Alpha-amylase variants with altered properties

Abstract

Disclosed are compositions comprising variants of alpha-amylase that have alpha-amylase activity and which exhibit altered properties relative to a parent AmyS-like alpha-amylase from which they are derived. The compositions comprise an additional enzyme such as a phytase. Also disclosed are methods of using the compositions, and kits related thereto.

1 - 49 . (canceled) 
     
     
         50 . An alpha-amylase variant comprising an amino acid sequence at least 95% identical to a parent hybrid alpha-amylase polypeptide comprising a combination of partial amino acid sequences deriving from at least two sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, 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: 15, and SEQ ID NO: 16, and having a substitution at an amino acid position corresponding to position 242 of SEQ ID NO: 1,
 wherein said variant has alpha-amylase activity and a T m  about 5-10° C. higher than said wild-type alpha-amylase; and   wherein the substitution at an amino acid position corresponding to position 242 of SEQ ID NO: 1 is selected from the group consisting of S242A, S242E, and S242Q.   
     
     
         51 . The alpha-amylase variant of  claim 50 , wherein the amino acid sequence is at least 98% identical to the AmyS-like alpha-amylase polypeptide sequence of SEQ ID NO: 1. 
     
     
         52 . An isolated polynucleotide encoding an alpha-amylase variant comprising an amino acid sequence at least 95% identical to a parent hybrid alpha-amylase polypeptide comprising a combination of partial amino acid sequences deriving from at least two sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, 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: 15, and SEQ ID NO: 16, and having a substitution at an amino acid position corresponding to position 242 of SEQ ID NO: 1,
 wherein said variant has alpha-amylase activity and a T m  about 5-10° C. higher than said wild-type alpha-amylase; and   wherein the substitution at an amino acid position corresponding to position 242 of SEQ ID NO: 1 is selected from the group consisting of S242A, S242E, and S242Q.   
     
     
         53 . A vector comprising the isolated polynucleotide of  claim 52 . 
     
     
         54 . An isolated host cell comprising the isolated polynucleotide of  claim 52  or the vector of  claim 53 . 
     
     
         55 . The isolated host cell of  claim 54  that is a  Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. thuringiensis, Streptomyces lividans, S. murinus; Escherichia coli , or a  Pseudomonas  spp. 
     
     
         56 . A composition comprising:
 a) the alpha-amylase variant of  claim 50 , and   b) at least one additional enzyme.   
     
     
         57 . The composition of  claim 56 , wherein the additional enzyme is a phytase. 
     
     
         58 . The composition of  claim 57 , wherein the alpha-amylase variant and the phytase are present in amounts such that the ratio of alpha-amylase units to phytase units (AAU:FTU) is about 1:15 to about 15:1. 
     
     
         59 . The composition of  claim 57 , wherein the alpha-amylase variant and the phytase are present in amounts such that the ratio of AAU:FTU is about 1:4 to about 3:1. 
     
     
         60 . The composition of  claim 57 , wherein the phytase has a sequence that is SEQ ID NO: 31. 
     
     
         61 . A method of treating a starch slurry comprising:
 a) adding to the starch slurry at least one phytase and at least one alpha-amylase variant of  claim 50 ;   wherein the phytase and the alpha-amylase variant are added at or about the same time, or separately in any order; and   b) incubating the starch slurry under conditions permissive of activity of the phytase and the alpha-amylase variant.   
     
     
         62 . The method of  claim 61 , wherein the phytase is added before the alpha-amylase variant. 
     
     
         63 . The method of  claim 62 , wherein the starch slurry is preincubated after adding the phytase and before adding the alpha-amylase variant. 
     
     
         64 . The method of  claim 61 , wherein the inclusion of the phytase results in an increase in thermostability of the alpha-amylase variant relative to a comparable method that does not include contacting the starch slurry with phytase. 
     
     
         65 . The method of  claim 61 , wherein the phytase and the alpha-amylase variant are present in a single blend before adding to the starch slurry. 
     
     
         66 . The method of  claim 61 , wherein the phytase has the amino acid sequence of SEQ ID NO: 31. 
     
     
         67 . A method of producing a fermentable substrate from a starch containing slurry comprising milled grain, the method comprising:
 a) contacting the starch-containing slurry with at least one phytase and at least one alpha-amylase variant of  claim 50  in an amount sufficient to produce a fermentable substrate from the starch-containing slurry;   wherein the contact with the phytase and the alpha-amylase variant is initiated at or about the same time, or separately in any order; and   b) incubating the starch-containing slurry under conditions permissive of activity of the phytase and the alpha-amylase variant for a time that allows production of the fermentable substrate;   wherein when the contact with the phytase is initiated before the alpha-amylase variant, the starch-containing slurry is incubated at a temperature that is 0-30° C. less than a gelatinization temperature prior to contacting the starch-containing slurry with the alpha-amylase variant, after which the temperature is raised above the gelatinization temperature for a time effective to hydrolyze starch.   
     
     
         68 . A method of treating a starch-containing material or a starch comprising contacting the starch-containing material or the starch with a composition comprising at least one alpha-amylase variant of  claim 50  under conditions sufficient to allow detectable activity of the alpha-amylase variant, wherein the starch-containing material or the starch is at least partially degraded by the alpha-amylase variant. 
     
     
         69 . The method of  claim 68  further comprising at least one additional enzyme which is a phytase, cellulase, protease, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, chitinase, cutinase, cyclodextrin glucanotransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, isomerase, laccase, lipase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, polyphenoloxidase, nuclease, ribonuclease, transglutaminase, xylanase, pullulanase, isoamylase, carrageenase, or a combination of two or more of the foregoing. 
     
     
         70 . The method of  claim 68  that is part of a process for starch degradation, liquefaction, fermentation, alcohol production, sweetener production, production of a fermentable substrate, cleaning, washing, stain removal, or baking process. 
     
     
         71 . A kit comprising, in one or more packages provided as a unit:
 i) at least one alpha-amylase variant of  claim 50 ; and   ii) at least one additional enzyme.   
     
     
         72 . The kit of  claim 71  further comprising instructions for using the alpha-amylase variant and the at least one additional enzyme in a useful process involving enzymatic cleavage of starch molecules. 
     
     
         73 . The kit of  claim 71 , wherein the additional enzyme is a phytase. 
     
     
         74 . The alpha-amylase variant of  claim 50 , further comprising an amino acid substitution at an amino acid position corresponding to position 97, 319, 358, or 443 of SEQ ID NO: 1. 
     
     
         75 . The alpha-amylase variant of  claim 50 , wherein the variant comprises an amino acid sequence at least 95% identical to the alpha-amylase polypeptide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. 
     
     
         76 . The alpha-amylase variant of  claim 50 , wherein the variant comprises an amino acid sequence at least 95% identical to the alpha-amylase polypeptide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. 
     
     
         77 . The alpha-amylase variant of  claim 50 , wherein the variant comprises an amino acid sequence at least 95% identical to the alpha-amylase polypeptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 16.