Variant Maltohexaose-Forming Alpha-Amylase Variants

Abstract

Disclosed are compositions and methods relating to variant maltohexaose-forming 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 variant α-amylase polypeptide derived from a parental α-amylase polypeptide, comprising at least one combinable mutation at a productive amino acid position; wherein:
 (i) the combinable mutation is the substitution of an amino acid residue present in the parental α-amylase with a different amino acid residue, which improves at least one desirable property of the variant α-amylase compared to the parental α-amylase, while not significantly decreasing either expression, activity, or stability of the variant α-amylase, compared to the parental α-amylase, 
 (ii) the productive position is an amino acid position that can be substituted with a plurality of different amino acid residues, each of which substitutions result in a variant α-amylase that meets the requirements of (i), and 
 (iii) the combinable mutation corresponds to a mutation listed in Lists A, B, C, or D, or in Table C or D, which use SEQ ID NO: 3 for numbering. 
 
     
     
         2 . The variant amylase of  claim 1 , wherein the combinable mutation produces a variant amylase wherein the minimum performance indices (PI) relative to the parental amylase for (i) protein expression, (ii) activity, and (iii) detergent stability or thermostability are greater than or equal to 0.9, and the PI for any one of (i), (ii), or (iii) that is greater than or equal to 1.0. 
     
     
         3 . The variant amylase of  claim 1 , wherein the combinable mutation produces a variant amylase wherein the minimum performance indices (PI) relative to the parental amylase for (i) protein expression, (ii) activity, and (iii) detergent stability or thermostability are greater than or equal to 0.8, and the PI for any one of (i), (ii), or (iii) that is greater than or equal to 1.2. 
     
     
         4 . The variant amylase of  claim 1 , wherein the combinable mutation produces a variant amylase wherein the minimum performance indices (PI) relative to the parental amylase for (i) protein expression, (ii) activity, and (iii) detergent stability or thermostability are greater than or equal to 0.5, and the PI for any one of (i), (ii), or (iii) that is greater than or equal to 1.5. 
     
     
         5 . The variant amylase of  claim 1 , wherein the combinable mutation has a suitability score of +++, ++++, or +++++, referring to Table B. 
     
     
         6 . The variant amylase of  claim 1 , wherein the combinable mutation has a suitability score of ++++, or +++++, referring to Table B. 
     
     
         7 . The variant amylase of  claim 1 , wherein the combinable mutation has a suitability score of +++++, referring to Table B. 
     
     
         8 . The variant amylase of  claim 1 , wherein the combinable mutation has a productivity score of 1 or 2. 
     
     
         9 . The variant amylase of  claim 1 , having a plurality of combinable mutations. 
     
     
         10 . The variant amylase of  claim 1 , further comprising a deletion corresponding to a residue selected from the group consisting of Arg-181, Gly-182, His-183, and Gly-184, using SEQ ID NO: 3 for numbering. 
     
     
         11 . The variant amylase of  claim 1 , further comprising deletions corresponding to residues Arg-181 and Gly-182, using SEQ ID NO: 3 for numbering. 
     
     
         12 . The variant amylase of  claim 1 , wherein the parental α-amylase or the variant α-amylase has at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variant α-amylase is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5. 
     
     
         13 . The variant amylase of  claim 1 , wherein the parental α-amylase or the variant α-amylase has at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variant α-amylase is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5. 
     
     
         14 . The variant amylase of  claim 1 , wherein the parental α-amylase or the variant α-amylase has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variant α-amylase is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5. 
     
     
         15 . The variant amylase of  claim 1 , wherein the parental α-amylase or the variant α-amylase has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or wherein the parental α-amylase or the variant α-amylase is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5. 
     
     
         16 . A composition comprising the variant amylase of  claim 1 . 
     
     
         17 . The composition of  claim 16 , wherein the composition is effective for removing starchy stains from laundry, dishes, or textiles. 
     
     
         18 . The composition of  claim 16 , further comprising a surfactant. 
     
     
         19 . The composition of  claim 16 , wherein the composition is a detergent composition. 
     
     
         20 . The composition of  claim 16 , wherein the composition is a laundry detergent or a laundry detergent additive. 
     
     
         21 . The composition of  claim 16 , wherein the composition is a manual or automatic dishwashing detergent. 
     
     
         22 . The composition of  claim 16 , further comprising one or more additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, and an amylase other than the amylase of any one of  claims 1 - 15 . 
     
     
         23 - 27 . (canceled) 
     
     
         28 . A method for removing a starchy stain or soil from a surface, comprising:
 contacting the surface in the presence of a aqueous composition comprising an effective amount of the variant amylase of  claim 1 ,   allowing the polypeptide to hydrolyze starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition, and   rinsing the surface,   thereby removing the starchy stain from the surface.   
     
     
         29 . The method of  claim 28 , wherein the aqueous composition further comprises a surfactant. 
     
     
         30 . The method of  claim 28 , wherein the surface is a textile surface. 
     
     
         31 . The method of  claim 28 , wherein the surface is on dishes. 
     
     
         32 . The method of  claim 28 , wherein the surface is a soiled hard surface. 
     
     
         33 . The method of  claim 28 , wherein the composition further comprises at least one additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, and an amylase other than the amylase of any one of  claims 1 - 15 . 
     
     
         34 - 72 . (canceled) 
     
     
         73 . A method of desizing a textile comprising contacting a desizing composition with a sized textile for a time sufficient to desize the textile, wherein the desizing composition comprises a variant α-amylase of  claim 1 . 
     
     
         74 . An isolated polynucleotide encoding a polypeptide of  claim 1 . 
     
     
         75 . An expression vector comprising the polynucleotide of  claim 74 . 
     
     
         76 . A host cell comprising the expression vector of  claim 75 .