METHOD TO IMPROVE THE STABILITY AND BROADEN THE PH RANGE OF FAMILY G/11 XYLANASES

Abstract: The present invention relates to protein engineering, and concerns especially family G/11 xylanases, and genes encoding said enzymes. In specific, the invention concerns <i>Trichoderma reesei XYNII</i> gene, which codes for endo-1,4- beta -xylanase (EC 3.2.1.8). The invention describes how site-directed mutagenesis can be used to improve the properties of an enzyme to match the industrial conditions where it is used. Protein engineering can be used to improve thermoactivity and thermostability of xylanases, as well as to broaden their pH range.

Description: Method to improve the stability and broaden the pH range of family G/l l xylanases Field of the invention This invention relates to protein engineering, and concerns especially family G/1 1 xylanases, and genes encoding said enzymes. In specific, the invention concerns Trichoderma reesei XYNII gene, which codes for endo-l,4-β-xylanase (EC 3.2.1.8). The invention describes how site-directed mutagenesis can be used to improve the properties of an enzyme to match the industrial conditions where it is used. Protein engineering can be used to improve thermoactivity and thermostability of xylanases, as well as to broaden their pH range. Background of the invention Xylanases are glycosyl hydrolases which hydrolyse β-l,4-linked xylopyranoside chains. Xylanases have been found in at least a hundred different organisms. Together with other glycosyl hydrolases they form a superfamily which includes more than 40 different enzyme families (Henrissat and Bairoch, 1993). Family 11 (previously G) xylanases are defined by the similarities in their gene sequences, protein structures, and catalytic mechanisms. Common features for the members of this family are high genetic homology, a size of about 20 kDa, and a double displacement catalytic mechanism (Tenkanen et α/., 1992; Wakarchuk et al., 1994). The family 1 1 xylanases mainly consist of β-strands which form two large β-sheets. and of one α-helix. These form a structure that resembles a partly-closed right hand, wherein the β-sheets are called A- and B-sheet. (Tόrrόnen & Rouvinen, 1997). The family 1 1 xylanases have special interest in industrial applications, because their structure is stable, and they are not susceptible to protease activity. In addition, xylanases can be produced economically on an industrial scale. Trichoderma reesei is known to produce three different xylanases of which xylanases I and II (Xynl and Xynll) are the best characterized (Tenkanen et ai, 1992). Xynl has a size of 19 kDa, and compared to Xynll it has low isoelectric point and pH optimum (pi 5.5, pH 3-4). Xynll has a size of 20 kDa and it has a pi of 9.0 and a pH optimum of 5.0-5.5 (Tόrrόnen and Rouvinen, 1995). The most important industrial applications of xylanases are pulp bleaching, modification of textile fibres, and biomass modification to improve its digestion in animal feeding (Prade, 1996). A common nominator in all these applications is the extreme conditions which face the enzyme High temperatures, and pH which substantially differs from the optimal pH of many xylanases decrease the effective utility of the presently available xylanases in industrial applications In feed applications the enzyme faces high temperature conditions for a short time (e g 2 - 5 mm at 90 °C) duπng feed preparation However, the actual catalytic activity of the enzyme is needed at lower temperatures (e g -37 °C) Consequently, the enzyme should not be inactivated irreversibly at high temperatures, while it has to be active at relatively low temperatures In pulp bleaching the material coming from alkaline wash has a high temperature (> 80 °C) and pH (> 10) None of the commercially available xylanases survives these conditions The pulp must be cooled and the alkaline pH neutralized in order to treat the pulp with presently available xylanases This means increased costs Protein engineering has been used - sometimes successively - to stabilise xylanases to resist the denaturing effect of the high temperature and pH Several thermostable, alkaliphihc and acidophi c xylanases have been found and cloned from thermophi c organisms (Bodie et al , 1995, Fukunaga et al , 1998) However, production of economical quantities of these enzymes has in most cases proved to be difficult Therefore the T reesei xylanase II, which is not as such thermostable, is in industπal use because it can be produced at low cost in large quantities As an alternative for isolating new xylanases, or developing production processes, one can envisage engineering the presently used xylanases to be more stable in extreme conditions The stability of Bacillus circulans xylanase has been improved by disulfide bridges, by binding the N-terminus of the protein to the C-terminus and the N-terminal part of the α- he x to the neighbouring β-strand (Wakarchuk et al , 1994) Also Campbell et al (1995) have modified Bacillus circulans xylanase by inter- and intramolecular disulfide bonds in order to increase thermo stability On the other hand, the stability of T reesei xylanase II has been improved by changing the N-terminal region to a respective part of a thermophihc xylanase (Sung et al , 1998) In addition to the improved thermostabihty, the activity range of the enzyme was broadened in alkaline pH Single point mutations have also been used to increase the stability of Bacillus pumilus xylanase (Arase et al , 1993) The influence of mutagenesis on stability has been studied on many other enzymes By compaπng the structures of thermophihc and mesophi c enzymes plenty of information has been obtained (Vogt et al , 1997) Structural information of thermophihc xylanases has also given information about factors influencing the thermostability of xylanases (Gruber et al , 1998, Harris et al , 1997) Summary of the invention The present invention relates to xylanases which belong to the family 1 1 (previously G) glycosyl hydrolases The invention provides xylanases modified so as to change their thermostability, thermoactivity, and/or broaden their pH range Vaπous modifications in the Trichoderma reesei xylanase structure, either alone or in combinations, result in the changes descπbed in this invention (1) the stability of the enzyme is increased by binding of the N-terminal region bv disulfide bridges (for example, the bridges formed by the mutation pairs T2C and T28C P5C and N19C, T7C and S16C, N10C and N29C) to the body of the protein, (2) the C-terminus is stabilised by extension with additional aspartic acid (+19 ID) which forms a salt bridge with argimne 58 (lysine 58 in the wild-type enzyme has been changed to argimne (K58R)), (3) the stability of the enzyme is increased by binding the α-hehx by a disulfide bridge to the body of the enzyme (e g L105C and Q162C), (4) point mutations have been made at different positions to improve the stability of xylanase (N11D, T26R, G30H, N67R, N97R, A132R, N157R, A160R, T165N, M169H, S186R) In specific, the present invention provides a modified Trichoderma reesei xvlanase in vvhich the ammo acids T2 and T28 have been changed to cysteines, K58 has been changed to argimne, and to the C-terminus of the enzyme an aspartic acid has been added (+19 ID), thereby forming a disulfide bridge between the amino acids T2C and T28C, and a salt bridge between the amino acids K58R and +191D Brief description of drawings Figure 1. A set of o gonucleotides used in the mutagenesis of xylanase (codon changes underlined) The sequences are also given in the appended Sequence Listing as sequences 1 to 12 Figure 2. A graph presenting the effect of the mutations T2C, T28C, K58R, and +191D on the thermal optimum of T reesei Xynll ( T = wild-type enzyme, Y5 = the mutated T reesei Xynll) Figure 3. A graph presenting the effect of the mutations T2C, T28C, K58R, and +191D on the pH-dependent activity of T. reesei Xynll ( WT and Y5 as in Figure 2) Figure 4. A graph presenting the effect of the mutations T2C, T28C, K58R, and +19 ID on the inactivation of T. reesei Xynll at different temperatures (WT and Y5 as in Figure 2) Figure 5. A graph presenting the effect of the mutations Q162C and L105C on the inactivation of T. reesei Xynll at different temperatures (W t. = wild-type enzyme) Detailed description of the invention The family G/1 1 xylanases originating from bacteria, yeast and fungi have common molecular structure. Examples of such xylanases are Aspergillus mger XynA Aspergύlus kawachn XynC Aspergillus tubigensts XynA Bacillus circulans XynA Bacillus pumilus XynA Bacillus subtihs XynA Neocallimastix patrwiarum XynA Streptomyces hvidans XynB Streptomyces hvidans XynC Streptomyces thermoviolaceus Xynll Thermomonospora fusca XynA Trichoderma harzianum Xyn Trichoderma reesei Xynl, Trichoderma reesei Xynll Trichoderma viride Xyn The invention deals with xylanases of the family G/1 1 with the following common features. (i) Enzymes in which the N-terminal sequence is a part of the double-layered β-sheet (in the family 1 1 xylanases the A- and the B-sheet, (Gruber, et al , 1998)) and in which the first β-strand (in T. reesei Xynll the amino acids 5-10) or the N-terminal end can be bound by disulfide bridges either to the adjacent β-strands (in T reesei Xynll the amino acids 13-19) or to other neighbouring regions (π) Enzymes in which the C-terminal peptide chain forms a β-strand (in T reesei Xynll amino acids 183-190), which is a part of a larger β-sheet and in which the C-terminal region can be bound by disulfide bridges to the adjacent β-strands or by salt bridges to the body of the enzyme (in) Enzymes which have an α-hehx on the other side of the enzyme structure with regard to the catalytic canyon, and wherein said α-hehx or the neighbouring regions can be bound more tightly by a disulfide bridge to the body of the protein The T reesei xylanase II has the above mentioned properties and in said enzyme thermostability, pH-stabi ty and thermo activity can be modified based on these properties The following changes have been made to the xylanase gene (XYNII) of T reesei 1 By site-directed mutagenesis disulfide bridges are formed in the N-terminal region * Threonines 2 and 28 are changed to cysteines resulting in a disulfide bridge being formed between them (T2C and T28C) * Prohne 5 and asparagme 19 are changed to cysteines resulting in a disulfide bridge being formed between them (P5C and N19C) * Threomne 7 and seπne 16 are changed to cysteines resulting in a disulfide bridge being formed between them (T7C and S 16C) * Asparagme 10 and asparagme 29 are changed to cysteines resulting in a disulfide bridge being formed between them (N10C and N29C) 2 By site-directed mutagenesis, the C-terminus is bound more tightly to the body of the enzyme by adding as a recombinant change one amino acid (e g aspartic acid or glutamic acid) to the C-terminus of the xylanase, which then forms a salt bridge from the C-terminus to the body of the enzyme If appropriate, a suitable amino acid replacement can be made in the body of the protein, so as to enable the formation of a salt bridge * An aspartic acid (+191D) is added to the C-terminal seπne (SI 90) This results in a salt bridge with argimne at position 58, where wild-type lysine has been replaced by argimne (K58R) 3 By site-directed mutagenesis at least one disulfide bridge is formed to stabilise the enzyme in the C-termmal part via the -hehx or a region near the α-hehx * Leucine 105 and glutamine 162 are changed to cysteines resulting in disulfide bridge between them (L105C and Q162C) 4 By site-directed mutagenesis point mutations are made to increase the stability of T reesei xylanase II N11D, T26R, G30H, N67R, N97R, A132R, N157R, A160R, T165N M169H, S186R Methods of the invention Production of mutated and recombinant XYNII genes were carried out by the following general procedures 1. Expression vector and production of the enzyme T reesei xylanase II was produced in E co strains XLl-Blue or Rv308 using the vector pKKtac (VTT, Espoo, Finland) or the vector pALK143 (ROAL, Rajamaki, Finland) T reesei XYNII gene was directly cloned by PCR from the cDNA of T reesei to the vector pKKtac (induction of expression by IPTG) Alternatively, the plasmid pALK143 was used which contains T reesei XYNII gene Both of the vectors secrete the xylanase into the medium, the vector pKKtac by pectate lyase (pelB) signal sequence and the vector pALK143 by amylase signal sequence 2. Site-directed mutagenesis and production of recombinant XYNII gene The production of mutated T reesei XYNII gene used in the Examples of this application, was effected as follows Mutations were produced by polymerase chain reaction (PCR) using ohgonucleotide primers which contained the sequences for the changed codons Examples of the used ohgonucleotides are given in Figure 1 , as well as in the appended Sequence Listing as sequences 1 to 12 PCR using the primers (containing the desired mutation) was carried out by Quick Change method (Stratagene, Westburg, Leusden, The Netherlands) and by generally known methods PfuTurbo was used as DNA polymerase (Stratagene, La Jolla, Ca, USA) The cloned E co strains were cultivated on plates containing xylan (birchwood xylan Sigma, Steinheim, Germany) coupled to Rhemazol Brilliant Blue The xylanase activity could be seen as halos around the colonies (Biely et al , 1985) 3. Determination of the activity of xylanases The xylanase activity of enzyme samples was determined by measuring the amount of reducing sugars released from the hydrolysed xylan fibre The reducing sugars were measured by DNS-method in 50 mM citrate-phosphate buffer (Bailey et al , 1992) Standard activity determination was carried out at pH 5 and 50 °C 4. Determination of the stability of the enzymes The stability of the xylanases was tested by measuring the half-life of the modified enzymes at different temperatures The enzyme was incubated for varying times at 55 or 65 °C and the residual activity was measured as described above The stability at high temperatures was also measured by incubating the enzymes for 10 mm at varying temperatures and subsequently measuring the residual activity by DNS-method The pH- dependent xylanase activity was measured by determining the enzyme activity in varying pH-values The temperature optimum of the enzyme was determined by measuring the activity at varying temperatures (10 min, pH 5) The properties of the mutated enzymes were compared to the wild-type T reesei Xynll enzyme Examples of mutations Example 1. The three-fold mutations (T2C, T28C and K58R) and the recombinant change (+191D) were made in T. reesei Xynll by using the methods described above The mutant enzyme was designated as Y5 Said mutant enzyme was expressed in E coh, which was cultivated at +37 °C in shake flasks using Luna Broth as growth medium After cultivation the cells were removed by centπfugation and the xylanase secreted into the medium was characterized in varying conditions, as described above Figure 2 shows the effect of the temperature to the enzyme activity when the mutant Y5 (T2C, T28C. K58R, +19 ID) and the wild-type (T. reesei Xynll) enzyme were incubated for 10 min with birchwood xylan in varying temperatures, and the relative amount of the reducing sugars as released were measured with DNS-method Said mutations improved the temperature optimum of xylanase by about 15 °C Example 2. The three-fold mutant xylanase (T2C, T28C, K58R, +191D) described in Example 1 was incubated for 10 min in 1% birchwood xylan at 50 °C in citrate-phosphate buffer in varying pH-values Figure 3 shows the relative amount of reducing sugars as released for the mutant and the wild-type xylanases The mutations broadened slightly the pH- dependent activity of the enzyme to alkaline direction The mutant enzyme was more active than the wild-type enzyme at pH 7 - 8, the activity of mutant enzyme was about 20% higher at pH 8 (50 °C) Example 3. The above-mentioned three-fold mutant (T2C, T28C, K58R +191D) and the wild-type enzyme were incubated for 10 min at varying temperatures After the incubation the samples were cooled and the residual activity was determined in standard conditions The wild-type enzyme was completely inactivated already at 55-60 °C The mutant enzyme retained about 50% of its activity even at 65 °C (Figure 4) Table 1 below shows the half-lives (Tl/2) of the mutant (Y5) and the wild-type xylanase at 55 °C and 65 °C Table 1. Example 4. With the above-mentioned methods a disulfide bridge was made (L105C and Q162C) to bind the C-terminus of the α-hehx to the neighbouring β-strand The enzyme was produced in E coh and its properties were determined Figure 5 shows the inactivation of the mutant enzyme at different temperatures compared to the wild-type enzyme At 55 °C the stability of the mutated enzyme increased about 20-fold, with regard to the wild- type enzyme, whereby the half-life increased from 5 min (the wild-type enzyme) up to about 1,5 hours (the mutated enzyme) Literature Arase, A., Yomo, T., Urabe, I., Hata, Y., Katsube, Y. & Okada, H. (1993). Stabilization of xylanase by random mutagenesis FEBS Letters 316, 123-7 Bailey, J. M., Biely, P. & Poutanen, K. (1992). Interlaboratory testing of methods for assay of xylanase activity J. Biotech. 23, 257-270 Biely, P., Mislovicova, D. & Toman, R. (1985). Soluble chromogenic substrates for the assay of endo-l,4-beta-xylanases and endo-l,4-beta-glucanases Analytical Biochemistry 144, 142-6 Bodie, E., Cuevas, W. A. & Koljonen, M. (1995). In United States Patent 5,437,992 Campbell, R.L., Rose, D.R., Sung, W.L., Yaguchi, M. & Wakarchuck, W. (1995). In United States Patent 5, 405, 769. Fukunaga, N., Iwasaki, Y., Kono. S., Kita, Y. & Izumi, Y. (1998). In United Stated Patent 5, 736,384 Gruber, K., Klintschar, G., Hayn, M., Schlacher, A., Steiner. W. & Kratky, C. (1998). Thermophihc xylanase from Thermomyces lanuginosus High-resolution X-ray structure and modeling studies Biochemistry 37, 13475-13485 Harris, G. W., Pickersgill, R. W., Connerton, I., Debeire, P., Touzel, J. P., Breton, C. & Perez, S. (1997). Structural basis of the properties of an industrially relevant thermophihc xylanase. Proteins 29, 77-86 Henrissat, B. & Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities BiochemicalJournal 293, 781-8 Prade, R. A. (1996). Xylanases from biology to biotechnology Biotechnology ά Genetic Engineering Reviews 13, 101-31 Sung, W. L., Yaguchi, M., Ishikawa, K., Huang, F., Wood, M. & Zahab, D. M. (1998). In United States Patent 5, 759,840 Tenkanen, M., Puls, J. & Poutanen, K. (1992). Two major Xylanases of Trichoderma reesei Enzyme Microb. Technol. 14, 566-574 Tδrrδnen, A. & Rouvinen, J. (1995). Structural comparison of two major endo-1,4- xylanases from Trichoderma reesei Biochemistry 34, 847-56 Tδrrδnen, A. & Rouvinen, J. (1997). Structural and functional properties of low molecular weight endo-l,4-beta-xylanases Journal of Biotechnology 57 ' , 137-49 Wakarchuk, W. W., Sung, W. L., Campbell, R. L., Cunningham, A., Watson, D. C. & Yaguchi, M. (1994). Thermostabilization of the Bacillus circulans xyla • ise by the introduction of disulfide bonds Protein Engineering 7 , 1379-86 Vogt, G., Woell, S. & Argos, P. (1997). Protein thermal stability, hydrogen bonds, and ion pairs. Journal of Molecular Biology 269, 63 1 -43.

Claims: Claims 1 A modified xylanase enzyme of family G/l l, having increased thermostability or pH- stability with respect to the corresponding wild-type xylanase, the wild-type enzyme having been modified by - binding the N-terminal region of the enzyme by disulfide bridges to the body of the protein, and/or - binding the C-terminal region of the enzyme by salt bridges to the body of the protein, or - binding the α-helix of the enzyme by disulfide bridges to the body of the protein, or - making single amino acid mutations in the protein 2 The modified xylanase according to claim 1, wherein the xylanase is a Trichoderma reesei xylanase. 3 The modified xylanase according to claim 2, wherein the xylanase is the Trichoderma reesei xylanase II (Xynll) 4 The modified xylanase according to any one of the claims 1 to 3, wherein binding the C-terminal region of the enzyme to the body of the protein has been effected by adding aspartic acid or glutamic acid to the C-terminus so that a salt bridge is formed between said added amino acid and a suitable amino acid in the body of the protein 5 The modified xylanase according to any one of the claims 1 to 4, wherein binding the N-terminal region of the enzyme to the body of the protein has been effected by forming a disulfide bridge between the amino acids T2C and T28C of the Trichoderma reesei xylanase II (Xynll) or between the corresponding amino acids of other xylanases of the family G/1 1 6 The modified xylanase according to claim 1, wherein in T. reesei xylanase II (Xynll) amino acids T2 and T28 have been changed to cysteines, K58 has been changed to arginine, and to the C-terminus of the enzyme an aspartic acid has been added (+19 ID), thereby forming a disulfide bridge between the amino acids T2C and T28C and a salt bridge between the amino acids K58R and +191D 7 The modified xylanase according to any one of the claims 1 to 3, wherein binding the C-terminal region of the enzyme by disulfide bridges to the body of the protein has been effected by binding the C-terminal part of the α-helix to the adjacent β-strand by a disulfide bridge between the amino acids L105C and Q162C of Trichoderma reesei xylanase II (Xynll) or between the corresponding amino acids of other xylanases of the family G/l l 8 A method for improving the thermostability and/or broadening the pH-range of family G/1 1 xylanases, comprising carrying out the steps of: - binding the N-terminal region of the enzyme by disulfide bridges to the body of the protein, and/or - binding the C-terminal region of the enzyme by salt bridges to the body of the protein, or - binding the α-helix of the enzyme by disulfide bridges to the body of the protein, or - making single amino acid mutations in the protein. 9 The method according to claim 8, wherein aspartic acid or glutamic acid is added to the C-terminus so that a salt bridge is formed between said added amino acid and a suitable amino acid in the body of the protein. 10 The method according to claim 9, wherein in T. reesei xylanase (Xynll) the additional aspartic acid or glutamic acid forms a salt bridge with the amino acid 58 which has been changed to lysine or arginine 1 1 The method according to claim 8, wherein in T. reesei xylanase (Xynll) amino acids T2 and T28 are changed to cysteines, K58 is changed to arginine, and to the C-terminus of the enzyme an aspartic acid is added (+191D), thereby forming a disulfide bridge between the amino acids T2C and T28C and a salt bridge between the amino acids K58R and +191D. 12. The method according to claim 8, wherein single amino acid mutations are made 13. The method according to claim 12, wherein in T. reesei Xynll the modification N11D is made.

Inventors: FENEL FRED, LEISOLA MATTI, TURUNEN OSSI, FENEL, FRED, LEISOLA, MATTI, TURUNEN, OSSI
Applicants: CARBOZYME OY, FENEL FRED, LEISOLA MATTI, TURUNEN OSSI, FENEL, FRED, LEISOLA, MATTI, TURUNEN, OSSI
Application year: 2000
Main classification: C12N9
IPC codes: C12N9, C12N15