Variants of Glycoside Hydrolases

Abstract

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

What is claimed is: 
     
         1 . An isolated variant of a parent glycoside hydrolase, comprising a substitution at a position corresponding to position 373 of SEQ ID NO: 2, wherein the variant comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 2, and wherein the variant has glycoside hydrolase activity. 
     
     
         2 . The variant of  claim 1 , which comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2. 
     
     
         3 . The variant of  claim 1 , which comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 2. 
     
     
         4 . The variant of  claim 1 , wherein the parent glycoside hydrolase comprises SEQ ID NO: 2, or a fragment thereof that has glycoside hydrolase activity. 
     
     
         5 . The variant of  claim 1 , wherein the parent glycoside hydrolase comprises SEQ ID NO: 2. 
     
     
         6 . The variant of  claim 1 , wherein the substitution is His. 
     
     
         7 . The variant of  claim 1 , wherein the substitution is N373H. 
     
     
         8 . The variant of  claim 1 , further comprising a substitution at a position corresponding to position 21, 94, 157, 205, 206, 247, 337, 350, 383, 438, 455, 467, or 486 of SEQ ID NO: 2. 
     
     
         9 . The variant of  claim 1 , further comprising a substitution of Pro at a position corresponding to 21; a substitution of Ser, Ala, Arg, or Gln at a position corresponding to 94; a substitution of Arg at a position corresponding to 157; a substitution of Arg at a position corresponding to 205; a substitution of Tyr at a position corresponding to 206; a substitution of Cys at a position corresponding to 247; a substitution of Val at a position corresponding to 337; a substitution of Ser at a position corresponding to 350; a substitution of Ala at a position corresponding to 383; a substitution of Leu at a position corresponding to 438; a substitution of Ala at a position corresponding to 455; a substitution of Ser at a position corresponding to 467; or a substitution of Trp at a position corresponding to 486. 
     
     
         10 . The variant of  claim 1 , further comprising the substitution S21P, G94S, G94A, G94R, G94Q, K157R, G205R, H206Y, Y247C, E337V, T350S, T383A, P438L, T455A, G467S, or C486W. 
     
     
         11 . The variant of  claim 1 , further comprising a substitution at a position corresponding to position 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, or 462 of SEQ ID NO: 2. 
     
     
         12 . The variant of  claim 1 , further comprising a substitution of Pro at a position corresponding to 8; a substitution of Asp at a position corresponding to 22; a substitution of Ile at a position corresponding to 41; a substitution of Ser at a position corresponding to 49; a substitution of Asn at a position corresponding to 57; a substitution of Asn at a position corresponding to 113; a substitution of Lys at a position corresponding to 193; a substitution of Pro, Thr, or Phe at a position corresponding to 196; a substitution of Ala at a position corresponding to 226; a substitution of Ala, Leu, or Gly at a position corresponding to 227; a substitution of Ile at a position corresponding to 246; a substitution of Lys at a position corresponding to 251; a substitution of Pro at a position corresponding to 255; a substitution of Asn at a position corresponding to 259; a substitution of Ser at a position corresponding to 301; a substitution of Ile at a position corresponding to 356; a substitution of Cys at a position corresponding to 371; a substitution of Phe at a position corresponding to 411; or a substitution of Ala at a position corresponding to 462 
     
     
         13 . The variant of  claim 1 , further comprising the substitution S8P, G22D, T41I, N49S, S57N, S113N, E193K, S196P, S196T, S196F, T226A, P227A, P227L, P227G, T246I, R251K, T255P, D259N, N301S, T356I, Y371C, S411F, or T462A. 
     
     
         14 . The variant of  claim 1 , which has improved activity on 4-methylumbelliferyl beta-D-lactoside at 64° C. for 45 minutes at pH 5.0 compared to the parent glycoside hydrolase. 
     
     
         15 . The variant of  claim 14 , wherein the improved activity is at least 2-fold compared to the parent glycoside hydrolase. 
     
     
         16 . The variant of  claim 14 , wherein the improved activity is at least 5-fold compared to the parent glycoside hydrolase. 
     
     
         17 . The variant of  claim 1 , wherein the parent glycoside hydrolase is a  Trichoderma reesei  glycoside hydrolase. 
     
     
         18 . A method for obtaining the variant of  claim 1 , comprising:
 (a) introducing into a parent glycoside hydrolase having at least 90% sequence identity to SEQ ID NO: 2, a substitution at a position corresponding to position 373; and   (b) recovering the variant.   
     
     
         19 . The method of  claim 18 , wherein the variant comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2. 
     
     
         20 . The method of  claim 18 , wherein the variant comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 2. 
     
     
         21 . The method of  claim 18 , wherein the parent glycoside hydrolase comprises SEQ ID NO: 2, or a fragment thereof that has glycoside hydrolase activity. 
     
     
         22 . The method of  claim 18 , wherein the substitution at a position corresponding to position 373 is His. 
     
     
         23 . The method of  claim 18 , wherein the substitution at a position corresponding to position 373 is N373H. 
     
     
         24 . The method of  claim 18 , wherein the variant has improved activity on 4-methylumbelliferyl beta-D-lactoside at 64° C. for 45 minutes at pH 5.0 compared to the parent glycoside hydrolase. 
     
     
         25 . A detergent composition comprising the variant of  claim 1  and a surfactant. 
     
     
         26 . A method for degrading cellulose- and hemicellulose-containing biomass, comprising treating the biomass with an effective amount of a variant of  claim 1  and recovering the degraded biomass.