Method for Screening Alpha-Amylases

Abstract

The present invention relates to variants of a parent alpha-amylase. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

1 . A method for screening alpha-amylases having high performance at low temperature, comprising the steps of
 a) determining the binding of the alpha-amylases to solid or immobilized substrate for the alpha amylase, such as starch;   b) selecting alpha-amylases having a low binding to the substrate.   
     
     
         2 . The method of  claim 1 , wherein the alpha-amylases has high wash performance at low temperature. 
     
     
         3 . The method of  claim 1 , wherein the alpha-amylases have less than 90% of the binding to starch than the alpha-amylase having SEQ ID NO: 8, preferably less than 80%, preferably less than 70%, preferably less than 65%, preferably less than 60%, preferably less than 50% of the binding, preferably less than 40% of the binding, preferably less than 30% of the binding, more preferred less than 20% of the binding and most preferred less than 10% of the binding. 
     
     
         4 . A method for selecting variants of a parent alpha-amylase comprising the steps of
 a) generating variants by substituting, deleting or inserting one or more amino acid residue in one or more amino acids located at the surface of the parent alpha-amylase;   b) testing the variants for the binding to solid or immobilized substrate for the alpha-amylase, such as starch;   c) selecting variants having a lower binding the substrate than the parent alpha-amylase.   
     
     
         5 . The method of  claim 3 , wherein the parent alpha-amylase is selected among amylases having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% identity to one of SEQ ID NO: 1-15. 
     
     
         6 . The method of  claim 4 , wherein the variants have less than 90% of the binding to starch than the parent alpha-amylase, preferably less than 80%, preferably less than 70%, preferably less than 65%, preferably less than 60%, preferably less than 50% of the binding, preferably less than 40% of the binding, preferably less than 30% of the binding, more preferred less than 20% of the binding and most preferred less than 10% of the binding. 
     
     
         7 . A variant polypeptide having alpha-amylase activity and having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to one of SEQ ID NO: 1-15, the variant is derived from a parent alpha-amylase by one or more (e.g. several) modifications selected from substitutions, insertions and deletions, wherein the variant alpha-amylase has lower binding to solid starch and higher wash performance at low temperature e.g. 15° C., compared with the patent alpha-amylase. 
     
     
         8 . The variant alpha-amylase of  claim 7 , wherein an amino acid residue in one or more substrate binding sites at the surface of the molecule has been modified. 
     
     
         9 . The variant alpha-amylase of  claim 7 , selected among:
 a) variant alpha-amylases having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to SEQ ID NO: 2, and comprising a deletion of positions 181+182, or 182+183, or 183+184 and further one or two or more modifications in any of positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 in SEQ ID NO: 2;   b) variant alpha-amylases having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to SEQ ID NO: 5, and comprising a deletion of positions 181+182, or 182+183, or 183+184 and further comprising one or two or more modifications in any of positions corresponding to W140, W159, W167, Q169, W189, E194, F262, W284, F289, G304, G305, K320, W347, W439, W469, G476 and G477 in SEQ ID NO: 5;   c) variant alpha-amylases having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to SEQ ID NO: 4, and comprising a deletion of positions 181+182, or 182+183, or 183+184 and further comprising one or two or more modifications in any of positions corresponding to W140, W159, W167, Q169, W189, E194, F262, W284, F289, G304, G305, K320, W347, W439, W469, G476 and G477 in SEQ ID NO: 4;   d) variant alpha-amylases having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to SEQ ID NO: 13, and comprising a modification in any of positions corresponding to W138, W157, W165, E167, W184, E189, E255, F257, F279, F284, G299, G300, K315, W342, R437, W467 and G475 in SEQ ID NO: 13;   e) variant alpha-amylases having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to SEQ ID NO: 14, and comprising a deletion of positions 176+177 or 177+178, or 178+179 and further comprising one or two or more modifications in any of positions corresponding to W136, W155, W163, E165, W184, E189, E255, F257, F279, F284, G299, G300, R315, W342, R437, W467 and G475 in SEQ ID NO: 14; and   f) variant alpha-amylases having having at least 80% sequence identity, preferably at least 85% sequence identity, more preferred at least 90% sequence identity, more preferred at least 95% sequence identity, more preferred at least 97% sequence identity, more preferred at least 98% sequence identity and most preferred at least 99% sequence identity to SEQ ID NO: 15, and comprising a deletion of positions 179+180 or 180+181, or 181+182 and further comprising one or two or more modifications in any of positions corresponding to W139, W158, W166, E168, W187, E192, F260, F287, G302, G303, W345, W437, W467, G474 and G475 in SEQ ID NO: 15,   wherein the modifications in a-f preferably are substitutions.   
     
     
         10 . The variant alpha-amylase of any one of  claim 7 , having at least 90% sequence identity to SEQ ID NO: 2 and comprising modifications in positions corresponding to the following positions in SEQ ID NO: 2:
 H183*+G184*+W140F;   H183*+G184*+Q169N;   H183*+G184*+Q169A;   H183*+G184*+W189Y+E190P;   H183*+G184*+N260D;   H183*+G184*+G477E;   H183*+G184*+G477Q;   H183*+G184*+G477K;   H183*+G184*+W189E+E190P;   H183*+G184*+A51I+W140Y;   H183*+G184*+W140Y+W189E;   H183*+G184*+W140Y+N260P;   H183*+G184*+W140Y+W284D;   H183*+G184*+W140Y+G476R;   H183*+G184*+W140Y+G477E;   H183*+G184*+W189E+W439R;   H183*+G184*+W284D+G477E;   H183*+G184*+W439R+G476R;   H183*+G184*+W439R+G477E;   H183*+G184*+E194D;   H183*+G184*+W439R+D467K;   H183*+G184*+R320M+W439R;   H183*+G184*+W439R+K485R;   H183*+G184*+Y160S;   H183*+G184*+W189F+E190P;   H183*+G184*+F262A;   H183*+G184*+Y363H;   H183*+G184*+G476E;   H183*+G184*+N260P+W439R;   H183*+G184*+N260P+G477E;   H183*+G184*+W439R+G476R;   H183*+G184*+K72S+W140Y;   H183*+G184*+G109A+M202L+Y203G;   H183*+G184*+E194S;   H183*+G184*+E345D+G477R;   H183*+G184*+K302N+W439R;   H183*+G184*+R320K+W439R   H183*+G184*+W159Y+W167Y+F262P+W439R+W469Y+G477Q;   H183*+G184*+W159Y+W167F+N260D+W439Y+W469Y+G476K+G477Q;   H183*+G184*+W159Y+W167Y+N260D+W439R+W469V+G476E+G477K;   H183*+G184*+W159Y+W167F+N260P+F262P+W439R+W469Y+G476K+G477E;   H183*+G184*+W159Y+W167F+F262P+W439V+W469Y+G476K+G477Q;   H183*+G184*+W159Y+W167F+F262P+W469Y+G476R;   H183*+G184*+W159Y+W167Y+N260G+W439R+W469Y;   H183*+G184*+W159Y+W167Y+N260G+W439R+W469Y;   H183*+G184*+W167Y+N260D+W439R+G476Q+G477E;   H183*+G184*+W167Y+N260P+F262P+W439R+G476E+G477R;   H183*+G184*+W159Y+W167F+N260G+W439R+W469Y+G476R;   H183*+G184*+W159Y+W167Y+N260D+F262P+W439Y;   H183*+G184*+W159Y+W167F+N260P+W439Y+W469V+G476Q+G477Q;   H183*+G184*+W167Y+N260D+W439R+W469V+G476Q+G477Q;   H183*+G184*+W159Y+W167Y+N260P+F262P+W439R+G476E+G477K;   H183*+G184*+W159Y+W167F+N260P+F262P+W439Y+W469Y+G476R;   H183*+G184*+W167F+N260D+F262P+P380Q+G477K;   H183*+G184*+W167Y+N260D+F262P+W439R+W469V+G476Q+G477K;   H183*+G184*+W167Y+N260D+F262P+W439V+W469Y;   H183*+G184*+W159Y+W167Y+N260D+W439R+W469V+G476E+G477K;   H183*+G184*+W167Y+N260D+W439R+W469Y+G476E+G477K;   H183*+G184*+W159Y+W167Y+N260D+G476E+G477Q;   H183*+G184*+W159Y+W167F+N260P+F262P+W439Y+G476K+G477Q;   H183*+G184*+N260D+F262P+W469Y+G476R+G477Q;   H183*+G184*+W167Y+L230I+N260P+W469Y;   H183*+G184*+W159Y+W167Y+N260P+E439Y+G476Q+G477Q; and   H183*+G184*+W167Y+F262P+W469Y+G476R+G477Q.   
     
     
         11 . The variant alpha-amylase of  claim 10 , which consists of SEQ ID NO: 2 with a modification selected among:
 H183*+G184*+W140F;   H183*+G184*+Q169N;   H183*+G184*+Q169A;   H183*+G184*+W189Y+E190P;   H183*+G184*+N260D;   H183*+G184*+G477E;   H183*+G184*+G477Q;   H183*+G184*+G477K;   H183*+G184*+W189E+E190P;   H183*+G184*+A51I+W140Y;   H183*+G184*+W140Y+W189E;   H183*+G184*+W140Y+N260P;   H183*+G184*+W140Y+W284D;   H183*+G184*+W140Y+G476R;   H183*+G184*+W140Y+G477E;   H183*+G184*+W189E+W439R;   H183*+G184*+W284D+G477E;   H183*+G184*+W439R+G476R;   H183*+G184*+W439R+G477E;   H183*+G184*+E194D;   H183*+G184*+W439R+D467K;   H183*+G184*+R320M+W439R;   H183*+G184*+W439R+K485R;   H183*+G184*+Y160S;   H183*+G184*+W189F+E190P;   H183*+G184*+F262A;   H183*+G184*+Y363H;   H183*+G184*+G476E;   H183*+G184*+N260P+W439R;   H183*+G184*+N260P+G477E;   H183*+G184*+W439R+G476R;   H183*+G184*+K72S+W140Y;   H183*+G184*+G109A+M202L+Y203G;   H183*+G184*+E194S;   H183*+G184*+E345D+G477R;   H183*+G184*+K302N+W439R;   H183*+G184*+R320K+W439R   H183*+G184*+W159Y+W167Y+F262P+W439R+W469Y+G477Q;   H183*+G184*+W159Y+W167F+N260D+W439Y+W469Y+G476K+G477Q;   H183*+G184*+W159Y+W167Y+N260D+W439R+W469V+G476E+G477K;   H183*+G184*+W159Y+W167F+N260P+F262P+W439R+W469Y+G476K+G477E;   H183*+G184*+W159Y+W167F+F262P+W439V+W469Y+G476K+G477Q;   H183*+G184*+W159Y+W167F+F262P+W469Y+G476R;   H183*+G184*+W159Y+W167Y+N260G+W439R+W469Y;   H183*+G184*+W159Y+W167Y+N260G+W439R+W469Y;   H183*+G184*+W167Y+N260D+W439R+G476Q+G477E;   H183*+G184*+W167Y+N260P+F262P+W439R+G476E+G477R;   H183*+G184*+W159Y+W167F+N260G+W439R+W469Y+G476R;   H183*+G184*+W159Y+W167Y+N260 D+F262P+W439Y;   H183*+G184*+W159Y+W167F+N260P+W439Y+W469V+G476Q+G477Q;   H183*+G184*+W167Y+N260D+W439R+W469V+G476Q+G477Q;   H183*+G184*+W159Y+W167Y+N260P+F262P+W439R+G476E+G477K;   H183*+G184*+W159Y+W167F+N260P+F262P+W439Y+W469Y+G476R;   H183*+G184*+W167F+N260D+F262P+P380Q+G477K;   H183*+G184*+W167Y+N260D+F262P+W439R+W469V+G476Q+G477K;   H183*+G184*+W167Y+N260D+F262P+W439V+W469Y;   H183*+G184*+W159Y+W167Y+N260D+W439R+W469V+G476E+G477K;   H183*+G184*+W167Y+N260D+W439R+W469Y+G476E+G477K;   H183*+G184*+W159Y+W167Y+N260D+G476E+G477Q;   H183*+G184*+W159Y+W167F+N260P+F262P+W439Y+G476K+G477Q;   H183*+G184*+N260D+F262P+W469Y+G476R+G477Q;   H183*+G184*+W167Y+L230I+N260P+W469Y;   H183*+G184*+W159Y+W167Y+N260P+E439Y+G476Q+G477Q; and   H183*+G184*+W167Y+F262P+W469Y+G476R+G477Q.   
     
     
         12 . The variant alpha-amylase of any one of  claim 7 , having at least 90% sequence identity to SEQ ID NO: 14 and comprising modifications in positions corresponding to the following positions in SEQ ID NO: 14:
 E178*+G179*;   E178*+G179*+T36N;   E178*+G179*+W136Y;   E178*+G179*+W155Y;   E178*+G179*+W155F;   E178*+G179*+W163Y;   E178*+G179*+G299E;   E178*+G179*+G299K;   E178*+G179*+G299R;   E178*+G179*+R315M;   E178*+G179*+R315A;   E178*+G179*+R315Q; and   E178*+G179*+W342Y.   
     
     
         13 . The variant of  claim 12  consisting of SEQ ID NO: 14 with a modification selected among:
 E178*+G179*; 
 E178*+G179*+T36N; 
 E178*+G179*+W136Y; 
 E178*+G179*+W155Y; 
 E178*+G179*+W155F; 
 E178*+G179*+W163Y; 
 E178*+G179*+G299E; 
 E178*+G179*+G299K; 
 E178*+G179*+G299R; 
 E178*+G179*+R315M; 
 E178*+G179*+R315A; 
 E178*+G179*+R315Q; and 
 E178*+G179*+W342Y. 
 
     
     
         14 . The variant alpha-amylase of  claim 7 , having at least 90% sequence identity to SEQ ID NO: 13 and comprising modifications in positions corresponding to position K315 and/or W467 in SEQ ID NO:13. 
     
     
         15 . The variant of  claim 14 , consisting of SEQ ID NO: 13 with a substitution K315M. 
     
     
         16 . A detergent composition comprising a variant alpha-amylase of any of the  claim 7 . 
     
     
         17 . Use of a variant alpha-amylase according to any of the  claim 7  in a cleaning process such as laundry or hard surface cleaning including automated dish wash.