Enzymes having pullulanase activity

Abstract

The present invention relates to thermostable pullulanases useful for industrial and scientific purposes. The present invention provides methods for producing the modified pullulanase, enzymatic compositions comprising the modified pullulanase, and methods for use of the enzymatic compositions.

What is claimed is: 
     
         1 . An isolated, synthetic, or recombinant nucleic acid encoding a polypeptide having pullulanase activity, selected from the group consisting of:
 (a) a nucleic acid encoding a polypeptide having pullunase activity comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:1;   (b) a nucleic acid encoding a polypeptide having pullulanase activity comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:1, or a fragment thereof, wherein the fragment encodes a polypeptide having pullulanase activity;   (c) a nucleic acid sequence encoding a polypeptide having pullulanase activity comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2;   (d) a nucleic acid sequence encoding a polypeptide having pullulanase activity comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, or a fragment thereof, wherein the fragment has pullulanase activity;   (e) the nucleic acid of (a), (b), (c), or (d) encoding a polypeptide having pullulanase activity but lacking a signal sequence or a carbohydrate binding module;   (f) the nucleic acid of (a), (b), (c), (d) or (e) encoding a polypeptide having pullulanase activity, and further comprising a heterologous sequence;   (g) the nucleic acid sequence of (g), wherein the heterologous sequence comprises a sequence encoding a heterologous signal sequence, carbohydrate binding module, catalytic domain (CD), or a combination thereof, or the heterologous signal sequence, carbohydrate binding module, or catalytic domain (CD) is derived from another pullulanase enzyme, or a non-pullulanase enzyme; or   (h) a nucleic acid sequence fully complementary to (a), (b), (c), (d), (e), (f), or (g).   
     
     
         2 . The isolated, synthetic, or recombinant nucleic acid of  claim 1 , wherein the pullulanase activity comprises the cleavage of both alpha-1,6 and alpha-1,4 bonds. 
     
     
         3 . The isolated, synthetic, or recombinant nucleic acid of  claim 1 , wherein the pullulanase activity comprises type II pullulanase activity. 
     
     
         4 . The isolated, synthetic, or recombinant nucleic acid of  claim 1 , wherein the pullulanase activity is thermostable and or thermotolerant. 
     
     
         5 . An isolated, synthetic, or recombinant polypeptide having pullulanase comprising
 (a) an amino acid sequence having at least 50% identity, or complete sequence identity to sequence of SEQ ID No. 2;   (b) having the amino acid sequence encoded by the nucleic acid of  claim 1 ;   (c) the amino acid sequence of (a) or (b), and comprising at least one conservative amino acid residue conservative substitutions;   (d) the amino acid sequence of (a) or (b) or (c) or a fragment thereof with pullulanase activity.   
     
     
         6 . The isolated, synthetic, or recombinant polypeptide of  claim 5 , where the pullulanase activity is thermostable. 
     
     
         7 . The isolated, synthetic, or recombinant polypeptide of  claim 5  wherein the polypeptide retains an pullulanase activity under conditions comprising a temperature range of between about 37° C. to about 84° C., or between about 55° C. to about 85° C., or between about 70° C. to about 84° C. 
     
     
         8 . The isolated, synthetic, or recombinant polypeptide of  claim 5 , where the pullulanase activity is thermotolerant. 
     
     
         9 . The isolated, synthetic, or recombinant nucleic acid of  claim 5 , wherein the polypeptide retains an pullulanase activity after exposure to a temperature in the range from greater than 37° C. to about 84° C., from greater than 55° C. to about 84° C. 
     
     
         10 . A method of hydrolyzing a starch linkage comprising contacting a substance containing the starch with a polypeptide of  claim 5 , and sequences substantially identical thereto. 
     
     
         11 . A detergent additive comprising with a polypeptide of  claim 5 . 
     
     
         12 . A method of producing ethanol comprising use of the polypeptide of  claim 5 . 
     
     
         13 . The method of  claim 12 , wherein the polypeptide is capable of digesting limit dextrans. 
     
     
         14 . The method of  claim 10  or  11  or  12 , further comprising addition of an pullulanase or a combination thereof. 
     
     
         15 . A detergent composition comprising the polypeptide of  claim 5 .