Alpha-Amylase Combinatorial Variants

Abstract

Disclosed are compositions and methods relating to variant alpha-amylases. The variant alpha-amylases are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing (e.g., desizing), in animal feed for improving digestibility, and for baking and brewing.

1 . A recombinant variant of a parent α-amylase comprising:
 a mutation at an amino acid residue corresponding to R377, and optionally S362; and 
 at least one mutation, and optionally at least two mutations, at an amino acid residue, or residues, corresponding to an amino acid residue selected from the group consisting of N126, F153, T180, E187, and I203; 
 wherein the variant α-amylase or the parent α-amylase has at least 60%, optionally 70%, optionally 80%, optionally 85%, optionally 90%, or optionally 95%, amino acid sequence identity relative to SEQ ID NO: 1, which is used for numbering; and 
 wherein the variant has increased low pH stability and/or starch liquifaction activity, compared to the parent α-amylase or a reference α-amylase differing from the variant α-amylase only by the absence of the mutations. 
 
     
     
         2 . The variant α-amylase of  claim 1 , comprising the mutation R377Y, and optionally S362A; and at least one mutation, and optionally at least two mutations, at an amino acid residue, or residues, corresponding to an amino acid residue selected from the group consisting of N126Y, F153W, T180H, T180D, E187P, and I203Y, using SEQ ID NO: 1 for numbering. 
     
     
         3 . The variant α-amylase of  claim 1 , comprising the mutations R377Y and S362A, using SEQ ID NO: 1 for numbering. 
     
     
         4 . The variant α-amylase of  claim 3 , further comprising the mutations N126Y, F153W, T180H, and E187P, using SEQ ID NO: 1 for numbering. 
     
     
         5 . The variant α-amylase of  claim 1 , further comprising a mutation as a position selected from the group consisting of A277, T89, S92 and Y303, using SEQ ID NO: 1 for numbering. 
     
     
         6 . The variant α-amylase of  claim 1 , further comprising a deletion of at least one amino acid residue corresponding to R178, G179, T180, and G181, using SEQ ID NO: 1 for numbering. 
     
     
         7 . The variant α-amylase of  claim 1 , further comprising deletions of amino acid residues corresponding to R178 and G179, or T180 and G181, using SEQ ID NO: 1 for numbering. 
     
     
         8 . The variant α-amylase of  claim 1 , further comprising a mutation at an amino acid residue corresponding to G476, G477, E132, Q167, A277, R458, T459, and/or D460, using SEQ ID NO: 1 for numbering. 
     
     
         9 . The variant α-amylase of  claim 1 , wherein the parental α-amylase is from a  Cytophaga  species or not from a  Bacillus  species. 
     
     
         10 . A method for converting starch to oligosaccharides, comprising contacting starch with effective amount of  claim 1 . 
     
     
         11 . A composition for liquefying starch comprising the variant amylase of  claim 1 . 
     
     
         12 . A recombinant variant of a parent α-amylase comprising:
 a mutation at at least one, and optionally a plurality, of amino acid residues corresponding to position T38, N126, F153, E187, I203, G476, and G477; and, optionally at least one mutation at an amino acid residue corresponding to R178, G179, T180, and G181, 
 wherein the variant α-amylase or the parent α-amylase has at least 60%, optionally 70%, optionally 80%, optionally 85%, optionally 90%, or optionally 95%, amino acid sequence identity relative to SEQ ID NO: 1, which is used for numbering; and 
 wherein the variant has increased detergent stability and/or cleaning performance compared to the parent α-amylase or a reference α-amylase differing from the variant α-amylase only by the absence of the mutations. 
 
     
     
         13 . The variant α-amylase of  claim 12 , comprising at least one, and optionally a plurality, of the mutations T38N, N126Y, F153W, E187P, I203Y, G476K, and G477E, using SEQ ID NO: 1 for numbering. 
     
     
         14 . The variant α-amylase of  claims 12 , further comprising a mutation at position T129, using SEQ ID NO: 1 for numbering. 
     
     
         15 . The variant α-amylase of  claim 14 , further comprising the mutation T129I, using SEQ ID NO: 1 for numbering. 
     
     
         16 . The variant α-amylase of  claim 12 , further comprising deletions of amino acid residues corresponding to R178 and G179, or T180 and G181, using SEQ ID NO: 1 for numbering. 
     
     
         17 . The variant α-amylase of  claim 12 , further comprising a mutation at an amino acid residue corresponding to E132, Q167, A277, R458, T459, and/or D460, using SEQ ID NO: 1 for numbering. 
     
     
         18 . The variant α-amylase of  claim 12 , lacking a mutation at an amino acid residue corresponding to position N88, N134, and/or L171, using SEQ ID NO: 1 for numbering. 
     
     
         19 . The variant α-amylase of  claim 12 , wherein the parental α-amylase is from a  Cytophaga  species or not from a  Bacillus  species. 
     
     
         20 . A method for removing a starchy stain or soil from a surface, comprising contacting the surface with an effective amount of the variant amylase of  claim 12 , and allowing the polypeptide to hydrolyze starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition, thereby removing the starchy stain from the surface. 
     
     
         21 . A detergent composition comprising the variant amylase of  claim 12 .