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.

What is claimed is: 
     
         1 . A variant α-amylase polypeptide derived from a parental α-amylase polypeptide, comprising at least two combinable mutations at productive amino acid positions; wherein:
 (i) each 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) a 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 wherein, 
 the combinable mutations produce 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, or 
 the combinable mutations produce 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. 
 
     
     
         2 . The variant amylase of  claim 1 , wherein the combinable mutation has a suitability score of +++, ++++, or +++++, referring to Table B. 
     
     
         3 . The variant amylase of  claim 1 , wherein the combinable mutation has a suitability score of ++++, or +++++, referring to Table B. 
     
     
         4 . The variant amylase of  claim 1 , wherein the combinable mutation has a suitability score of +++++, referring to Table B. 
     
     
         5 . The variant amylase of  claim 1 , wherein the combinable mutation has a productivity score of 1 or 2. 
     
     
         6 . The variant amylase of  claim 1 , having a plurality of combinable mutations. 
     
     
         7 . 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. 
     
     
         8 . The variant amylase of  claim 1 , further comprising deletions corresponding to residues Arg-181 and Gly-182, using SEQ ID NO: 3 for numbering. 
     
     
         9 . 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. 
     
     
         10 . 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. 
     
     
         11 . 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. 
     
     
         12 . 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. 
     
     
         13 . A composition comprising the variant amylase of  claim 1 . 
     
     
         14 . The composition of  claim 13 , wherein the composition is effective for removing starchy stains from laundry, dishes, or textiles. 
     
     
         15 . The composition of  claim 13 , further comprising a surfactant.