Geobacillus stearothermophilus alpha amylase (amys) variants with improved properties

Abstract

Described are variants of a parent α-amylase that exhibits an alteration in at least one of the following properties relative to said parent α-amylase: specific activity, substrate specificity, substrate binding, substrate cleavage, thermal stability, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, pI, and wash performance. The variants are suitable for starch conversion, ethanol production, laundry washing, dish washing, hard surface cleaning, textile desizing, and/or sweetener production.

1 .- 28 . (canceled) 
     
     
         29 . A method for hydrolyzing a soluble starch substrate using an α-amylase variant, comprising contacting the starch substrate with an α-amylase variant having alpha amylase activity and at least one altered characteristic that improves enzyme performance, the variant comprising:
 an amino acid sequence having at least 90% sequence identity to the parental α-amylase polypeptide of SEQ ID NO: 2, and 
 having a substitution that introduces the amino acid residue E188P, wherein the α-amylase variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide. 
 
     
     
         30 . The method of  claim 29 , further comprising contacting the starch substrate with a phytase. 
     
     
         31 . The method of  claim 30 , wherein the contacting with a phytase is prior to or simultaneous with the contacting with the α-amylase variant. 
     
     
         32 . A method for liquefying starch in a slurry, the slurry having been produced from a substrate comprising plant material in either a dry or wet milling process, said method comprising:
 a primary and/or secondary liquefaction step comprising adding to the slurry in the primary or secondary liquefaction step, in any order, a combination of at least one phytic acid hydrolyzing enzyme and at least one α-amylase variant, either simultaneously or separately to produce liquefied starch, wherein the α-amylase variant comprises   an amino acid sequence having at least 90% sequence identity to the parental α-amylase polypeptide of SEQ ID NO: 2, and   having a substitution that introduces the amino acid residue E188P, wherein the α-amylase variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide.   
     
     
         33 . The method of  claim 32 , wherein the starch is granular starch. 
     
     
         34 . The method of  claim 32 , further comprising saccharifying the liquefied starch to obtain fermentable sugars. 
     
     
         35 . The method of  claim 34 , further comprising fermenting the fermentable sugars under suitable fermentation conditions to obtain end-products such as alcohol. 
     
     
         36 . The method of  claim 35 , wherein the alcohol end-product is ethanol. 
     
     
         36 . The method of  claim 32 , wherein the phytic acid hydrolyzing enzyme is phytase. 
     
     
         37 . The method of  claim 32 , wherein the primary or secondary liquefaction step does not include pH adjustment of the slurry. 
     
     
         38 . The method of  claim 37 , wherein the pH of the primary or secondary liquefaction is in the range of pH 4.5 to 5.4. 
     
     
         39 . The method of  claim 37 , wherein the pH of the primary or secondary liquefaction is in the range of 4.8 to 5.8. 
     
     
         40 . A method of producing alcohol from starch slurry comprising:
 a primary and/or secondary liquefaction step comprising adding to the starch slurry in the primary or secondary liquefaction step, in any order, a combination of a phytic acid hydrolyzing enzyme and an α-amylase variant, either simultaneously or separately to produce liquefied starch, wherein the α-amylase variant comprises   an amino acid sequence having at least 90% sequence identity to the parental α-amylase polypeptide of SEQ ID NO: 2, and   having a substitution that introduces the amino acid residue E188P, wherein the α-amylase variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide,   a saccharification step comprising saccharifying the liquefied starch to obtain fermentable sugars to produce fermentable sugars,   fermenting the fermentable sugars under suitable fermentation conditions using a fermenting microorganism to produce alcohol.   
     
     
         41 . The method of  claim 40  wherein the phytic acid hydrolyzing enzyme is phytase. 
     
     
         42 . The method of  claim 40  wherein the alcohol is ethanol. 
     
     
         43 . The method of  claim 40 , wherein the primary or secondary liquefaction step does not include pH adjustment of the slurry. 
     
     
         44 . The method of  claim 43 , wherein the pH of the primary or secondary liquefaction is in the range of pH 4.5 to 5.4. 
     
     
         45 . The method of  claim 43 , wherein the pH of the primary or secondary liquefaction is in the range of 4.8 to 5.8.