Enzymes Having Alpha Amylase Activity and Methods of Making and Using them

Abstract

The invention relates to alpha amylases and to polynucleotides encoding the alpha amylases. In addition methods of designing new alpha amylases and methods of use thereof are also provided. The alpha amylases have increased activity and stability at acidic, neutral and alkaline pH and increased temperature.

What is claimed is: 
     
         1 . An isolated, synthetic, or recombinant polypeptide comprising:
 (a) an amino acid sequence having at least 85% sequence identity to SEQ ID NO:102, wherein the polypeptide has alpha-amylase activity;   (b) a polypeptide encoded by a nucleic acid having at least 85% sequence identity to SEQ ID NO:101, wherein the polypeptide has alpha-amylase activity; or   (c) an amino acid sequence comprising an enzymatically active fragment of the polypeptide of (a) or (b), wherein the fragment has alpha-amylase activity.   
     
     
         2 . (canceled) 
     
     
         3 . An isolated, synthetic, or recombinant polypeptide having alpha amylase activity comprising the amino acid sequence of SEQ ID NO:102, or encoded by the nucleic acid sequence of SEQ ID NO:101. 
     
     
         4 . A method of hydrolyzing a polysaccharide or oligosaccharide linkage comprising
 (a) providing the polypeptide of  claim 1 , and   (b) contacting a substance containing the polysaccharide or oligosaccharide with the polypeptide of  claim 1 , under conditions which facilitate the hydrolysis of the polysaccharide or oligosaccharide linkage.   
     
     
         5 . A method for washing an object comprising
 (a) providing the polypeptide of  claim 1 , and   (b) contacting said object with the polypeptide of  claim 1  under conditions sufficient for said washing.   
     
     
         6 . A method for textile processing comprising
 (a) providing the polypeptide of  claim 1 , and   (b) contacting a textile with the polypeptide of (a) under conditions effective for said desizing; or   (c) the method of (a) and (b), wherein the method is for textile desizing.   
     
     
         7 . A method for treatment of lignocellulosic fibers comprising
 (a) providing the polypeptide of  claim 1 , and   (b) contacting the lignocellulosic fibers with the polypeptide of (a), under conditions effective for hydrolyzing the lignocellulosic fibers, thereby modifying a property of the lignocellulosic fiber.   
     
     
         8 . A method for producing a high-maltose, a high-glucose syrup or a mixed syrup comprising
 (a) providing the polypeptide of  claim 1 , and   (b) liquefying a polysaccharide or oligosaccharide using an effective amount of the polypeptide of (a) to obtain a soluble polysaccharide or oligosaccharide hydrolysate; and saccharifying the soluble polysaccharide or oligosaccharide hydrolysate, thereby producing a high-maltose, a high-glucose syrup or a mixed syrup.   
     
     
         9 . A method for making an alcohol comprising
 (a) providing the polypeptide of  claim 1 ,   (b) contacting a polysaccharide-comprising or oligosaccharide-comprising composition with the polypeptide of (a), to produce a sugar, and   (c) fermenting the sugar of (b) to produce alcohol; or   (d) the method of (a) to (d), wherein the alcohol is ethanol.   
     
     
         10 . A method for corn wet milling comprising
 (a) providing the polypeptide of  claim 1 , and   (b) contacting a corn or a corn starch with the polypeptide of (a) under conditions sufficient for the polypeptide to hydrolyze an oligosaccharide or a polysaccharide in the corn or corn starch.   
     
     
         11 . A method for dry milling comprising:
 (a) providing the polypeptide of  claim 1 , and   (b) contacting a whole ground grain with the polypeptide of (a), under conditions sufficient for the polypeptide to hydrolyze an oligosaccharide or a polysaccharide in the whole ground grain.   
     
     
         12 . A method for baking comprising contacting a dough, a baking premix, a baking additive or a baked product with the polypeptide of  claim 1 . 
     
     
         13 . The isolated, synthetic or recombinant polypeptide of  claim 1 , further comprising at least one glycosylation site or wherein the polypeptide is glycosylated. 
     
     
         14 . A composition comprising the polypeptide of  claim 1 . 
     
     
         15 . The composition of  claim 14 , wherein the composition is a detergent, a detergent additive, a food, a food supplement, a feed supplement, or a feed. 
     
     
         16 . An isolated, synthetic, or recombinant polypeptide comprising:
 (a) an amino acid sequence having at least 85% sequence identity to SEQ ID NO:102, and having at least one or more conservative amino acid substitutions, deletions or insertions, wherein the polypeptide has alpha amylase activity, wherein the conservative amino acid substitutions comprise: substitution of a isoleucine, valine, leucine or methionine for another; substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine, and having an alpha amylase activity; or   (b) an amino acid sequence comprising an enzymatically active fragment of (a), wherein the fragment has alpha-amylase activity.   
     
     
         17 . A method for liquefying or removing a polysaccharide or an oligosaccharide from a composition comprising:
 (a) providing the polypeptide of  claim 1 ;   (b) providing a composition comprising a polysaccharide or an oligosaccharide; and   (c) contacting the composition of (b) with the polypeptide of (a), under conditions wherein the polypeptide removes or liquefies the polysaccharide or the oligosaccharide.   
     
     
         18 . A method for making a food or a feed comprising contacting a food, a feed or an oligosaccharide or polysaccharide comprising composition with the polypeptide of  claim 1 . 
     
     
         19 . A method of producing sugar comprising contacting a composition comprising an oligosaccharide or polysaccharide with the polypeptide of  claim 1 . 
     
     
         20 . A method for pulp, paper or fiber processing comprising
 (a) contacting a pulp, a paper, a recycled paper or a fiber with the polypeptide of  claim 1 , or   (b) the method of (a), wherein the process is for deinking of the pulp, paper, recycled paper, or fiber.   
     
     
         21 . A method for drilling comprising contacting a drilling or a production fluid with the polypeptide of  claim 1 . 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The isolated, synthetic, or recombinant polypeptide of  claim 1 , wherein the sequence identity to SEQ ID NO: 101 or to SEQ ID NO: 102, is at least 97%. 
     
     
         25 . (canceled)