

[00141] PCR was used to generate a DNA fragment encoding (95-190) region with the S99C and Yl 18C mutation.

[00142] With the appropriate PCR template pTrX-118C (see Table 5, WO 03/046169), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 10).

TABLE 10

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(g) TX-99C-1 TX-Cl KpnVHindlll

[00143] The cut PCR product (g) (Table 10) was ligated into a Kpnl/Hindlll-lineaήzed plasmid pOmpTrX(l-l 13), (described in 1.3) to generate the plasmid pOmpTrX-99C-l 18C.

1.10 Construction of plasmid pOmpTrX-58R-99C-118C

[00144] The pOmpTrX-58R-99C-118C plasmid, with additional mutations of K58R, S99C and Y118C, and a secretion leading signal sequence, compared to the precursor plasmid pTrX, was prepared.

[00145] PCR was used to generate a DNA fragment encoding the (54-190) region with the K58R, S99C and Yl 18C mutations.

[00146] With the appropriate PCR template, pOmpTrX-99C-118C (described in 1.9), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 11).

TABLE 11

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

CO TX-58R-1 TX-Cl XmaVHindm

[00147] The cut PCR product (h) (Table 11) was ligated into a XmαI/Hm<fflI-linearized plasmid pOmpTrX(l-l 13) (described in 1.3) to generate the plasmid pOmpTrX-58R-99C-l 18C.

1.11 Construction ofplasmidspOmpTrX-40H-99C-118C and pOmpTrX-40H-58R-99C-118C

[00148] The pOmpTrX-40η-99C-118C plasmid, with additional mutations of S40H, S99C and Y118C, and a secretion leading signal sequence, compared to the precursor plasmid pTrX, was prepared. The pOmρTrX-40H-58R-99C-l 18C plasmid has an extra mutation of K58R

[00149] PCR was used to generate DNA fragments encoding the (39-190) region with the S40H, S99C and Y1 18C mutations, with or without the mutation K58R, as determined by the appropriate plasmid templates.

[00150] For the creation of pOmpTrX-40H-99C-118C with the appropriate PCR template, pOmpTrX-99C-l 18C (described in 1.9), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 12).

TABLE 12

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(i) TX-40H-1 TX-Cl Munl/Hindlϊl

[00151] For the creation of ρOmpTrX-40H-58R-99C-l 18C, with the appropriate PCR template pOmpTrX-58R-99C-l 18C (described in 1.10), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 13).

TABLE 13

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

G) TX-40H-1 TX-Cl MunVHindlll

[00152] The cut PCR product (i) (Table 12) and (j) (Table 13) were ligated into a Munl/Hindlϊl- linearized plasmid, pOmpTrX(l-1 13) (described in 1.3), to generate plasmids pOmpTrX-40H- 99C-118C and pOmpTrX-40H-58R-99C-l 18C, respectively.

1.12 Construction of plasmid pOmpTrX-HML-40R-58R-99C-118C

[00153] The pOmpTrX-HML-40R-58R-99C-l 18C plasmid, with additional mutations of S40R, K58R, S99C and Y118C, and a secretion leading signal sequence, compared to the precursor plasmid pTrX-HML, was produced.

[00154] With the appropriate PCR template, pOmpTrX-58R-99C-l 18C (described in 1.10), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 14).

TABLE 14

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(k) TX-40R-1 TX-Cl MunVHindllϊ

[00155] The cut PCR product (k) (Table 14) was ligated into a MunVHindlll-lineaήzed plasmid pOmpTrX-HML(l-113) (described in 1.5) to generate plasmid pOmpTrX-10H-27M-29L-40R- 58R-99C-1 18C.

1.13 Construction of plasmid pOmpTrX-10H-27M-29L-40R-58R-75A-99C-118C

[00156] The pOmpTrX-10H-27M-29L-40R-58R-75A-99C-118C plasmid, with the additional mutation S75A compared to the plasmid pOmpTrX-10H-27M-29L-40R-58R-99C-118, was prepared. The PCR primers with mutation S75A (in bold type) are shown below.

TX-75A-1 (SEQ ID NO: 52) 69 70 71 72 73 74 75 76 77 78 79 80 81

N G N S Y L A V Y G W S R 5'-T GGG AAT TCA TAC TTA GCC GTC TAT GGC TGG TCT AG EcoRI

[00157] With the appropriate PCR templates pOmpTrX-10H-27M-29L-40R-58R-99C-118C (described in 1.12) for both PCR products (1) and (m), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 15).

TABLE 15

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(1) TX-I OH-I TX-Cl PinAVEcoRl

(m) TX-75A-1 TX-Cl EcoRl/Hindlll

[00158] The cut PCR products (1 and m) (Table 15) were ligated into a PmAI////«λH-linearized plasmid, pOmpTrX(l-l 13) (described in 1.3), to generate the plasmid pOmpTrX-10H-27M-29L- 40R-58R-75A-99C-118C.

/.14 Construction of plasmid pOmpTrX-10H-27M-29L-75A-99C-105H-118C-125A-129E

[00159] The pOmpTrX-10H-27M-29L-75A-99C-105H-118C-125A-129E plasmid, was prepared via two PCR reactions, involving the following primers:

TX-118C-1 (SEQ ID NO:53)

111 112 113 114 115 116 117 118 119 120 121 122 D G S V Y D I C R T Q R 5'-GAC GGA TCC GTA TAT GAT ATC TGC CGT ACC CAA CGC BamHI

TX-99C105H-lr (SEQ ID NO: 54)

114 113 112 111 110 109 108 107 106 105 104 103 102 101 100 V S G D S T V E G H K T A G T 5'-TAC GGA TCC ATC ACT AGT GAC TTC GCC GTG TTT TGT GGC GCC GGT BamHI Kasl

99 98 97 96 C P N Y ACA CGG ATT GTA Kasl

[00160] With plasmid pTrX-10H-27M-29L-75A-105H-125A-129E as a PCR template (Table 5), one PCR product (n) was synthesized to encode the (8-112) sequence, and another PCR product (o) to encode the (113-190) region.

[00161] With the appropriate PCR template plasmid pTrX-10H-27M-29L-75A-105H-125A- 129E (Table 5), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 16).

TABLE 16

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(n) TX-I OH-I TX-99C-105H- PinAVBamHI

Ir

(o) TX-I l 8C- 1 TX-Cl BammiHindm

[00162] The cut PCR products (n and o) (Table 16) were ligated into a PmAI/HmdIII-linearized plasmid, pOmpTrX-HML(l-113) (described in 1.5) to generate plasmid pOmpTrX-10H-27M- 29L-75A-99C-105H-118C-125A-129E.

1.15 Construction of plasmid pOmpTrX-10H-27M-29L-58R-75A-99C-105H-H8C-125λ-l29E

[00163] The pOmpTrX-10H-27M-29L-58R-75A-99C-105H-l 18C-125A-129E plasmid, with an additional mutation K58R compared to the precursor plasmid pOmpTrX-10H-27M-29L-75A- 99C- 105H- 118C- 125 A- 129E (in 1.14), was created.

[00164] With the appropriate PCR template pOmpTrX-10H-27M-29L-75A-99C-105H-118C- 125A-129E (in 1.14), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 17).

TABLE 17

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(P) TX-58R-1 TX-Cl XmaVHindHl

[00165] The cut PCR product (p) (Table 17) was ligated into a Xmal/HindUl-lineaήzed plasmid pOmpTrX-HML(l-113) (described in 1.5) to generate the plasmid pOmpTrX-10H-27M-29L- 58R-75A-99C-105H-118C-125A-129E.

1.16 Construction of plasmid pOmpTrX-lOH-llDηM^L-SSR-JSAWC-lOSH-lUC-nSA- 129E

[00166] The pOmpTrX-10H-HD-27M-29L-58R-75A-99C-105H-118C-125A-129E plasmid, with an extra NIlD mutation, was prepared using the same strategy as for the plasmid pOmpTrX-10H-27M-29L-58R-75A-99C-105H-118C-125A-129E in 1.15.

[00167] The cut PCR product (p) (Table 17) which was prepared for the construction of pOmpTrX-10H-27M-29L-58R-75A-99C-105H-118C-125A-129E (in 1.15), was ligated into a Xmαl/HiWIII-linearized plasmid pOmpTrX-HDML(l-113) (described in 1.5) to generate the plasmid pOmpTrX- 1 OH- 11 D-27M-29L-58R-75 A-99C- 105H- 118C- 125 A- 129E.

1.17 Construction of plasmid pOmp-TrX-lOH-im-UM^L-iOX-Sm-lSAWC-lOSH-mC- 125A-129E, where X is C, F, H, R, Y, A or T

[00168] The plasmids pOmpTrX-10H-HD-27M-29L-40X-58R-75A-99C-105H-118C-125A- 129E, where X is C, F, H, R, Y, A or T, were prepared using the same strategy, with a PCR primer harbouring the appropriate mutation:

TX-40C-1 (SEQ ID NO: 55)

39 40 41 42 43 44 45 46 47 48 W C N S G N F V G G

5 ' - GTC AAT TGG TGT AAC TCC GGA AAC TTC GTA GGT GGA Mun I

TX-40Y-1 (SEQ ID NO:56)

39 40 41 42 43 44 45 46 47 48

W Y N S G N F V G G

5 ' - GTC AAT TGG TAT AAC TCC GGA AAC TTC GTA GGT GGA

Mun I

TX-40F-1 (SEQ ID NO:57)

39 40 41 42 43 44 45 46 47 48 W F N S G N F V G G

5 ' - GTC AAT TGG TTT AAC TCC GGA AAC TTC GTA GGT GGA Mun I

TX-40T-1 (SEQ ID NO:58)

39 40 41 42 43 44 45 46 47 48 W T N S G N F V G G

5 ' - GTC AAT TGG ACT AAC TCC GGA AAC TTC GTA GGT GGA Mun I

TX-40A-1 (SEQ ID NO: 59)

39 40 41 42 43 44 45 46 47 48 W A N S G N F V G G

5 ' - GTC AAT TGG GCT AAC TCC GGA AAC TTC GTA GGT GGA Mun I

[00169] TX-40H-1 and TX-40R-1 have been described in 1.8.

[00170] With the appropriate PCR template plasmid pOmpTrX-10H-27M-29L-58R-75A-99C- 105H-118C-125A-129E (described in 1.15), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 18).

TABLE 18

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(q) TX-40H-1 TX-Cl Munl/Hindlll

(r) TX-40R-1 TX-Cl MunVHindm

(S) TX-40C-1 TX-Cl MunVHindm

(t) TX-40F-1 TX-Cl Munl/Hindlll

(U) TX-40Y-1 TX-Cl MunVHindm

(V) TX-40T-1 TX-Cl Munl/Hindlll

(w) TX-40A-1 TX-Cl MunVHindill

[00171] The cut PCR products (q, r, s, t, u, v and w) (Table 18) were ligated into a Mml/HzWIII-linearized plasmid pOmpTrX-ηDML(l-113) (described in 1.5) to generate the plasmids pOmp-TrX-lOH-11D-27M-29L-40H-58R-75A-99C-105H-1 18C-125A-129E, pOmp- TrX- 1 OH- 11 D-27M-29L-40R-58R-75 A-99C- 105H- 118C- 125 A- 129E, pOmp-TrX- 1 OH- 11 D- 27M-29L-40C-58R-75A-99C-105H-118C-125A-129E, pOmp-TrX-lOH-11D-27M-29L-40F- 58R-75A-99C-105H-118C-125A-129E, pOmp-TrX-10H-l lD-27M-29L-40F-58R-75A-99C- 105H- 118C- 125 A- 129E, pOmp-TrX- 1 OH- 11 D-27M-29L-40T-58R-75 A-99C- 105H- 118C- 125 A- 129E and pOmp-TrX- 1 OH- 11 D-27M-29L-40A-58R-75 A-99C- 105H- 118C- 125 A- 129E, respectively.

1.18 Construction of plasmid pOmp-TrX-10H-llD-27M-29L-40H-52C-58R-75λ-99C-105H- 118C-125A-129E

[00172] The pOmpTrX-lOH-11D-27M-29L-40H-52C-58R-75A-99C-105H-118C-125A12 9E plasmid was prepared via ligation of two PCR sequences, namely a PinAVXmal-lineaήzed (8- 53) fragment and an XmαI////«<λII-linearized (54-190) fragment. The former sequence was synthesized via a PCR with the following primer harbouring the mutation Q52C:

TX-52C-lr (SEQ ID NO: 60)

54 53 52 51 50 49 48 47 46 45 44 G P C W G K G G V F N 5'-GT CCC GGG ACA CCA ACC TTT TCC ACC TAC GAA GT Xmal

[00173] With the appropriate PCR template plasmid, pOmpTrX-lOH-11D-27M-29L-40H-58R- 75A-99C-105H-118C-125A-129E (described in 1.17), the primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 19).

TABLE 19

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR

product

(x) TX-IOHl ID-I TX-52C-lr PinAl/Xmal

[00174] The (54-190) fragment has already been prepared as the Xmal/Hindlll-cut PCR product (p) ofTable l7 in l.l5.

[00175] Ligation of the PinAVXmal-cut (8-53) fragment (x) (Table 19) and the Xmal/Hindlll- cut (54-190) fragment (p) (Table 17) into the PmAI/HzWIII-linearized precursor plasmid pOmp- TrX-(I-1 13) (described in 1.3), yielded the new plasmid pOmpTrX-10η-HD-27M-29L-40η- 52C-58R-75A-99C-105H-118C-125A-129E.

1.19 Construction of plasmid pOmp-TrX-10H-llD-27M-29L-40R-58R-75A-99C-105H-118C- 125A-129E-144R-161R

[00176] Plasmid pOmp-TrX-lOH-11D-27M-29L-40R-58R-75A-99C-105H-118C- 125 A-129E- 144R-161R was created, which differed from plasmid pOmp-TrX-lOH-11D-27M-29L-40R-58R- 75A-99C-105H-118C-125A-129E of 1.18 by two mutations: H144R and Q161R.

[00177] It was synthesized via a ligation of two appropriately cut PCR products. The first insert encoding region (39-112) was generated through a Munl/BamUl-cuttmg of the PCR product (r) already described in Table 18 of 1.17.

[00178] The second insert encoding the (113-190) region was prepared via PCR with the appropriate PCR template plasmid pTrX-10H-27M-29L-75A105H-125A129E-144R-161R (WO 03/046169). The primers and the restriction enzymes to cut the ends of the PCR products are listed below (Table 20).

TABLE 20

PCR PCR upstream PCR reverse Restriction product primer primer enzymes for PCR product

(y) TX-I l 8C- 1 TX-Cl BamHl/Hmdlll

[00179] The two appropriately cut PCR products (y) (Table 20) and the Munl/BamHl-cut (r) were ligated into a Munl/Hindlll-linQaήzed plasmid pOmpTrX-HDML(l-113) (described in 1.5) to generate the plasmid pOmp-TrX-lOH-l lD^M^L^OR-SδR^SA-QQC-lOSH-l lδC-^SA- 129E-144R-161R.

EXAMPLE 2: Characterization of mutant xylanases

2.1 Production ofxylαnαses

[00180] The culture conditions comprised a 5 mL culture of overnight innoculant in 2YT medium (16 g bacto-tryptone, 10 g yeast extract, 5 g NaCl, 1 L of water) containing ampicillin (100 mg/L). The culture was spread out on a tray (32 x 25 cm) evenly covered by 0.5 L of solidified YT agar (8 g yeast extract, 5 g bacto-tryptone, 5 g NaCl, 15 g of agar in 1 L of water) containing ampicillin (100 mg/L). The cultures were grown at 37 ⁰ C. After 40 hr, the cells (2 g) were harvested for extraction of xylanase.

2.2 Purification of mutant xylanases

[00181] The harvested cells were put into a tube for a freeze-thaw extraction of xylanase. The procedure comprised a freezing period in a dry ice/ethanol bath for 5 minutes, followed by water/ice bath for 10 minutes. The procedure was repeated thrice. The cells were extracted with buffer (5 mL, 100 mM Na citrate, pH 5.5). Centrifuging at 8000 x g for 30 minutes yielded a supernatant containing xylanase. The xylanase solution was adjusted to pH 5.2. The precipitate which appeared was removed through centrifuging at the same condition. The supernatant was heated at a range of 50-60 ⁰ C, depending of the thermostability of the recombinant xylanase, for 30 minutes to convert more undesirable bacterial proteins into precipitate, which was removed by centrifugation.

[00182] Prior to column chromatography, the supernatant was adjusted to pH 4.6 by acetic acid and centrifuged to remove any precipitate. The subsequent method for column chromatography was identical for all mutant xylanases.

[00183] Following acidification and centrifugation, the xylanase sample was pumped onto a 50 mL bed volume, CM-sepharose fast flow, cation exchange column (Pharmacia Biotech, Uppsala), equilibrated in 10 mM sodium acetate (pH 5.1). The xylanase was eluted with a 250 mL linear gradient (0 to 0.6 M NaCl in 10 mM sodium acetate, pH 5.1) at a flow rate of 1 mL/min. The xylanases elute at 150 to 200 mL of the gradient. Aliquots from the collected fractions are examined by SDS-PAGE, and those fractions having most of the xylanase present were pooled. The purified xylanase was quantified by spectrophotometry at 280 nm using an extinction coefficient between 54,600 and 53,400 M ^"1 , for most mutant TrX xylanases. A typical purification from 1O g of cells yielded 25 mg of xylanase.

2.3 Standard assay for the measurement of enzymatic activity

[00184] 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 dessicator. The measurement of activity was performed as follows. Reaction mixtures containing 100 μL of 30 mg/mL xylan previously diluted in assay buffer (50 mM sodium citrate, pH 5.5 or the pH optimum of the tested xylanase), 150 μL assay buffer, and 50 μL of enzyme (15 μg/mL) diluted in assay buffer were incubated at 4O ⁰ C. 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, which is incorporated herein by reference).

EXAMPLE 3: Thermophilicity of mutant xylanases

[00185] Thermophilicity was examined to test the effect of different temperatures on the enzymatic hydrolysis of soluble xylan by different mutant xylanases.

[00186] The assay procedure was similar to the standard assay with changes in the incubation temperature and time. The xylanases (15 μg/mL) and soluble birchwood xylan substrate, in 50 mM sodium citrate buffer of pH 5.5, or stated otherwise, were mixed and incubated in a circulating water bath at different temperatures. After a 30-minute incubation, the amount of

reducing sugars released from xylan was determined by HBAH analysis and was calculated as a relative activity, with the value at 4O ⁰ C or the temperature optimum representing 100%.

[00187] The effect of temperature on the hydrolysis of xylan by Trichoderma reesei xylanase TrX with individual mutations like S40H, K58R, S99C or Yl 18C is shown in Figure 3.

[00188] The mutation S40H in xylanase TrX-40H showed a moderately improved enzymatic activity at higher temperature as compared to TrX (Figure 3). In the case of the xylanase TrX- 58R, the mutation K58H by itself showed no improvement over TrX (Figure 3), as already reported by Turunen et al. (2002).

[00189] The increase of thermophilicity by the single mutation Yl 18C as in xylanase TrX-118C (Figue 3) has been described in the art (WO 03/046169), but the possibility of a disulfide bond created through introduction of a cysteine-118 has never been studied. This mutation can potentially form a disulfide linkage with a cysteine replacement at residue 99.

[00190] Initially the single mutation S99C was tested in the form of a mutant xylanase, TrX- 99C (Figure 3), with no apparent improvement of enzymatic activity at higher temperature, therefore demonstrating this mutation has no effect on the temperature/activity profile of TrX. However, when the S99C and Y118C mutations were incorporated in the form of the double mutant xylanase TrX-99C-l 18C there was a dramatic enhancement of thermophilicity (Figure 3), even when compared to the single mutant TrX-118C. The improvement of the temperature optima of the double mutant TrX-99C-l 18C over the natural xylanase, TrX, is about 7 ⁰ C (Figure 4). In addition to higher temperature optimum (Figure 4), TrX-99C-l 18C also exhibited higher optimal activity than TrX at their respective temperature optima (Figure 3).

[00191] The additive effect of the mutations S40H and K58R on the 99C/118C mutations to increase enzymatic activity at higher temperatures was confirmed in the form of the mutant xylanases TrX-58R-99C-118C, TrX-40H-99C-1 18C and TrX-40H-58R-99C-118C (Figures 3 and 4). Although the mutation K58R by itself failed to improve the activity of xylanase at higher temperature (Figure 3), it has demonstrated a positive effect in combination with other mutations S40H and S99C/Y118C.

[00192] The new mutations are compatible with other advantageous mutations which have been described in the art. This was demonstrated below in the creation of xylanase variants with higher temperature optimum and optimal activity.

[00193] The mutations of NlOH, Y27M and N29L to increase the thermophilicity of TrX, in the form of the mutant TrX-10H-27M-29L (or TrX-HML), have been described (U.S. Patent No. 5,759,840). Incorporation of the new mutations S40H, K58R, and S99C/Y118C into TrX-IOH- 27M-29L created the variant xylanases TrX-10H-27M-29L-40R-58R-99C-118C (Figure 5) and TrX-10H-27M-29L-40R-58R-75A-99C-l 18C with further improved thermophilicity (Figure 5).

[00194] The mutations of NlOH, Y27M, N29L, S75A, L105H, Q125A and I129E to increase the thermophilicity of TrX, in the form of the mutant TrX-10H-27M-29L-75A-105H-125A-129E (TrX-HML-AHAE), are described in WO 01/92487. Incorporation of the mutations S40H, K58R, and S99C/Y118C to TrX-10H-27M-29L-75S-105H-125A-129E created the variant xylanases TrX-10H-27M-29L-75A-99C-105H-118C-125A-129E (Figures 6 and 8) and TrX- 10H-27M-29L-58R-75A-99C-105H-118C-125A-129E (Figure 6), both with further enhanced thermophilicity.

[00195] The mutation of NI lD is described in WO 03/046169. Addition of this mutation created a variant TrX-10H-l lD-27M-29L-58R-75A-99C-105H-118C-125A-129E (Figures 6, 7 and 8). Variations of mutation 40 (S40C, A, F, H, R, Y or T) were introduced into this variant to create the new mutants TrX-10H-l lD-27M-29L-40X-58R-75A-99C-105H-118C-125A-129E (where X = C, A, F, H, R, Y and T). As indicated in the study above, introduction of mutations S40H or S40R moderately improved the relative activity at higher temperature as compared to the host enzyme (Figures 6, 7 and 8). Furthermore, other mutations S40C, S40F and S40Y exhibited the same enhancing effect (Figure 7), while S40T and S40A showed no such enhancing effect on the temperature/activity profile (Figure 7).

[00196] Another mutation, Q52C, was introduced into TrX-I OH-11D-27M-29L-40H-58R-75A- 99C-105H-1 18C-125A-129E. The mutant xylanase TrX- 1 OH- 11D-27M-29L-40H-52C-58R- 75A-99C-105H-1 18C-125A-129E was able to retain significant relative activity at higher temperatures of 80 and 85°C (Figures 6, 7 and 8).

[00197] The mutations H144R and Q161R (described in WO 03/046169) have been shown to increase the pH optimum of the xylanase TrX-10H-l lD-27M-29L-75A-105H-118C-125A- 129E-144R-161R (or TrX-lOH-11D-27M-29L-AH-118C-AE-RR). Addition of mutations S40R, K58R and S99C allowed the mutant xylanase TrX-10H-l lD-27M-29L-40R-58R-75A-99C- 105H-1 18C-125A-129E-144R-161R to retain greater activity at higher temperatures of 80 and 85°C (Figure 9).

[00198] The above results demonstrate that the enhancing effect of the mutations S40X (X = C, F, H, R or Y), Q52C, K58R, and the disulfide S99C/Y118C mutation on the thermophilicity of the mutant xylanase are not only complementary or additive to each other, but also to other mutations disclosed in the art (U.S. Patent No. 5,759,840, WO 01/92487 and WO 03/046169).

EXAMPLE 4: Alkalophilicity of mutant xylanases

[00199] The alkalophilicity of genetically modified xylanases was examined to test the effect that different pH conditions had on the enzymatic hydrolysis of soluble birchwood xylan by mutant xylanases. The assay procedure was similar to the standard assay with changes in the incubation temperature and time. Aliquots of genetically modified xylanases (15 μg/mL) and soluble xylan substrate in 50 raM sodium citrate buffers which varied between pH 4-8 were incubated together at 55° or 65 ⁰ C as stated. Following 30 minute incubations, the amount of reducing sugars released from the xylan substrate was determined by HBAH analysis and the enzymatic activity as a function of pH was calculated for a variety of mutant xylanases with the maximal activity taken as 100%.

[00200] The effect of the S99C/Y118C mutations on the pH/activity profile of xylanase was investigated. The new disulfide mutant TrX-99-C118C maintained greater activity at high pH values of 6.5-7.5 (Figure 10), as compared to the natural xylanase TrX. The pH range to maintain 80% optimal activity is 4.8-7.0 for the disulfide mutant xylanase TrX-99C-118C and only 4.8-6.0 for natural xylanase TrX, indicating a broader effective pH range for the former.

[00201] Xylanases with the individual mutations TrX-99C and TrX-118C were also compared to TrX-99C-l 18C and the natural xylanase TrX. Both TrX-99C and TrX-118C have the same pH/activity profile as TrX (Figure 10). This confirmed that the improvement of activity at higher

pH is a result of the combination of S99C and Yl 18C mutations to form the disulfide bond, and not the single Cys mutations.

[00202] The effect of the mutations S40X (X is H or R), K58R and the disulfide S99C/Y118C on the pH/activity profile of xylanase was also studied in two groups of mutants constructed above.

[00203] The first group was derived from the mutant TrX-10H-27M-29L-75A-105H-125A- 129E (or TrX-HML-AH-AE) and are described in WO 01/92487. Derivatives like TrX-IOH- 27M-29L-75 A-99C- 105H- 118C- 125 A- 129E, TrX- 10H-27M-29L-58R-75 A-99C- 105H- 118C- 125A-129E, TrX-10H-l lD-27M-29L-58R-75A-99C-105H-118C-125A-129E and TrX-IOH- 11D-27M-29L-40H-58R-75A-99C-105H-118C-125A-129E showed greater activity at higher pH (Figure 11), as compared to the parent TrX-10H-27M-29L-75A-105H-125A-129E. However, the greatest improvement of activity by TrX-10H-27M-29L-75A-99C-105H-118C-125A-129E, over the parent xylanase TrX-10H-27M-29L-75A-105H-125A-129E, was via the addition of the 99C/118C disulfide. The other mutant xylanases in this series (Figure 11), with mutations S40H or K58R, showed no additional effect on the activity of xylanase.

[00204] The enhancing effect of the S99C/Y118C mutations was further demonstrated in the second group based on TrX-10H-27M-29L-75A-105H-125A129E-144R-161R, a xylanase containing two mutations H144R and Ql 61 R which has been shown to successfully increase the pH optimum of xylanase (see WO 01/92487). This construct, TrX- 1 OH- 1 1D-27M-29L-40R- 58R-75A-99C-105H-118C-125A-129E-144R-161R, exhibited greater activity at higher pH than its parent TrX-10H-27M-29L-75A-105H-125A-129E-144R-161R (Figure 12). It also outperformed another xylanase TrX- 1 OH- 11 D-27M-29L-75A- 105H- 116G- 118C- 125 A- 129E- 144R-161R (Figure 12), a mutant xylanase which previously showed the most improved pH/activity profile among mutant xylanases in WO 03/046169.

EXAMPLE 5: Thermostability of mutant xylanases

[00205] The tolerance of xylanase to incubation at different temperatures in the absence of substrate was investigated. The xylanase (150 μg/mL) in assay buffer (50 mM sodium citrate, pH 5.0) was incubated for 30 minutes at 48, 52, 56 and 6O ⁰ C. Aliquots were cooled to room

temperature (around 2O ⁰ C) and the residual enzymatic activity of the samples was determined via the HBAH assay at 55°C for 30 minutes. The residual enzymatic activity at 48 ⁰ C was normalized to 100%.

[00206] The disulfide mutant, TrX-99C-118C, retained greater residual activity than the natural xylanase TrX (Figure 13) after incubation at higher temperatures. The T ₅₀ was 58 ⁰ C for the disulfide xylanase, as compared to a T ₅₀ of 51 ⁰ C for the natural xylanase TrX (Figure 13), which is an increase in the thermostability of the former by about 7°C.

[00207] While the present invention has described mutant xylanases which exhibit improved thermophilicity, alkalophilicity and thermostability, and the benefits associated with these enzymes in the production of paper pulp, these mutant xylanases may also be of use in other industrial processes, for example, but not limited to, the washing of precision devices and semiconductors. Furthermore, by virtue their increased thermophilicity and thermostability the mutant xylanases may be used in chemical processes that employ small quantities of denaturants or detergents or in the presence of solvents, for example, but not limited to, small amounts of apolar solvents, such as, but not limited to, hexane, dioxanes, carbon tetrachloride, benzene, ethers, chloroform, acetic acid and methylene chloride, and polar solvents, such as, but not limited to, acetone, alcohols, dimethylformamide, acetonitrile, sulfolane, dimethylsulfoxide and water.

EXAMPLE 6: Isolation of Trichoderma reesei Genomic DNA and Construction of T. reesei Genomic Libraries

[00208] Trichoderma reesei strain M2C38 is a proprietary strain of Iogen Corporation derived from Trichoderma reesei RutC30 (ATCC #56765; Montenecourt and Eveleigh, 1979), which was, in turn, derived from Trichoderma reesei Qm6A (ATCC # 13631; Mandels and Reese, 1957). It is well understood by those skilled in the art that the procedures described herein, the genetic constructs from these strains, and the expression of the genetic constructs in these strains are applicable to all Trichoderma strains derived from Qm6A.

[00209] To isolate genomic DNA, 50 mL of Potato Dextrose Broth (Difco) was inoculated with T. reesei spores collected from a Potato Dextrose Agar plate with a sterile inoculation loop. The

cultures were shaken at 200 rpm for 2-3 days at 28°C. The mycelia was filtered onto a GFA glass microfibre filter (Whatman) and washed with cold, deionized water. The fungal cakes were frozen in liquid nitrogen crushed into a powder with a pre-chilled mortar and pestle; 0.5 g of powdered biomass were resuspended in 5 mL of 100 mM Tris, 50 mM EDTA, pH 7.5 plus 1% sodium dodecyl sulphate (SDS). The lysate was centrifuged (5000 g for 20 min, 4 ⁰ C) to pellet cell debris. The supernatent was extracted with 1 volume buffer (10 mM Tris, 1 mM EDTA, pH 8.0) saturated phenol followed by extraction with 1 volume of buffer-saturated phenol:chloroform:isoamyl alcohol (25:24:1) in order to remove soluble proteins. DNA was precipitated from the solution by adding 0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes of cold 95% ethanol. After incubating for at least 1 h at -20 ⁰ C, the DNA was pelleted by centrifugation (5000 g for 20 min, 4 ⁰ C), rinsed with 10 mL 70% ethanol, air-dried and resuspended in 1 mL 10 mM Tris, 1 mM EDTA, pH 8.0. RNA was digested by the addition of Ribonuclease A (Boehringer Mannheim) added to a final concentration of 0.1 mg/mL and incubation at 37°C for 1 hour. Sequential extractions with 1 volume of buffer-saturated phenol and 1 volume of buffer-saturated phenol:chloroform:isoamyl alcohol (25:24:1) was used to remove the ribonuclease from the DNA solution. The DNA was again precipitated with 0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes of cold 95% ethanol, pelleted by centrifugation, rinsed with 70% ethanol, air-dried and resuspended in 50 μL of 10 mM Tris, 1 mM EDTA, pH 8.0. The concentration of DNA was determined by measuring the absorbance of the solution at 260 nm (p. Cl in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

[00210] Two plasmid libraries and one phage library were constructed using genomic DNA isolated from T. reesei strain M2C38. The plasmid libraries were constructed in the vector pUCl 19 (Viera and Messing, 1987) as follows: 10 μg genomic DNA was digested for 20 hrs at 37°C in a 100 μL volume with 2 units/μg of Barήαλ or EcoRl restriction enzymes. The digested DNA was fractionated on a 0.75% agarose gel run in 0.04 M Tris-acetate, 1 mM EDTA and stained with ethidium bromide. Gel slices corresponding to the sizes of the genes of interest (based on published information and Southern blots) were excised and subjected to electro- elution to recover the DNA fragments (Sambrook et al., pp. 6.28-6.29). These enriched fractions of DNA were ligated into pUC119 in order to create gene libraries in ligation reactions containing 20-50 μg/mL DNA in a 2:1 molar ratio of vectoπinsert DNA, 1 mM ATP and 5 units

T4 DNA ligase in a total volume of 10-15 μL at 4°C for 16 h. Escherichia coli strain HBlOl was electroporated with the ligation reactions using the Cell Porator System (Gibco/BRL) following the manufacturer's protocol and transformants selected on LB agar containing 70μg/mL ampicillin.

[00211] The phage library was constructed in the lambda vector λDASH (Stratagene, Inc.) as follows: genomic DNA (3 μg) was digested with 2, 1, 0.5 and 0.5 units/μg Bamlil for 1 hour at 37 ⁰ C to generate fragments 9-23 kB in size. The DNA from each digest was purified by extraction with 1 volume Tris-staturated phenol :choro form :isoamyl alcohol (25:24:1), followed by precipitation with 10 μL 3 M sodium acetate, pH 5.2 and 250 μl 95% ethanol (-20 ⁰ C). The digested DNA was pelleted by microcentrifugation, rinsed with 0.5 mL cold 70% ethanol, air- dried and resuspended in 10 μL sterile, deionized water. Enrichment of DNA fragments 9-23 kB in size was confirmed by agarose gel electrophoresis (0.8% agarose in 0.04 M Tris-acetate, 1 mM EDTA). Digested DNA (0.4 μg) was ligated to 1 μg λDASH arms predigested with Bamlil (Stratagene) in a reaction containing 2 units T4 DNA ligase and 1 mM ATP in a total volume of 5 μl at 4°C overnight. The ligation mix was packaged into phage particles using the GigaPack® II Gold packaging extracts (Stratagene) following the manufacturer's protocol. The library was titred using the E. coli host strain XLl -Blue MRA (P2) and found to contain 3 x 10 ⁵ independent clones.

EXAMPLE 7: Isolation of genomic clones from T. reesei M2C38 libraries

7.1 Cloning the cellobiohydrolase I (cbhl) and cellobiohydrolase II (cbh2) genes from pUC119 libraries

[00212] E. coli HBlOl transformants harboring cbhl or cbh2 clones from recombinant pUC1 19-ZfamHlor -EcoRI libraries were identified by colony lift hybridization: 1-3 x 10 ⁴ colonies were transferred onto HyBond™ nylon membranes (Amersham); membranes were placed colony-side up onto blotting paper (VWR 238) saturated with 0.5 M NaOH, 1 M NaCl for 5 min to lyse the bacterial cells and denature the DNA; the membranes were then neutralized by placing them colony-side up onto blotting paper (VWR 238) saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 min; the membranes were allowed to air-dry for 30 min and the DNA was then fixed to the membranes by baking at 80 ⁰ C for 2 h.

[00213] ³² P-labelled probes were prepared by PCR amplification of short (0.7-1.5 kB) fragments of the cbhl and cbh2 coding regions from the enriched pool of BamHl or EcoRl fragments, respectively, in a labelling reaction containing 10-50 ng target DNA, 0.2 mM each d(GCT)TP, 0.5 μM dATP, 20-40 μCi α- ³² P-dATP, 10 pmole oligonucleotide primers and 0.5 units Taq polymerase in a total volume of 20 μL. The reaction was subjected to 6-7 cycles of amplification (95°C, 2 min; 56°C, 1.5 min; 70 ⁰ C, 5 min). The amplified, ³² P-labelled DNA was precipitated by the addition of 0.5 mL 10% (w/v) trichloroacetic acid and 0.5 mg yeast tRNA. The DNA was pelleted by microcentrifugation, washed twice with 1 mL 70% ethanol, air-dried and resuspended in 1 M Tris pH 7.5, 1 mM EDTA.

[00214] Nylon membranes onto which the recombinant pUCl 19 plasmids had been fixed were prehybridized in heat-sealed bags for 1 h at 60-65°C in 1 M NaCl, 1% SDS, 50 mM Tris, 1 mM EDTA pH 7.5 with 100 μg/mL denatured sheared salmon sperm DNA. Hybridizations were performed in heat-sealed bags in the same buffer with only 50 μg/mL denatured sheared salmon sperm DNA and 5 x 10 ⁶ - 5 x 10 ⁷ cpm of denatured cbhl or cbh2 probe for 16-20 h at 60-65°C. Membranes were washed once for 15 min with 1 M NaCl, 0.5% SDS at 60°C, twice for 15 min each with 0.3M NaCl, 0.5% SDS at 60 ⁰ C and once for 15 min with 0.03M NaCl, 0.5% SDS at 55°C. Membranes were again placed in heat-sealed bags and exposed to Kodak RP X-ray film to 16-48 h at -70 ⁰ C. The X-ray film was developed following the manufacturer's protocols. Colonies giving strong or weak signals were picked and cultured in 2xYT media supplemented with 70 μg/mL ampicillin. Plasmid DNA was isolated from these cultures using the alkaline lysis method (Sambrook, et al., pp. 1.25-1.28) and analyzed by restriction digest, Southern hybridization (Sambrook, et al., pp. 9.38-9.44) and PCR analysis (Sambrook, et al., pp. 14.18- 14,19).

[00215] Clones carrying the cbhl gene were identified by colony lift hybridization of the pUC119-2?αmHl library with a 0.7 kb cbhl probe prepared using oligonucleotide primers designed to amplify bp 597-1361 of the published cbhl sequence (Shoemaker et al., 1983.). A cbhl clone, pCOR132, was isolated containing a 5.7 kb BamHl fragment corresponding to the promoter (4.7 kb) and 1 kb of the cbhl structural gene (2.3 kb). From this, a 2.5 kb EcoRl fragment containing the cbhl promoter (2.1 kb) and 5' end of the cbhl coding region (0.4 kb) was subcloned into pUCl 19 to generate pCB152. Clones carrying the cbh2 gene were identified

by colony lift hybridization of the pUC119-£cøRl library with a 1.5 kb cbh2 probe prepared using oligonucleotide primers designed to amplify bp 580-2114 of the published cbh2 sequence (Chen et al. 1987). A cbh2 clone, pZUK.600 was isolated containing a 4.8 kb EcoRl fragment corresponding to the promoter (600 bp), structural gene (2.3 kb) and terminator (1.9 kb).

7.2 Cloning cbhl terminator andxylanase II (xln2) gene from λDASH libraries

[00216] Digoxigen-11-dUTP labelled probes were prepared from PCR amplified coding regions of the cbhl and xln2 genes by random prime labeling using the DIG Labeling and Detection kit (Boehringer Mannheim) and following the manufacturer's protocols. Genomic clones containing the cbhl and xln2 genes were identified by plaque-lift hybridization of the λDASH library. For each gene of interest, 1 x 10 ⁴ clones were transferred to Nytran® (Schleicher and Schull) nylon membranes. The phage particles were lysed and the phage DNA denatured by placing the membranes plaque-side up on blotting paper (VWR238) saturated with 0.5 M NaOH, 1 M NaCl for 5 min; the membranes were then neutralized by placing them plaque-side up onto blotting paper saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 min; the membranes were allowed to air-dry for 30 min and the DNA was then fixed to the membranes by baking at 80 ⁰ C for 2 h. The membranes were prehybridized in heat-sealed bags in a solution of 6X SSPE, 5X Denhardt's, 1% SDS plus 100 μg/mL denatured, sheared salmon sperm DNA at 65°C for 2 h. The membranes were then hybrized in heat-sealed bags in the same solution containing 50 μg/mL denatured, sheared salmon sperm DNA and 0.5 μg of digoxigen-dUTP labelled probes at 65°C overnight. The membranes were washed twice for 15 min in 2X SSPE, 0.1% SDS at RT, twice for 15 min in 0.2XSSPE, 0.1% SDS at 65°C and once for 5 min in 2X SSPE. Positively hybridizing clones were identified by reaction with an anti-digoxigenin/alkaline phosphatase antibody conjugate, 5-bromo-4-chloro-3-indoyl phosphate and 4-nitro blue tetrazolium chloride (Boehringer Mannheim) following the manufacturer's protocol. Positively hybridizing clones were further purified by a second round of screening with the digoxigen-dUTP labelled probes.

[00217] Individual clones were isolated and the phage DNA purified as described in Sambrook et al. (1989) pp. 2.118-2.121 with the exception that the CsCl gradient step was replaced by extraction with 1 volume of phenol :choroform:isoamyl alcohol (25:24:1) and 1 volume of chloroform:isoamyl alcohol (24:1). The DNA was precipitated with 0.1 volumes of 3 M sodium

acetate, pH 5.2 and 2.5 volumes cold 95% ethanol. The precipitated phage DNA was washed with 0.5 niL cold 70% ethanol, air-dried and resuspended in 50 μL 10 rnM Tris, 1 mM EDTA pH 8.0. Restriction fragments containing the genes of interest were identified by restriction digests of the purified phage DNA and Southern blot hybridization (Sambrook, et al., pp. 9.38- 9.44) using the same digoxigen-dUTP labelled probes used to screen the λDASH library. The membranes were hybridized and positively hybridizing fragments visualized by the same methods used for the plaque lifts. Once the desired restriction fragments from each λDASH clone were identified, the restriction digests were repeated, the fragments were resolved on a 0.8% agarose gel in TAE and the desired bands excised. The DNA was eluted from the gel slices using the Sephaglas BandPrep Kit (Pharmacia) following the manufacturer's protocol.

[00218] Clones carrying the cbhl gene were identified by colony lift hybridization of the λDASH library (Example 7) with a cbhl probe comprising bp 45-2220 of the published cbhl sequence (Shoemaker et al.). A 1.8 kb BamHl fragment containing the 3' end of the cbhl coding region (0.5 kb) and the cbhl terminator (1.3 kb) was isolated by restriction digestion of phage DNA purified from a λDASH cbhl clone. This fragment was subcloned into the BamKl site of the E.coli plasmid vector pUC119 to generate the plasmid pCBITa. Clones carrying the xln2 gene were identified by colony lift hybridization of the λDASH library (Example 7) with a xln2 probe comprising bp 100-783 of the published xln2 sequence (Saarelainen et al., 1993, MoI. Gen. Genet. 241 :497-503). A 5.7 kb Kpnl fragment containing the promoter (2.3 kb), coding region (0.8 kb) and terminator (2.6 kb) the xln2 gene was isolated by restriction digestion of phage DNA purified from a λDASH xln2 clone. This fragment was subcloned into the Kpnl site of pUCl 19 to generate the plasmid pXYN2K-2.

EXAMPLE 8: Construction of a vector directing the expression of modified Family 11 xylanases in Trichoderma reesei.

[00219] A 2.4 kb fragment containing the promoter and secretion signal of the xln2 gene (bp - 2150 to +195 where +1 indicates the ATG start codon and +193-195 represent codon 32) was amplified with Pwo polymerase from the genomic xln2 subclone pXYN2K-2 (Example 7) using a x//τ2-specific primer containing a Pin Al at bp 190-195 or codons 31 and 32) and the pUC reverse primer (Cat. No. 18432-013, Gibco/BRL) which anneals downstream of the Kpnl site at

the 5' end of the xln2 gene. This xln2 PCR product was inserted as a blunt-ended fragment into the Smal site of the pUC119 polylinker in such an orientation that the BamHl site of the polylinker is 3' to the PmAI site; this generated the plasmid pUC/XynPSS(Pin). The same xln2 PCR product was reisolated from pUC/XynPSS(Pin) by digestion with EcoRl (which was amplified as part of the pUCl 19 polylinker from pXYN2K-2) and BamHl and inserted into the plasmid pBR322L (a derivative of pBR322 containing an Sphl-Notl-Sall adaptor between the original Sphl and Sail sites at bp 565 and 650), also digested with EcoRl and BamHl, to generate the plasmid pBR322LXP. To facilitate high level expression of the modified xylanases, a 1.3 kb Hindlll fragment comprising bp -1400 to -121 of the xln2 promoter in pBR322LXP was replaced with a 1.2 kb Hindlll fragment comprising bp -1399 to -204 of the cbhl promoter which was isolated by Hindlll digestion of pCOR132; this generated the plasmid pBR322LXC. Finally, the EcoRl site of pBR322LXC was then blunted with Klenow and Spel linkers (Cat. No. 1086, New England Biolabs) were added to generate pBR322SpXC.

[00220] A fragment containing codons 1-190 of the xylanase gene containing the mutations N27H, Y27M, N29L was isolated from the plasmid pUC/HML (described in Example 9.1 below) by digestion with Nhel and BamHl inserted into pCB219N-N digested with Nhel and BamHl to generate pHML/C2ter. To make pCB219N-N, a cbh2 terminator fragment was amplified from the pZUK600 (described in Example 7, above) template using a primer homologous to bp 2226-2242 of the published 3' untranslated region of the cbh2 gene (Chen et al., 1987) containing a short polylinker comprising Xbal-Nhel-BamHl-Smal-Kpnl sites at the 5' end and the pUC forward primer (Cat. No. 1224, New England Biolabs) which anneals upstream of the EcoRl site at the 3' end of cbh2 in pZUK600. This fragment was digested at the engineered Xbal and EcoRl sites and inserted into the corresponding sites of pUC119 to generate pCB219. An EcoRl -Notl adaptor (Cat. No. 35310-010, Gibco/BRL) was inserted into the unique EcoRl site of pCB219 to generate pCB219N. A 2.7 kb fragment comprising codons 9-190 of the HTX4 gene and the cbh2 terminator was isolated from pHTX4/C2ter by digestion with PinAl and Notl and inserted into pBR322SpXC digested with PinPλ and Notl to generate the expression cassette pc/xHML-εC.

EXAMPLE 9: Mutagenesis of T. reesei xylanase II to generate the variant TRX-10H-11D- 27M-29L-40R-58R-75A-99C-105H-118C-125A-129E-131ν

9.1 Introduction of mutations NlOH, 27M, Y29L

[00221] The synthetic DNA comprising codons 32-190 in pTrX-HML (Example 1.4) was replaced by the corresponding genomic fragment of T. reesei xln2, containing a 108 bp intron at codon 58, which was amplified using genomic T. reesei DNA as a template and introducing a unique FmAI site at codons 31 and 32 and a unique BamHl directly downstream of the TAG stop codon. This generates pUC/HML.

9.2 Introduction of mutations ISA, 105H, 12SA, 129E

[00222] A 3.2 kb Sstl fragment containing the promoter regions, the xln2 gene, and part of the cbh2 terminator was isolated from pc/xHML-EC (Example 8) and cloned into the Sstl site in the polylinker of the mutagenesis vector, pALTER®-l (Promega). Four sequential rounds of mutagenesis were performed to alter specific amino acids using primers specifically designed to incorporate the desired mutations:

S75A: AGCTACCTCG CCGTGTACGG (SEQ ID NO: 83)

L105H: CCACCAAGCA CGGCGAGGT (SEQ ID NO.84)

S125A: ACGCAGCGCG TCAACGCCCC GTCCATCATC GGC (SEQ ID NO:85)

I129E: AACGCCCCGT CCATCGAGGG CACCGCCACC TTT (SEQ ID NO:86)

(see WO 01/92487 and WO 03/046169; which are incorporated herein by reference, for associated methods); this generated the plasmid pALT-TrX-10H-27M-29L-75A-105H-125A- 129E. The incorporation of all mutations was verified by DNA sequence analysis.

9.3 Introduction of mutations K58R, S99C, Y118C

[00223] One round of mutagenesis was performed on the plasmid pALT-TrX-10H-27M-29L- 75A-105H-125A-129E using the Promega Altered Sites® II in vitro Mutagenesis System System and the primer sequences:

K58R: GGC ACC AAG AAC CGC TAA GAC TAC CTA (SEQ ID NO:87)

S99C: ACC TAC AAC CCG TGC ACG GGC GCC ACC (SEQ IDNO:88)

Yl 18C: C TAC GAC ATT TGC CGC ACG C (SEQ ID NO:89)

to introduce the K58R, S99C, and Y118C mutations and generate pALT-TrX-10H-27M-29L- 58R-75A-99C-105H-118C-125A-129E. The incorporation of all mutations was verified by DNA sequence analysis.

9.4 Introduction of mutations NIlD, S40R

[00224] One round of mutagenesis was performed on the plasmid pALT-TrX-10H-27M-29L- 58R-75A-99C-105H-118C-125A-129E using the Promega Altered Sites ® II in vitro Mutagenesis System and the primer sequences:

Nl ID: GGT TAC CAC GAC GGT TAC T (SEQ ID NO:90)

S40R: TCC GTC AAC TGG CGC AAC TCG GGC AAC (SEQ ID NO:91)

to introduce the NI lD and S40R mutations and generate pALT-TrX-10H-HD-27M-29L-40R- 58R-75A-99C-105H-118C-125A-129E. The incorporation of all mutations was verified by DNA sequence analysis.

9.5 Introduction of mutation Tl 3 IN

[00225] One round of mutagenesis was performed on the plasmid pALT-TrX-10H-HD-27M- 29L-40R-58R-75A-99C-105H-118C-125A-129E using the Promega Altered Sites® II in vitro Mutagenesis System and the primer sequence:

Tl31N: CCG TCC ATC GAG GGC AAC GCC ACC TTT TAC (SEQ ID NO:92)

to introduce the Tl 3 IN mutations and generate ρALT-TRX-10H-HD-27M-29L-40R-58R-75A- 99C-105H-118C-125A-129E-131N. The incorporation of the mutation was verified by DNA sequence analysis

EXAMPLE 10: Construction of a vector directing the expression of TrX-10H-llD-27M- 29L-40R-58R-75A-99C-105H-118C-125A-129E-131N in Trichoderma reesei.

[00226] The 3640 bp Sad fragment containing the promoter regions, the modified xln2 gene and part of the cbh2 terminator from pALT-TRX-lOH-11D-27M-29L-40R-58R-75A-99C-105H-

118C- 125 A-129E- 13 IN was cloned into the Sad site of a plasmid containing the remaining cbh2 terminator sequence in pSP72. This step generates the expression cassette containing plasmid pV _x TRX-lOH-l lD^M^L^OR-SδR^SA^C-lOSH-l lδC-nSA- E-mN-PSP. The selection cassette containing plasmid, pNCBglNSNB(r), was derived from a N. crassa pyr4 containing plasmid, pFB6 (Radford et al, 1985). A 3.2 kb BgIW fragment from pFB6 containing the N. crassa pyr4 gene (GenBank accession Ml 3448) as well as its promoter, terminator and some 5' UTR sequences was cloned into the BamHl site of pUC119 modified to contain Notl, Smal, Nhel and BgIW sites in the polylinker (between EcoRI and Sad) to generate pNCBgl- NSNB(r). A 2238bp Kpnl fragment containing the entire N. crassa pyr4 coding region, promoter and terminator sequences was isolated from pNCBgl-NSNB(r) and cloned into the unique Kpnl site of the expression cassette-containing plasmid to generate p7 _x TRX-10H-HD- 27M-29L-40R-58R-75 A-99C- 105H- 118C- 125 A- 129E- 13 IN-TV.

EXAMPLE 11: Transformation of the Tήchoderma ra?se/ M2C38.

[00227] 5 x 10 ⁶ spores of M2C38aux5 were plated onto sterile cellophane on Potato Dextrose agar supplemented with 5 mM undine and are incubated for 20 hours at 30°C to facilitate spore germination and mycelial growth. Cellophane discs with mycelia were transferred to 10 mL of a protoplasting solution containing 7.5 g/L Driselase and 125 units of protease free β-glucanase (InterSpex Products Inc., Cat. Nos. 0465-1 and 0410-3, respectively) in 50 mM potassium phosphate buffer, pH 6.5 containing 0.6 M ammonium sulfate (Buffer P). The mycelial mat was digested for 5 hours with shaking at 60 rpm. Protoplasts were separated from undigested mycelia by filtration through sterile No. 30 MIRACLOTH™ and collected into a sterile 50 mL round-bottom centrifuge tube and recovered by centrifugation at 1000-1500 x g for 10 min at room temperature. Protoplasts were washed with 5 mL of Buffer P and centrifuged again at 1000-1500 x g for 10 min at room temperature. Protoplasts were resuspended in 1 mL of STC buffer (1.2 M sorbitol, 10 mM CaCl ₂ , 10 mM Tris-HCL, pH 7.5). For transformation, 0.1 mL of resuspended protoplasts were combined with 10 μg of vector DNA and 25 μL of PEG solution (25% PEG 4000, 50 mM CaCl ₂ , 10 mM Tris-HCl, pH 7.5). After incubation in an ice water bath for 30 min, 1 mL of PEG solution was added and the mixture incubated for 5 min at room temperature. Transformation mix was diluted with 2 mL of 1.2 M sorbitol in PEG solution and the entire mix was added to 25 mL of molten MMSS agar media (see below) cooled to about

47°C and the protoplast suspension poured over MMSS agar. Plates were incubated at 3O ⁰ C until colony growth was visible. Transformants were transferred to individual plates containing MM agar and allowed to sporulate. Spores were collected and plated at high dilution on MM agar to isolate homokaryon transformants, which were then plated onto PDA to allow for growth and sufficient sporulation to inoculate the screening cultures as described in Example 12 below.

[00228] Minimal medium (MM) agar contains the components set forth in Table 21.

TABLE 21

Reagent Per L

KH ₂ PO ₄ 10 g

(NH ₄ ) ₂ SO ₄ 6 g

Na ₃ Citrate-2H ₂ O 3 g

FeSO ₄ 7H ₂ O 5 mg

MnSO ₄ H ₂ O 1.6 mg

ZnSO ₄ -7H ₂ O 1.4 mg

CaCl ₂ -2H ₂ O 2 mg

Agar 2O g

20% Glucose f.s. 5O mL

1 M MgSO ₄ -7H ₂ O f.s. 4 mL pH to 5.5

[00229] MMSS agar contains the same components as MM agar plus 1.2 M sorbitol, 1 g/L YNB (Yeast Nitrogen Base w/o Amino Acids from DIFCO Cat. No.291940) and 0.12 g/L amino acids (-Ura DO Supplement from BD Biosciences Cat. No. 630416).

EXAMPLE 12: Detection of thermophilic xylanase activity in T. reesei culture filtrates

[00230] The presence of thermophilic xylanase activity in culture filtrates of T. reesei transformants was determined by measuring the release of reducing sugars from a soluble wheat arabinoxylan substrate at 65°C. Specifically, 30 μL of an appropriate dilution of culture filtrate was pre-incubated at 65°C for 5 min. Subsequently, 300 μL of a solution of 1.5% wheat arabinoxylan (Megazyme International) redissolved in pH 7.0 phosphate buffer containing 0.04% Tween, also pre-incubated at 65°C for 5 min, was added to the enzyme sample in a

microcentrifuge tube. The tubes were vortexed briefly to facilitate mixing and then the reaction was incubated at 65 °C for 20 min. The enzymatic hydrolysis reaction was stopped by the addition of 150 μL of the stopping solution containing 43.64 mM 2-hydroxy-3,5-dinitrobenzoic acid, 0.93 M sodium potassium tartrate, 0.4 M sodium hydroxide and 0.4 M potassium hydroxide. The resulting solution was then boiled for 10 minutes to facilitate reaction of the 2- hydroxy-3,5-dinitrobenzoic acid with the reducing sugars released from the arabinoxylan substrate by the enzyme. The tubes were cooled on ice for 5 minutes and then 1.5 mL of deionized water was added. The absorbance of the solution was measured at 530 nm. The amount of reducing sugar released by the thermophilic xylanases during the incubation was calculated from a standard curve of A530 measurements of several dilutions of a pure xylose solution reacted with the same stopping solution.

EXAMPLE 13: Production of modified xylanases in liquid cultures

[00231] Individual colonies of Tήchoderma were transferred to PDA plates for the propagation of each culture. Sporulation was necessary for the uniform inoculation micro-cultures which were used in testing the ability of the culture to produce the thermophilic xylanases and cellulase. The culture media is composed of the following:

TABLE 22

Component g/L

(NH ₄ ) ₂ SO ₄ 12.7

KH ₂ PO ₄ 8.00

MgSO ₄ VH ₂ O 4.00

CaCl ₂ -2H ₂ O 1.02

Corn Steeped Liquor 5.00

CaCO ₃ 20.00

Carbon source** 30-35

Trace elements* 2 mL/L

^♦ Trace elements solution contains 5 g/L FeSO ₄ -7H ₂ 0; 1.6 g/L MnSO ₄ H ₂ O; 1.4 g/L ZnSO ₄ -7H ₂ 0.

** glucose, Solka floe, lactose, cellobiose, sophorose, corn syrup, or Avicel. The carbon source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the remaining media initially or through the course of the fermentation.

[00232] Individual transformants were grown in the above media in 1 mL cultures in 24-well micro-plates. The initial pH was 5.5 and the media sterilized by steam autoclave for 30 minutes at 121 ⁰ C prior to inoculation. For both native and transformed cells, spores were isolated from the PDA plates, suspended in water and 10 ⁴ -10 ⁶ spores per mL were used to inoculate each culture. The cultures were shaken at 500 rpm at a temperature of 3O ⁰ C for a period of 6 days. The biomass was separated from the filtrate containing the secreted protein by centrifugation at 12,000 rpm. The protein concentration was determined using the Bio-Rad Protein Assay (Cat. No. 500-0001). Xylanase activity was determined as described in Example 12. Strains expressing the highest xylanase activity and exhibiting high overall protein production were selected for growth in 30-litre pilot fermentations.

EXAMPLE 14: Production of xylanases in 30 L fed-batch fermentations.

[00233] T. reesei strains were grown on Potato Dextrose Agar at 28-30 ⁰ C until a confluent lawn of spores was obtained. Spores were collected and used to inoculate 750 mL of Berkeley media (10 g/L glucose, 1.4 g/L (NH ₄ ) ₂ SO ₄ , 2.0 g/L KH ₂ PO ₄ , 0.31 g/L MgSO ₄ -7H ₂ 0, 0.53 g/L CaCl ₂ ; 5.1 g/L dry corn steep, 5 mg/L FeSO ₄ -7H ₂ O; 0.8 mg/L MnSO ₄ -H ₂ O, 0.7 mg/L ZnSO ₄ -7H ₂ O) in a 2 L baffled flask. After 3 days of growth at 28 ⁰ C and 150 rpm, this culture was used to inoculate 23 L of fermentation medium with the following initial composition: 31 g/L glucose, 4.4 g/L (NH ₄ ) ₂ SO ₄ , 2.77 g/L KH ₂ PO ₄ , 1.4 g/L MgSO ₄ -7H ₂ 0, 0.37 g/L CaCl ₂ , 12 g/L dry corn steep, 3.5 mg/L FeSO ₄ -7H ₂ O, 1.12 mg/L MnSO ₄ H ₂ O, 0.98 g/L ZnSO ₄ -7H ₂ O. A fed-batch aerobic fermentation using one or more of the inducing carbohydrate sources listed in Example 13 was run for 6 days at pH 4.5 and 28 ⁰ C in a 30 L New Brunswick Microferm fermentor. After 6 days, the culture was filtered over Harborlite and the culture filtrate adjusted to pH 4.5 and preserved with 0.5% benzoate to prevent microbial growth.

[00234] The protein concentration in daily fermentor samples was determined using the Bio- Rad Protein Assay (Cat. No. 500-0001). Xylanase activity was determined as described in Example 12.

[00235] The expression of TrX-lOH-l lD^M^L^OR-SδR^SA^C-lOSH-l lSC-^SA- transformed T. reesei strains (biomass and xylanase activity) in 30 L fermentations is presented in Table 23 below.

TABLE 23

Strain Enzyme Protein Xylanase (mg/mL) Activity (XU/g)

P345A TrX-10H-l lD-27M-29L-40R-58R- 63-88 12021

75A-99C-105H-118C-125A-129E-

131N

P275H TrX-I OH-1 1D-27M-29L-40R-58R- 69 7145

75 A-99C- 105H- 118C- 125A- 129E

EXAMPLE 15: λlkalophilicitv and thermophilicity of the modified xylanases TrX-IOH- 11D-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E-131N and TrX-10H-llD-27M- 29L-40R-58R-75A-99C-105H-118C-125A-129E

[00236] The pH and temperature profiles of the modified xylanases TrX- 1 OH- 11D-27M-29L- 40R-58R-75A-99C-105H- 1 18C- 125A-129E- 131 N and TrX- 1 OH- 11 D-27M-29L-40R-58R-75A- 99C-105H-118C-125A-129E made by Trichoderma strains P345A and P275H are shown in Figures 14, 15, and 16. The data was generated by measuring reducing sugar release from wheat arabinoxylan, hardwood pulp, or softwood pulp with variable conditions.

[00237] Figure 14 shows that the temperature optimum of TrX-10H-l lD-27M-29L-40R-58R- 75A-99C-105H-118C-125A-129E-131N is slightly higher than the optimum of TrX- 1 OH- 11 D- 27M-29L-40R-58R-75A-99C-105H-118C-125A-129E. Temperature profiles for each enzyme were generated on a 1 % wheat arabinoxylan (Megazyme International) substrate at pH 7 for 60 minutes.

[00238] Figures 15 and 16 show that the thermophilic/alkalophilic enzyme TrX- 1 OH- 11D-27M- 29L-40R-58R-75A-99C-105H-118C-125A-129E-131N from P345A has a slightly broader pH optimum range on both hardwood and softwood pulp than TrX- 1 OH- 11D-27M-29L-40R-58R- 75A-99C-105H-1 18C-125A-129E from strain P275H. The pH profiles were generated at 70 ⁰ C

for 60 minutes on 10% consistency pulp. Enzyme TrX-I OH-11D-27M-29L-40R-58R-75A-99C- 105H- 118C- 125A- 129E- 13 IN was added at a dose of 400 mL/t of pulp and enzyme TrX-IOH- 11D-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E was added at a dose of 800 mL/t of pulp.

EXAMPLE 16: Thermostability testing of the modified xylanases TrX-10H-llD-27M-29L- 40R-58R-75A-99C-105H-118C-125A-129E-131N, TrX-10H-llD-27M-29L-40R-58R-75A- 99C-105H-118C-125A-129E and TrX-10H-27M-29L

[00239] The tolerance of modified xylanases TrX-10H-l lD-27M-29L-40R-58R-75A-99C- 105H-118C-125A-129E-131N, TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A- 129E and TrX-10H-27M-29L to incubation at different temperatures in the absence of substrate was investigated. The modified xylanases were diluted 10- to 50- fold in 200 mM bis-tris propane buffer at pH 8.0 and incubated for 30 min at 50°C, 6O ⁰ C, 70 ⁰ C and 80 ⁰ C. At the end of the incubation period, the residual enzyme activity was determined as described in Example 12 with the following exception: an aliquot of the treated enzyme solution was added at a 100-fold dilution to a 1% birchwood xylan solution in 200 mM bis-tris propane buffer at pH 7.0 that had been pre-incubated to 7O ⁰ C (for TrX-I OH-11D-27M-29L-40R-58R-75A-99C-105H-118C-125A- 129E-131N and TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-l 18C-125A-129E) or pH 6.5 pre-incubated to 55°C. The residual activity was normalized to the activity measured for each enzyme after 0 min pre-incubation at 50 ⁰ C.

[00240] Both TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E-131N and TrX-10H-l lD-27M-29L-40R-58R-75A-99C-105H-118C-125A-129E containing the 99C-118C disulfide show superior thermostability to TrX-HML, which lacks the 99C-118C disulfide. The T ₅₀ was determined to be 71-72 ⁰ C for the disulfide xylanases, as compared to a T ₅₀ of 65 ⁰ C for the TrX-10H-27M-29L (Figure 17).

[00241] The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described herein.

[00242] All references and citations are herein incorporated by reference.

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