Enzymes having glycosidase activity and methods of use thereof

Abstract

The invention relates to a polypeptide having glycosidase activity. In addition methods of designing new glycosidases and method of use thereof are also provided. The glycosidases have increased activity and stability at increased pH and Temperature.

1 . An isolated, synthetic or recombinant polypeptide having glycosidase activity comprising:
 (a) a sequence having the sequence of SEQ ID NO:28, or an enzymatically active fragment thereof;   (b) a polypeptide comprising a sequence having at least 70% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (c) a polypeptide comprising a sequence having at least 75% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (d) a polypeptide comprising a sequence having at least 80% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (e) a polypeptide comprising a sequence having at least 85% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (f) a polypeptide comprising a sequence having at least 90% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (g) a polypeptide comprising a sequence having at least 95% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof   (h) a polypeptide comprising a sequence having at least 96% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (i) a polypeptide comprising a sequence having at least 97% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof;   (j) a polypeptide comprising a sequence having at least 98% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof; or   (k) a polypeptide comprising a sequence having at least 99% sequence identity to the sequence SEQ ID NO:28, or an enzymatically active fragment thereof.   
     
     
         2 . A method of producing a polypeptide having a glycosidase activity comprising: (a) introducing a nucleic acid that encodes the polypeptide of  claim 1  into an isolated host cell, (b) culturing the host cell, (c) expressing from the host cell a polypeptide, wherein the polypeptide has glycosidase activity, and (d) isolating the polypeptide. 
     
     
         3 . A method for degrading a starch comprising use of the polypeptide of  claim 1 . 
     
     
         4 . A method for generating a glucose or a maltose comprising use of the polypeptide of  claim 1 . 
     
     
         5 . A method for conversion of biomass into a fuel or a chemical comprising use of the polypeptide of  claim 1 . 
     
     
         6 . A method for hydrolyzing a guar gum comprising use of the polypeptide of  claim 1 . 
     
     
         7 . A method for waste treatment comprising use of the polypeptide of  claim 1 . 
     
     
         8 . A method for making low lactose content milk comprising use of the polypeptide of  claim 1 . 
     
     
         9 . A method for drilling or well stimulation comprising use of the polypeptide of  claim 1 . 
     
     
         10 . A method for making a fuel comprising use of the polypeptide of  claim 1 . 
     
     
         11 . A detergent comprising a polypeptide of  claim 1 . 
     
     
         12 . A composition comprising a polypeptide of  claim 1 , wherein optionally, the composition comprises a starch, a juice, a food, a feed, a fuel, or a textile. 
     
     
         13 . A juice comprising a polypeptide of  claim 1 . 
     
     
         14 . A feed or food comprising a polypeptide of  claim 1 . 
     
     
         15 . A fuel comprising a polypeptide of  claim 1 . 
     
     
         16 . An oil- or gas-comprising composition comprising a polypeptide of  claim 1 . 
     
     
         17 . A waste composition comprising a polypeptide of  claim 1 . 
     
     
         18 . The isolated, synthetic, or recombinant polypeptide of  claim 1 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         19 . The method of  claim 2 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         20 . The method of  claim 3 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         21 . The method of  claim 4 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         22 . The method of  claim 5 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         23 . The method of  claim 6 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         24 . The method of  claim 7 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         25 . The method of  claim 8 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         26 . The method of  claim 9 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         27 . The method of  claim 10 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         28 . A method of generating a variant of a polypeptide having glycosidase activity comprising:
 (a) providing a template nucleic acid comprising a sequence encoding the polypeptide of  claim 1 , and   (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid,   wherein the variant nucleic acid encodes a polypeptide having glycosidase activity, and expressing the variant nucleic acid, thereby generating a variant polypeptide having glycosidase activity.   
     
     
         29 . The method of  claim 28 , wherein the modifications are introduced by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site specific mutagenesis, gene reassembly, gene site saturated mutagenesis or a combination thereof. 
     
     
         30 . The method of  claim 28 , wherein the method is iteratively repeated. 
     
     
         31 . A method for comparing a first sequence to a reference sequence wherein said first sequence is a polypeptide sequence of  claim 1  comprising: reading the first sequence and the reference sequence through use of a computer program which compares sequences; and determining differences between the first sequence and the reference sequence with the computer program. 
     
     
         32 . A method for identifying a feature in a sequence wherein the sequence is a polypeptide sequence of  claim 1  comprising: reading the sequence through the use of a computer program which identifies features in sequences; and identifying features in the sequences with the computer program. 
     
     
         33 . The isolated, synthetic or recombinant polypeptide of  claim 1 , wherein the polypeptide is an enzyme which is stable to heat, is heat resistant and catalyzes the hydrolysis of glycoside bonds, and wherein the enzyme is able to renature and regain activity after exposure to temperatures of from about 60° C. to 105° C. 
     
     
         34 . A method of catalyzing the hydrolysis of glycoside bonds comprising contacting a sample containing glycoside bonds with the polypeptide of  claim 1  under conditions which facilitate the hydrolysis of the glycoside bonds. 
     
     
         35 . An enzyme preparation comprising the polypeptide of  claim 1 , wherein optionally, the enzyme preparation is liquid or is dry. 
     
     
         36 . A method for modifying small molecules, comprising mixing the polypeptide of  claim 1  with a small molecule to produce a modified small molecule. 
     
     
         37 . The method of  claim 36  wherein a library of modified small molecules is tested to determine if a modified small molecule is present within the library which exhibits a desired activity. 
     
     
         38 . The method of  claim 36  wherein a specific biocatalytic reaction which produces the modified small molecule of desired activity is identified by systematically eliminating each of the biocatalytic reactions used to produce a portion of the library, and then testing the small molecules produced in the portion of the library for the presence or absence of the modified small molecule with the desired activity. 
     
     
         39 . The method of  claim 38  wherein the specific biocatalytic reactions which produce the modified small molecule of desired activity is optionally repeated. 
     
     
         40 . The method of  claim 38  or  39  wherein (a) the biocatalytic reactions are conducted with a group of biocatalysts that react with distinct structural moieties found within the structure of a small molecule, (b) each biocatalyst is specific for one structural moiety or a group of related structural moieties; and each biocatalyst reacts with many different small molecules which contain the distinct structural moiety. 
     
     
         41 . The method of  claim 28 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         42 . The isolated, synthetic or recombinant polypeptide of  claim 33 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         43 . The composition of  claim 12 , wherein the glycosidase activity comprises a pullulanase activity. 
     
     
         44 . The enzyme preparation of  claim 35 , wherein the glycosidase activity comprises a pullulanase activity.