Compositions and Methods Comprising Alpha-Amylase Variants with Altered Properties

Abstract

Described are compositions and methods relating to variant alpha-amylasess having altered biochemical properties and advantageous performance characteristics as compared to a reference alpha-amylase. The variants are suitable for use in various industrial applications such as starch conversion, ethanol production, laundry, dishwashing, pulp and paper production, textile desizing, and/or sweetener production.

1 . An isolated alpha-amylase variant, wherein said variant is a mature form of alpha-amylase having amylase activity, and comprising a substitution at one or more positions selected from the group consisting of 1, 2, 3, 4, 5, 7, 15, 16, 17, 18, 19, 22, 25, 26, 28, 29, 30, 32, 35, 36, 37, 50, 51, 52, 53, 54, 55, 56, 59, 60, 70, 71, 72, 73, 75, 78, 83, 87, 90, 91, 93, 94, 95, 104, 105, 107, 108, 110, 112, 113, 116, 118, 125, 126, 128, 129, 130, 131, 134, 136, 138, 142, 144, 147, 149, 150, 152, 154, 156, 158, 160, 161, 162, 165, 166, 168, 169, 170, 172, 174, 177, 178, 182, 183, 185, 189, 192, 195, 197, 201, 202, 203, 207, 210, 214, 217, 221, 228, 234, 236, 237, 246, 250, 254, 255, 257, 264, 267, 269, 270, 272, 275, 279, 283, 284, 298, 301, 303, 305, 306, 310, 311, 314, 318, 319, 320, 322, 323, 336, 337, 338, 339, 340, 344, 359, 374, 375, 376, 377, 379, 381, 382, 393, 394, 399, 401, 407, 408, 419, 433, 436, 438, 444, 447, 448, 451, 453, 459, 465, 470, 475, 476, 483, and 484;
 wherein the positions correspond to amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2; and   wherein the substitution of the naturally-occurring amino acid residue at the one or more positions for a different amino acid residue produces an alpha-amylase variant having a performance index >1.0 for a measure of stability, and a performance index >1.0 for a measure of activity.   
     
     
         2 . The isolated alpha-amylase variant of  claim 1 , comprising a substitution at one or more positions selected from the group consisting of 7, 29, 35, 53, 60, 72, 87, 108, 116, 126, 128, 129, 130, 131, 134, 136, 138, 142, 156, 161, 165, 178, 182, 185, 189, 192, 195, 197, 202, 210, 214, 217, 221, 234, 246, 269, 303, 310, 337, 340, 374, 401, and 438, and wherein the substitution of the naturally-occurring amino acid residue for a different amino acid residue, produces an alpha-amylase variant having a performance index >1.5 for a measure of activity and a performance index >1.0 for a measure of stability. 
     
     
         3 . The isolated alpha-amylase variant of  claim 1 , comprising a substitution at one or more positions selected from the group consisting of 2, 7, 22, 25, 28, 30, 37, 70, 75, 83, 87, 91, 93, 108, 128, 160, 165, 178, 182, 183, 217, 269, 270, 279, 283, 298, 305, 306, 310, 320, 374, 375, 376, 407, 419, 475, and 476, wherein the substitution of the naturally-occurring amino acid residue for a different amino acid residue, produces an alpha-amylase variant having a performance index >1.5 for a measure of stability and a performance index >1.0 for a measure of activity. 
     
     
         4 . The isolated alpha-amylase variant of  claim 1 , comprising a substitution at one or more positions selected from the group consisting of 83, 125, 128, 131, 160, 178, 182, 183, 185, 189, 279, 305, 319, 320, 379, 407, 433, 453, 475, 476, and 483. 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The isolated alpha-amylase variant of  claim 1 , further comprising a substitution at position 243 corresponding to the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
         9 . The isolated alpha-amylase variant of  claim 1 , further comprising a deletion at position 180 and/or position 181, corresponding to the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
         10 . The alpha-amylase variant of  claim 1 , wherein the alpha-amylase variant is derived from a parent alpha-amylase having an amino acid sequence that is at least 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. 
     
     
         11 . The isolated alpha-amylase variant of  claim 1 , wherein the alpha-amylase variant has at least 75% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
         12 . The isolated alpha-amylase variant of  claim 1 , wherein the alpha-amylase variant has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
         13 . The isolated alpha-amylase variant of  claim 1 , wherein the alpha-amylase variant has at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
         14 . The alpha-amylase variant of  claim 1 , wherein the variant comprises a substitution at one or more positions selected from the group consisting of 128, 178, 182, 185, and 189 corresponding to the amino acid sequence set forth in SEQ ID: 2, wherein the substitution provides improved cleaning performance or improved detergent stability. 
     
     
         15 . The alpha-amylase variant of  claim 1 , wherein the alpha-amylase variant comprises:
 (a) an alanine at position 125,
 a cysteine at position 128, 
 an isoleucine at position 131, 
 an isoleucine at position 165, 
 a leucine at position 178, 
 a glycine at position 182, 
 a tyrosine at position 202, 
 an arginine at position 305, 
 a threonine at position 319, or 
 an arginine at position 475; 
   (b) the substitutions N128C+K178L+T182G+Y305R+G475K, and
 at least one additional substitution selected from the group consisting of S125A, T131I, T165I, F202Y, and D319T; or 
   (c) the substitutions
 N128C+K178L+T182G+F202Y+Y305R+D319T+G475K, 
 S125A+N128C+K178L+T182G+Y305R+G475K, or 
 S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K; 
   wherein the variant optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181; and   wherein the positions correspond to the amino acid sequence set forth in SEQ ID NO: 2.   
     
     
         16 . The alpha-amylase variant of  claim 1 , wherein said alpha-amylase variant comprises a substitution at position 475. 
     
     
         17 - 22 . (canceled) 
     
     
         23 . An isolated nucleic acid encoding the alpha-amylase variant of  claim 1 . 
     
     
         24 . An expression vector comprising the isolated nucleic acid of  claim 23 , in operable combination with a promoter. 
     
     
         25 . A host cell comprising the expression vector of  claim 24 . 
     
     
         26 . A cleaning composition comprising the alpha-amylase variant of  claim 1 . 
     
     
         27 . The cleaning composition of  claim 26 , further comprising at least one additional enzyme selected from the group consisting of a protease, a lipase, a cutinase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a perhydrolase, a pectate lyase, and a peroxidase. 
     
     
         28 . The cleaning composition of  claim 27 , wherein the at least one additional enzyme is a protease. 
     
     
         29 . The cleaning composition of  claim 28 , wherein the at least one additional enzyme is a subtilisin. 
     
     
         30 . The cleaning composition of  claim 29 , wherein the at least one additional enzyme is subtilisin BPN′ or a variant, thereof. 
     
     
         31 . The cleaning composition of  claim 30 , wherein the at least one additional enzyme is subtilisin BPN′Y217L or a variant, thereof. 
     
     
         32 - 38 . (canceled)