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  Trichoderma reesei  XYNII gene, which codes for endo-1,4-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.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a Continuation of U.S. patent application Ser. No. 13/849,465, filed Mar. 22, 2013, which is a divisional of U.S. patent application Ser. No. 11/963,285, filed Dec. 21, 2007 (now U.S. Pat. No. 8,426,181), which is a Continuation of U.S. patent application Ser. No. 10/912,272, filed Aug. 5, 2004 (now U.S. Pat. No. 7,718,411), which is a Continuation of U.S. patent application Ser. No. 10/110,079, filed Apr. 29, 2002, abandoned, which is a National Phase under 37 CFR 371 of International application PCT/FI00/00877, filed Oct. 12, 2000, which claims priority to U.S. provisional application 60/163,283, filed Nov. 3, 1999, the contents of all of the above are incorporated by reference in their entirety. 
     
    
     SEQUENCE LISTING  
       [0002]    The sequence listing submitted via EFS, in compliance with C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “30760-US-CNT-3_SeqListing.txt”, created on Sep. 16, 2014, which is 4.79 KB (4,906 bytes) in size. 
       FIELD OF THE INVENTION 
       [0003]    This invention relates to protein engineering, and concerns especially family G/11 xylanases, and genes encoding said enzymes. In specific, the invention concerns  Trichoderma reesei  XYNII gene, which codes for endo-1,4-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 
       [0004]    Xylanases are glycosyl hydrolases which hydrolyse β-1,4-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 al., 1992; Wakarchuk et al., 1994). 
         [0005]    The family 11 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. (Törrönen &amp; Rouvinen, 1997). The family 11 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 (XynI and XynII) are the best characterized (Tenkanen et al., 1992). XynI has a size of 19 kDa, and compared to XynII it has low isoelectric point and pH optimum (pI 5.5, pH 3-4). XynII has a size of 20 kDa and it has a pI of 9.0 and a pH optimum of 5.0-5.5 (Törrönen and Rouvinen, 1995). 
         [0006]    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. 
         [0007]    In feed applications the enzyme faces high temperature conditions for a short time (e.g. 2-5 min at 90° C.) during 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. 
         [0008]    In pulp bleaching the material coming from alkaline wash has a high temperature (&gt;80° C.) and pH (&gt;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. 
         [0009]    Several thermostable, alkaliphilic and acidophilic xylanases have been found and cloned from thermophilic 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 industrial 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. 
         [0010]    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-helix 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 thermostability. 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 thermophilic xylanase (Sung et al., 1998). In addition to the improved thermostability, 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 comparing the structures of thermophilic and mesophilic enzymes plenty of information has been obtained (Vogt et al., 1997). Structural information of thermophilic xylanases has also given information about factors influencing the thermostability of xylanases (Gruber et al., 1998; Harris et al., 1997). 
       SUMMARY OF THE INVENTION 
       [0011]    The present invention relates to xylanases which belong to the family 11 (previously G) glycosyl hydrolases. The invention provides xylanases modified so as to change their thermostability, thermoactivity, and/or broaden their pH range. 
         [0012]    Various modifications in the  Trichoderma reesei  xylanase structure, either alone or in combinations, result in the changes described in this invention: 
         [0013]    (1) the stability of the enzyme is increased by binding of the N-terminal region by 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; 
         [0014]    (2) the C-terminus is stabilised by extension with additional aspartic acid (+191D) which forms a salt bridge with arginine 58 (lysine 58 in the wild-type enzyme has been changed to arginine (K58R)); 
         [0015]    (3) the stability of the enzyme is increased by binding the a-helix by a disulfide bridge to the body of the enzyme (e.g. L105C and Q162C); 
         [0016]    (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). 
         [0017]    In specific, the present invention provides a modified  Trichoderma reesei  xylanase in which the 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 (+191D), 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 
         [0018]      FIG. 1 . A set of oligonucleotides used in the mutagenesis of xylanase (codon changes underlined). The sequences are also given in the appended Sequence Listing as sequences 1 to 12. More specifically, the oligonucleotides correspond to the sequences in the sequence listing as follows: T2C is SEQ ID NO:1, T28C is SEQ ID NO:2, K58R is SEQ ID NO:3, 191D is SEQ ID NO:4, P5C is SEQ ID NO:5, N19C is SEQ ID NO:6, T7C is SEQ ID NO:7, S16C is SEQ ID NO:8, N10C is SEQ ID NO:9, N29C is SEQ ID NO:10, L105C is SEQ ID NO:11, and Q162C is SEQ ID NO:12. 
           [0019]      FIG. 2 . A graph presenting the effect of the mutations T2C, T28C, K58R, and +191D on the thermal optimum of  T. reesei  XynII (WT=wild-type enzyme; Y5=the mutated  T. reesei  XynII). 
           [0020]      FIG. 3 . A graph presenting the effect of the mutations T2C, T28C, K58R, and +191D on the pH-dependent activity of  T. reesei  XynII (WT and Y5 as in  FIG. 2 ). 
           [0021]      FIG. 4 . A graph presenting the effect of the mutations T2C, T28C, K58R, and +191D on the inactivation of  T. reesei  XynII at different temperatures (WT and Y5 as in  FIG. 2 ). 
           [0022]      FIG. 5 . A graph presenting the effect of the mutations Q162C and L105C on the inactivation of  T. reesei  XynII at different temperatures (W.t.=wild-type enzyme). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0023]    The family G/11 xylanases originating from bacteria, yeast and fungi have common molecular structure. Examples of such xylanases are:
     Aspergillus niger  XynA     Aspergillus kawachii  XynC     Aspergillus tubigensis  XynA     Bacillus circulans  XynA     Bacillus pumilus  XynA     Bacillus subtilis  XynA     Neocallimastix patriciarum  XynA     Streptomyces lividans  XynB     Streptomyces lividans  XynC     Streptomyces thermoviolaceus  XynII     Thermomonospora fusca  XynA     Trichoderma harzianum  Xyn     Trichoderma reesei  XynI,  Trichoderma reesei  XynII     Trichoderma viride  Xyn   
 
         [0038]    The invention deals with xylanases of the family G/11 with the following common features: 
         [0039]    Enzymes in which the N-terminal sequence is a part of the double-layered β-sheet (in the family 11 xylanases the A- and the B-sheet, (Gruber, et al., 1998)) and in which the first β-strand (in  T. reesei  XynII 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  XynII the amino acids 13-19) or to other neighbouring regions. 
         [0040]    (ii). Enzymes in which the C-terminal peptide chain forms a β-strand (in  T. reesei  XynII 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. 
         [0041]    (iii). Enzymes which have an α-helix on the other side of the enzyme structure with regard to the catalytic canyon, and wherein said α-helix or the neighbouring regions can be bound more tightly by a disulfide bridge to the body of the protein. 
         [0042]    The  T. reesei  xylanase II has the above mentioned properties and in said enzyme thermostability, pH-stability and thermoactivity can be modified based on these properties. The following changes have been made to the xylanase gene (XYNII) of  T. reesei:    
         [0043]    By site-directed mutagenesis disulfide bridges are formed in the N-terminal region: 
         [0044]    Threonines 2 and 28 are changed to cysteines resulting in a disulfide bridge being formed between them (T2C and T28C). 
         [0045]    Proline 5 and asparagine 19 are changed to cysteines resulting in a disulfide bridge being formed between them (P5C and N19C). 
         [0046]    Threonine 7 and serine 16 are changed to cysteines resulting in a disulfide bridge being formed between them (T7C and S16C). 
         [0047]    Asparagine 10 and asparagine 29 are changed to cysteines resulting in a disulfide bridge being formed between them (N10C and N29C). 
         [0048]    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. 
         [0049]    An aspartic acid (+191D) is added to the C-terminal serine (S190). This results in a salt bridge with arginine at position 58, where wild-type lysine has been replaced by arginine (K58R). 
         [0050]    By site-directed mutagenesis at least one disulfide bridge is formed to stabilise the enzyme in the C-terminal part via the α-helix or a region near the α-helix. 
         [0051]    Leucine 105 and glutamine 162 are changed to cysteines resulting in disulfide bridge between them (L105C and Q162C). 
         [0052]    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 
       [0053]    Production of mutated and recombinant XYNII genes were carried out by the following general procedures: 
       Expression Vector and Production of the Enzyme 
       [0054]      T. reesei  xylanase II was produced in  E. coli  strains XL1-Blue or Rv308 using the vector pKKtac (VTT, Espoo, Finland) or the vector pALK143 (ROAL, Rajamäki, 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 
       [0055]    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 oligonucleotide primers which contained the sequences for the changed codons. Examples of the used oligonucleotides are given in  FIG. 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, Calif., USA). The cloned  E. coli  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 
       [0056]    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 
       [0057]    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 min 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  XynII enzyme. 
       EXAMPLES OF MUTATIONS 
     Example 1 
       [0058]    The three-fold mutations (T2C, T28C and K58R) and the recombinant change (+191D) were made in  T. reesei  XynII by using the methods described above. The mutant enzyme was designated as Y5. Said mutant enzyme was expressed in  E. coli , which was cultivated at +37° C. in shake flasks using Luria Broth as growth medium. After cultivation the cells were removed by centrifugation and the xylanase secreted into the medium was characterized in varying conditions, as described above.  FIG. 2  shows the effect of the temperature to the enzyme activity when the mutant Y5 (T2C, T28C, K58R, +191D) and the wild-type ( T. reesei  XynII) 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 
       [0059]    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.  FIG. 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 
       [0060]    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. ( FIG. 4 ). Table 1 below shows the half-lives (T½) of the mutant (Y5) and the wild-type xylanase at 55° C. and 65° C. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                 pH 5 
                 pH 8 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 55° C. 
                   
                   
               
               
                   
                 Y5 
                 stable 
                 stable 
               
             
          
           
               
                   
                 Wild-type XynII 
                 ~5 
                 min 
                 ~2 min 
               
               
                   
                 65° C. 
               
               
                   
                 Y5 
                 20-25 
                 min 
                 ~10 min  
               
               
                   
                 Wild-type XynII 
                 40 
                 sec 
               
               
                   
                   
               
             
          
         
       
     
       Example 4 
       [0061]    With the above-mentioned methods a disulfide bridge was made (L105C and Q162C) to bind the C-terminus of the α-helix to the neighbouring β-strand. The enzyme was produced in  E. coli  and its properties were determined  FIG. 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 
       [0062]    Arase, A., Yomo, T., Urabe, I., Hata, Y., Katsube, Y. &amp; Okada, H. (1993). Stabilization of xylanase by random mutagenesis.  FEBS Letters  316, 123-7. 
         [0063]    Bailey, J. M., Biely, P. &amp; Poutanen, K. (1992). Interlaboratory testing of methods for assay of xylanase activity.  J. Biotech.  23, 257-270. 
         [0064]    Biely, P., Mislovicova, D. &amp; Toman, R. (1985). Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases.  Analytical Biochemistry  144, 142-6. 
         [0065]    Bodie, E., Cuevas, W. A. &amp; Koljonen, M. (1995). In U.S. Pat. No. 5,437,992. 
         [0066]    Campbell, R. L., Rose, D. R., Sung, W. L., Yaguchi, M. &amp; Wakarchuck, W. (1995). In U.S. Pat. No. 5,405,769. 
         [0067]    Fukunaga, N., Iwasaki, Y., Kono, S., Kita, Y. &amp; Izumi, Y. (1998). In U.S. Pat. No. 5,736,384. 
         [0068]    Gruber, K., Klintschar, G., Hayn, M., Schlacher, A., Steiner, W. &amp; Kratky, C. (1998). Thermophilic xylanase from Thermomyces lanuginosus: High-resolution X-ray structure and modeling studies.  Biochemistry  37, 13475-13485. 
         [0069]    Harris, G. W., Pickersgill, R. W., Connerton, I., Debeire, P., Touzel, J. P., Breton, C. &amp; Perez, S. (1997). Structural basis of the properties of an industrially relevant thermophilic xylanase.  Proteins  29, 77-86. 
         [0070]    Henrissat, B. &amp; Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.  Biochemical Journal  293, 781-8. 
         [0071]    Prade, R. A. (1996). Xylanases: from biology to biotechnology.  Biotechnology &amp; Genetic Engineering Reviews  13, 101-31. 
         [0072]    Sung, W. L., Yaguchi, M., Ishikawa, K., Huang, F., Wood, M. &amp; Zahab, D. M. (1998). In U.S. Pat. No. 5,759,840. 
         [0073]    Tenkanen, M., Puls, J. &amp; Poutanen, K. (1992). Two major Xylanases of Trichoderma reesei.  Enzyme Microb. Technol.  14, 566-574. 
         [0074]    Törrönen, A. &amp; Rouvinen, J. (1995). Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei.  Biochemistry  34, 847-56. 
         [0075]    Törrönen, A. &amp; Rouvinen, J. (1997). Structural and functional properties of low molecular weight endo-1,4-beta-xylanases.  Journal of Biotechnology  57, 137-49. 
         [0076]    Wakarchuk, W. W., Sung, W. L., Campbell, R. L., Cunningham, A., Watson, D. C. &amp; Yaguchi, M. (1994). Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds.  Protein Engineering  7, 1379-86. 
         [0077]    Vogt, G., Woell, S. &amp; Argos, P. (1997). Protein thermal stability, hydrogen bonds, and ion pairs.  Journal of Molecular Biology  269, 631-43.