Chimeric alpha-amylase variants

Abstract

Chimeric alpha amylases having the characteristics of high thermostability and good performance in starch degradation, especially high-temperature liquefaction processes, are provided. The alpha-amylases are chimeras of AmyL and AmyS enzymes, and are useful in starch degradation processes. Methods of making the chimeric enzymes, and methods of using the chimeric alpha-amylases for liquefaction, cleaning starch residue from a surface, and treating woven material to remove coatings. Kits for practicing the methods are provided. Polynucleotides encoding the chimeric amylases, vectors, and expression hosts also are provided.

1 . A chimeric polypeptide having a length of about 480-515 amino acid residues, the chimeric polypeptide having (a) an amino-terminal domain comprising 180 or more contiguous amino acid residues of an N-terminal portion of an AmyL amylase, and (b) a carboxy-terminal domain comprising a carboxy-terminal portion of an AmyS amylase; with the proviso that the chimeric polypeptide is not a wild-type AmyL amylase or AmyS amylase, and wherein said chimeric polypeptide has enhanced thermostability relative at least to the AmyS amylase. 
     
     
         2 . The chimeric polypeptide of  claim 1  comprising at least one substituted amino acid residue in the amino-terminal domain, relative to the AmyL amylase, so as to provide at least a part of a stabilizing structure that enhances thermostability of the polypeptide. 
     
     
         3 . The chimeric polypeptide of  claim 2 , wherein the stabilizing structure is a salt bridge formed, at least in part, by the substituted amino acid residue. 
     
     
         4 . The chimeric polypeptide of  claim 2 , wherein the substituted amino acid residue corresponds to position 187 or 188, or both, in the AmyL or AmyS amylase. 
     
     
         5 . The chimeric polypeptide of  claim 4 , wherein an Asp residue and a Thr residue are substituted in the chimeric polypeptide for two consecutive Ser residues in the AmyL amylase. 
     
     
         6 . The chimeric polypeptide of  claim 1 , wherein the AmyL amylase has the amino acid sequence of SEQ ID NO: 1. 
     
     
         7 . The chimeric polypeptide of  claim 1 , wherein the AmyS amylase has the amino acid sequence of SEQ ID NO: 2. 
     
     
         8 . The chimeric polypeptide of  claim 1  having at least about 95% sequence identity to any of SEQ ID NOS: 1-17, with the proviso that the chimeric polypeptide is not SEQ ID NOS: 1 or 2. 
     
     
         9 . The chimeric polypeptide of  claim 8  wherein an Asp residue and a Thr residue are substituted in the chimeric polypeptide for two consecutive Ser residues at position 187 in the AmyL amylase. 
     
     
         10 . The chimeric polypeptide of  claim 1  comprising a catalytic activity of an α-amylase that retains at least 50% of its activity after incubation at 95° C. for 30 minutes. 
     
     
         11 . The chimeric polypeptide of  claim 10  that retains at least about 60% of its catalytic activity after incubation at 95° C. for 60 minutes. 
     
     
         12 . The chimeric polypeptide of  claim 11  which retains at least about 80% of its catalytic activity after incubation at 95° C. for 60 minutes. 
     
     
         13 . The chimeric polypeptide of  claim 1  comprising a catalytic activity of an α-amylase that has greater specific activity than the AmyL amylase. 
     
     
         14 . A thermostable chimeric α-amylase comprising an N-terminal portion and a C-terminal portion; the N-terminal portion comprising a contiguous amino acid sequence from an N-terminal portion of an AmyL amylase, and the C-terminal portion comprising a contiguous amino acid sequence from a C-terminal portion of an AmyS amylase; wherein the chimeric α-amylase has a specific activity greater than the AmyL amylase, and greater thermostability at 95° C. than the AmyS amylase, and having a primary amino acid sequence that is about 475-520 residues long. 
     
     
         15 . The chimeric amylase of  claim 14  which, when used in a liquefaction process for a starch slurry, has about equivalent peak viscosity reduction of the starch slurry as AmyS amylase in a comparable liquefaction process, and reduces final viscosity of the starch slurry as well as the AmyL amylase in a comparable liquefaction process. 
     
     
         16 . The chimeric amylase of  claim 15  that retains at least 50% of its specific activity after incubation at 95° C. for 30 minutes. 
     
     
         17 . The chimeric amylase of  claim 16  that retains at least 80% of it specific activity after incubation at 95° C. for 60 minutes. 
     
     
         18 . The chimeric amylase of  claim 15 , wherein the AmyL amylase has the sequence of SEQ ID NO: 1 and the AmyS amylase has the sequence of SEQ ID NO: 2. 
     
     
         19 . The chimeric amylase of  claim 17  having at least about 95% sequence identity to any of SEQ ID NOS: 1-17, with the proviso that the chimeric amylase is not SEQ ID NO: 1 or 2. 
     
     
         20 . The chimeric amylase of  claim 19  further comprising at least one substituted amino acid residue in the N-terminal domain, relative to the AmyL amylase, so as to provide at least a part of a stabilizing structure that enhances thermostability of the polypeptide. 
     
     
         21 . The chimeric polypeptide of  claim 20 , wherein the stabilizing structure is a salt bridge formed, at least in part, by the substituted amino acid residue. 
     
     
         22 . The chimeric polypeptide of  claim 21 , wherein the substituted amino acid residue corresponds to position 187 or 188 or both in the AmyL or AmyS amylase. 
     
     
         23 . The chimeric polypeptide of  claim 22 , wherein an Asp residue and a Thr residue are substituted in the chimeric polypeptide for two consecutive Ser residues in the AmyL amylase. 
     
     
         24 . A composition comprising one or more of:
 a chimeric polypeptide having a length of about 480-515 amino acid residues, having an amino-terminal domain comprising about 180 or more contiguous amino acid residues of an N-terminal portion of an AmyL amylase, and a carboxy-terminal domain comprising a carboxy-terminal portion of an AmyS amylase; said chimeric polypeptide having enhanced thermostability relative at least to the AmyS amylase; or   a thermostable chimeric α-amylase about 475-520 amino acid residues long, having an N-terminal portion comprising a contiguous amino acid sequence from an N-terminal portion of an AmyL amylase, and a C-terminal portion comprising a contiguous amino acid sequence from a C-terminal portion of an AmyS amylase, said chimeric α-amylase having a specific activity greater than the AmyL amylase and greater thermostability at 95° C. than the AmyS amylase; or   a combination thereof.   
     
     
         25 . The composition of  claim 24  further comprising one or more additional polypeptides. 
     
     
         26 . The composition of  claim 25 , wherein the one or more additional polypeptides is an enzyme. 
     
     
         27 . The composition of  claim 24  that includes one or more detergents or cleaning agents. 
     
     
         28 . The composition of  claim 24  that is formulated for use in food or food processes. 
     
     
         29 . A food-grade lyophilized composition comprising the composition of  claim 24 . 
     
     
         30 . A polynucleotide encoding:
 a chimeric polypeptide having a length of about 480-515 amino acid residues, and having an amino-terminal domain comprising about 180 or more contiguous amino acid residues of an N-terminal portion of an AmyL amylase, and a carboxy-terminal domain comprising a carboxy-terminal portion of an AmyS amylase; said chimeric polypeptide having enhanced thermostability relative at least to the AmyS amylase; or   a thermostable chimeric α-amylase about 475-520 amino acid residues long, and having an N-terminal portion comprising a contiguous amino acid sequence from an N-terminal portion of an AmyL amylase, and a C-terminal portion comprising a contiguous amino acid sequence from a C-terminal portion of an AmyS amylase, said chimeric α-amylase having a specific activity greater than the AmyL amylase, and greater thermostability at 95° C. than the AmyS amylase.   
     
     
         31 . The polynucleotide of  claim 30  that encodes a polypeptide having at least about 95% sequence identity to any of SEQ ID NOS: 1-17, with the proviso that said polypeptide is not SEQ ID NO: 1 or 2. 
     
     
         32 . The polynucleotide of  claim 31 , wherein the codon usage is optimized for expression of the chimeric polypeptide in a microorganism or a plant. 
     
     
         33 . A vector comprising the polynucleotide of  claim 32 . 
     
     
         34 . A bacterial cell comprising the vector of  claim 33 , wherein the vector is an expression vector. 
     
     
         35 . A host cell that expresses the polynucleotide of  claim 30 . 
     
     
         36 . The host cell of  claim 35 , wherein the host cell is a bacteria or a plant. 
     
     
         37 . The host cell of  claim 36  that is from an organism acceptable for the production of food processing aids. 
     
     
         38 . The host cell of  claim 35  that is a  Bacillus licheniformis, Bacillus subtilis , or  Bacillus stearothermophilus.    
     
     
         39 . The host cell of  claim 36 , wherein the host cell is a plant and the plant is used for ethanol production. 
     
     
         40 . A method of producing a composition comprising a chimeric polypeptide or a thermostable α-amylase the method comprising:
 utilizing a host cell selected from the group consisting of  Bacillus licheniformis, B. subtilis , and  B. stearothermophilus , for a fermentation process wherein a protein is expressed, said protein comprising:
 (a) a chimeric polypeptide having a length of about 480-515 amino acid residues, having an amino-terminal domain comprising about 180 or more contiguous amino acid residues of an N-terminal portion of an AmyL amylase, and a carboxy-terminal domain comprising a carboxy-terminal portion of an AmyS amylase; said chimeric polypeptide having enhanced thermostability relative at least to the AmyS amylase; or 
 (b) a thermostable chimeric α-amylase about 475-520 amino acid residues long, having an N-terminal portion comprising a contiguous amino acid sequence from an N-terminal portion of an AmyL amylase, and a C-terminal portion comprising a contiguous amino acid sequence from a C-terminal portion of an AmyS amylase, said chimeric α-amylase having a specific activity greater than the AmyL amylase and greater thermostability at 95° C. than the AmyS amylase, and 
 
 at least partially purifying the expressed polypeptide, thereby producing the composition. 
 
     
     
         41 . The method of  claim 40 , wherein the fermentation process is a fed-batch fermentation process. 
     
     
         42 . The method of  claim 40  further comprising the step of further purifying the polypeptide expressed to make a purified composition having no genotoxic potential in in vitro assays; and no toxic effects in acute and sub-chronic dosing studies in animals. 
     
     
         43 . The method of  claim 42 , wherein the purified composition comprises not more than 40 ppm total heavy metals, not more than 5 ppm arsenic, not more than 10 ppm lead, not more than 5×104 total viable organisms (CFU/g), not more than 30 coliforms (CFU/g), and no detectable  Salmonella , mycotoxins or antibacterial activity by standard tests. 
     
     
         44 . The method of  claim 40 , wherein the composition comprises more than one α-amylase activity. 
     
     
         45 . A method of liquefying a starch slurry comprising:
 making a slurry comprising a starch,   heating the slurry to an acceptable temperature for liquefaction,   adding to the slurry, a composition comprising one or more of:
 a chimeric polypeptide having a length of about 480-515 amino acid residues, having an amino-terminal domain comprising about 180 or more contiguous amino acid residues of an N-terminal portion of an AmyL amylase, and a carboxy-terminal domain comprising a carboxy-terminal portion of an AmyS amylase; said chimeric polypeptide having enhanced thermostability relative at least to the AmyS amylase; or 
 a thermostable chimeric α-amylase about 475-520 amino acid residues long, having an N-terminal portion comprising a contiguous amino acid sequence from an N-terminal portion of an AmyL amylase, and a C-terminal portion comprising a contiguous amino acid sequence from a C-terminal portion of an AmyS amylase, said chimeric α-amylase having a specific activity greater than the AmyL amylase, and greater thermostability at 95° C. than the AmyS amylase, or 
 a combination thereof, and 
   incubating the slurry with the composition for a time and at a temperature sufficient to liquefy the starch slurry.   
     
     
         46 . The method of  claim 26 , wherein the addition of the composition
 reduces the peak viscosity of the slurry as well as the addition of an AmyS amylase used in a comparable liquefaction, and   reduces the final viscosity of the slurry as well as the addition of an AmyL amylase used in a comparable liquefaction.   
     
     
         47 . The method of  claim 46 , wherein the temperature is at least about 80° C. to about 100° C. 
     
     
         48 . The method of  claim 45 , wherein the slurry comprises about 15-40% starch on a dry-weight basis. 
     
     
         49 . The method of  claim 48 , wherein the liquefaction is part of a fermentation. 
     
     
         50 . The method of  claim 49 , wherein the fermentation is used to produce a food product, a food additive, a fuel, or a fuel additive. 
     
     
         51 . The method of  claim 50 , wherein the fuel or fuel additive is an alcohol. 
     
     
         52 . The method of  claim 51 , wherein the alcohol is ethanol. 
     
     
         53 . A method of cleaning a surface to remove starch residue comprising the steps of providing a surface that has starch residue to be removed, contacting the surface with a composition of  claim 24 , for a time and at a temperature sufficient to result in removal of the starch residue. 
     
     
         54 . The method of  claim 53 , wherein the composition comprises one or more of a protease, a lipase, an additional amylase, or a combination thereof. 
     
     
         55 . The method of  claim 54  further comprising a step of rinsing or bulk removal of residue prior to the contacting step. 
     
     
         56 . The method of  claim 53 , wherein the temperature during the contacting step reaches at least 50-100° C. 
     
     
         57 . A method of treating a woven material that has been previously subjected to contact with a coating comprising starch or a starch-derivative, the method comprising contacting the woven material with a solution comprising a composition according to  claim 24  for a time and under conditions sufficient to substantially remove the coating from the woven material. 
     
     
         58 . The method of  claim 57 , wherein the woven material is a fabric. 
     
     
         59 . The method of  claim 57 , wherein the contacting step is performed at a pressure that is greater then ambient atmospheric pressure. 
     
     
         60 . A kit for facilitating liquefaction of starch slurry, said kit comprising at least one of:
 a chimeric polypeptide having a length of about 480-515 amino acid residues, and comprising an amino-terminal domain comprising about 180 or more contiguous amino acid residues of an N-terminal portion of an AmyL amylase, and a carboxy-terminal domain comprising a carboxy-terminal portion of an AmyS amylase; said chimeric polypeptide having enhanced thermostability relative at least to the AmyS amylase; or   a thermostable chimeric α-amylase about 475-520 amino acid residues long, and comprising an N-terminal portion comprising a contiguous amino acid sequence from an N-terminal portion of an AmyL amylase, and a C-terminal portion comprising a contiguous amino acid sequence from a C-terminal portion of an AmyS amylase, said chimeric α-amylase having a specific activity greater than the AmyL amylase and greater thermostability at 95° C. than the AmyS amylase, and   instructions for use of the kit in the liquefaction of a starch slurry.