Modified xylanases exhibiting increased thermophilicity and alkalophilicity

Abstract

The present invention pertains to modified xylanase enzymes that exhibit increased thermostability and alkalophilicity, when compared with their native counterparts. Several modified xylanases exhibiting these properties are disclosed including xylanases with at least one modification at amino acid position 10, 27, 29, 75, 104, 105, 125, 129, 132, 135, 144, 157, 161, 162, or 165, or a combination thereof. Also included within the present invention is a modified xylanase that comprises at least one substituted amino acid residue and that may be characterized as having a maximum effective temperature (MET) between about 69° C. and about 78° C., wherein the modified xylanase is a Family 11 xylanase obtained from a Trichoderma sp.. The present invention also includes a modified Family 1 p 1 xylanase obtained from a Trichoderma sp. characterized as having a maximum effective pH (MEP) between about 5.8 and about 7.6. Modified xylanases characterized as having a MET between about 69° C. and about 78° C. and a MEP between about 5.8 and 7.6 are also disclosed.

The embodiments of the invention in which an exclusive property of privilege is claimed are defined as follows:  
     
         1 . A modified xylanase comprising at least one substituted amino acid residue at a position selected from the group consisting of amino acid 75, 104, 105, 125, 129, 132, 135, 144, 157, 161, 162,and 165, said position determined from sequence alignment of said modified xylanase with  Trichoderma reesei  xylanase II amino acid sequence defined in SEQ ID NO:16.  
     
     
         2 . The modified xylanase of  claim 1 , wherein said modified xylanase exhibits improved thermophilicity, alkalophilicity, or a combination thereof, in comparison to a corresponding native xylanase.  
     
     
         3 . The modified xylanase of  claim 1 , wherein said substituted amino acid is at position 75 and is selected from the group consisting of a non-polar and a polar amino acid.  
     
     
         4 . The modified xylanase of  claim 3 , wherein said at least one substituted amino acid is selected from the group consisting of Ala, Cys, Gly, and Thr.  
     
     
         5 . The modified xylanase of  claim 4 , wherein said modified xylanase is derived from a Family 11 xylanase.  
     
     
         6 . The modified xylanase of  claim 5 , wherein said Family 11 xylanase is a  Trichoderma ressei  xylanase.  
     
     
         7 . The modified xylanase of  claim 4 , further comprising a His at position 10, Met at position 27 and Leu at position 29 (HML).  
     
     
         8 . The modified xylanase of  claim 1  wherein said at least one substituted amino acid residue is a polar amino acid at position 105, said modified xylanase further comprising a His at position 10, Met at position 27; Leug at position 29  
     
     
         9 . The modified xylanase of  claim 8;  wherein said polar amino acid is selected from the group consisting of His, Lys, and Arg.  
     
     
         10 . The modified xylanase of  claim 9 , wherein the xylanase is a Family 11 xylanase.  
     
     
         11 . The modified xylanase of  claim 10 , wherein said Family 11 xylanase is a  Trichoderma ressei  xylanase.  
     
     
         12 . The modified xylanase of  claim 11 , further comprising a second substituted amino acid residue at position 75, said second substituted amino acid is selected from the group consisting of a non-polar and a polar amino acid.  
     
     
         13 . The modified xylanase of  claim 12 , wherein said second substituted amino acid is selected from the group consisting of Ala, Cys, Gly, and Tbr.  
     
     
         14 . The modified xylanase of  claim 13 , wherein the xylanase is a Family 11 xylanase.  
     
     
         15 . The modified xylanase of  claim 14 , wherein said Family 11 xylanase is a  Trichoderma reesei  xylanase.  
     
     
         16 . The modified xylanase of  claim 1  wherein said at least one substituted amino acid residue is a polar amino acid at position 157, 161, 162 and 165, said modified xylanase further comprising a His at position 10, Met at position 27, Leu at position 29.  
     
     
         17 . The modified xylanase of  claim 16 , wherein said polar amino acid at positions 161, 162, and 165 is selected from the group consisting of Arg, Lys and His, and said polar amino acid at position 157 is selected from the group consisting of Asp and Glu.  
     
     
         18 . The modified xylanase of  claim 17 , wherein the xylanase is a Family 11 xylanase.  
     
     
         19 . The modified xylanase of  claim 18 , wherein said Family 11 xylanase is a  Trichioderma reesei  xylanase.  
     
     
         20 . The modified xylanase of  claim 1  wherein said at least one substituted amino acid residue is a non-polar amino acid at position 125, said modified xylanase further comprising a His at position 10, Met at position 27 Leu at position 29.  
     
     
         21 . The modified xylanase of  claim 20 , wherein said at least one substituted amino acid is Ala.  
     
     
         22 . The modified xylanase of  claim 21 , wherein said modified xylanase is derived from a Family 11 xylanase.  
     
     
         23 . The modified xylanase of  claim 22 , wherein said Family 11 xylanase is a  Trichoderma reesei  xylanase.  
     
     
         24 . The modified xylanase of  claim 20 , further comprising a second substituted amino acid at position 129, said second substituted amino acid is an acidic amino acid.  
     
     
         25 . The modified xylanase of  claim 24 , wherein said second substituted amino acid is Glu.  
     
     
         26 . The modified xylanase of  claim 24 , further comprising a third substituted amino acid residue at position 75, said third substituted amino acid is a nonpolar amino acid, and a fourth substituted amino acid residue at position 105, said fourth amino acid is a polar amino acid.  
     
     
         27 . The modified xylanase of  claim 26 , wherein said third substituted amino acid is selected from the group consisting of Ala, Cys, Gly, and Thr, and wherein said fourth substituted amino acid is selected from the group consisting of His, Lys, and Arg.  
     
     
         28 . The modified xylanase of  claim 26 , further comprising a-fifth substituted amino acid residue at position 104, said fifth substituted amino acid is a non polar amino acid.  
     
     
         29 . The modified xylanase of claim 28, wherein said fifth substituted amino acid is a Pro.  
     
     
         30 . The modified xylanase of  claim 26 , further comprising a fifth substituted amino acid residue at position 132, and a sixth substituted amino acid residue at position 135, said fifth and sixth substituted amino acid each being a polar amino acid.  
     
     
         31 . The modified xylanase of  claim 30 , further comprising a seventh substituted amino acid residue at position 144, said seventh substituted amino acid is a polar amino acid.  
     
     
         32 . The modified xylanase of  claim 31 , further comprising an eighth substituted amino acid residue at position 157, a ninth substituted amino acid residue at position 161, a tenth substituted amino aicd at position 162, and an eleventh substituted amino acid residue at position 165, each of said eighth, ninth, tenth and eleventh substituted amino acid is a polar amino acid.  
     
     
         33 . The modified xylanase of  claim 26 , further comprising a fifth substituted amino acid residue at position 157, a sixth substituted amino acid at position 161, a seventh substituted amino acid at position 162, and an eighth substituted amino acid at position 165, each of said fifth, sixth seventh and eighth substituted amino acid is a polar amino acid.  
     
     
         34 . A use of the modified xylanase in  claim 1  in an industrial process.  
     
     
         35 . The use as defined in  claim 34  wherein said industrial process is a pulp manufacturing.  
     
     
         36 . A modified xylanase comprising at least one substituted amino acid residue, wherein said modified xylanase is characterized as having a maximum effective temperature ( between about 69 ° C. to about 78° C., and wherein said modified xylanase is a Family 11 xylanase obtained from a Trichoderma sp.  
     
     
         37 . The modified xylanase of  claim 36 , wherein said MET is between about 70° to about 75° C.  
     
     
         38 . A modified xylanase comprising at least one substituted amino acid residue, wherein said modified xylanase is characterized as having a maximum effective pH (MEP) between about pH 5.8 to about pH 7.6, and wherein said modified xylanase is a Family 11 xylanase obtained from a Trichoderma sp.  
     
     
         39 . The modified xylanase of  claim 38 , wherein said MEP is between about pH 6.5 to about pH 7.4.  
     
     
         40 . The modified xylanase of  claim 36 , wherein said modified xylanase is further characterized as having a maximum effective pH (REP) is between about pH 5.8 to about pH 7.6.  
     
     
         41 . The modified xylanase of  claim 37 , wherein said modified xylanase is further characterized as having a maximum effective pH (MEP) is between about pH 6.5 to about pH 7.4.  
     
     
         42 . A modified xylanase selected from the group consisting of: 
 TrX-75A    TrX-161R-162H-165H;    TrX-HML-75A;    TrX-HML-10511H;    TrX-HML-105R;    TrX-HML-105K;    TrX-HML-75A-105H;    TrX-HML-75A-105R;    TrX-HML-75C-105R;    TrX-HML-75G-105R;    TrX-HML-75T-105R    TrX-HML-125A;    TrX-HML-125A-129E;    TrX-HML-75G-105R-125A-129E (TrX-HML-GRAE);    TrX-HML-75A-105H-125A-129E (TrX-HML-AHAE);    TrX-HML-75G-105H-125A-129E (TrX-HML-GHAE);    TrX-HML-75A-105R-125A-129E (TrX-HML-ARAE);    TrX-HML-75G-104P-105R-125A-129E (TrX-HML GPRAE);    TrX-HML-75G-104P-105H-125A-129E (TrX-HML-GPHAE);    TrX-HML-AHAE-RR;    TrX-HML-AHAE-RRR;    TrX-HML-AHAE-RRR-DRHH;    TrX-HML-AHA-RR-DRHH; and    TrX-HML-AHAE-RR-DRHH