3-epimerase

Abstract

A protein comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6, SEQ ID NO: 2 or SEQ ID NO: 4. The protein has ketose 3-epimerase activity.

1 . A protein comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6, SEQ ID NO: 2 or SEQ ID NO: 4, wherein the protein has ketose 3-epimerase activity. 
     
     
         2 . A protein according to  claim 1 , wherein the polypeptide sequence has at least 90% sequence identity to SEQ ID NO: 6, SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         3 . A protein according to  claim 1 , wherein the polypeptide sequence comprises the sequence of SEQ ID NO: 13. 
     
     
         4 . A protein according to  claim 1 , wherein the protein is immobilized on a solid substrate. 
     
     
         5 . A nucleic acid molecule comprising a polynucleotide sequence encoding a protein according to  claim 1 . 
     
     
         6 . A nucleic acid molecule according to  claim 5 , comprising a polynucleotide sequence which:
 i) has at least 70% sequence identity to SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3; or   ii) hybridizes under highly stringent conditions to a polynucleotide having a sequence complementary to the sequence set forth in SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3.   
     
     
         7 . A vector comprising a nucleic acid molecule according to  claim 5 . 
     
     
         8 . A host cell comprising a recombinant nucleic acid molecule according to  claim 5 . 
     
     
         9 . A host cell according to  claim 8 , wherein the host cell is a yeast, bacterium or other microorganism, or is a mammalian, plant or other cell culture. 
     
     
         10 . A host cell according to  claim 9 , wherein the host cell is  E. coli.    
     
     
         11 . Allulose produced by a protein according to  claim 1 . 
     
     
         12 . A method of producing allulose comprising contacting a protein according to  claim 1  with a fructose substrate under conditions such that the fructose substrate is converted into allulose. 
     
     
         13 . A method according to  claim 12 , wherein the protein is present in a host cell. 
     
     
         14 . A method according to  claim 12 , wherein the protein is in isolated form. 
     
     
         15 . A method according to  claim 12 , wherein the conditions comprise maintaining the protein and the fructose substrate at a temperature between 25° C. and 75° C. 
     
     
         16 . A method according to  claim 12 , wherein the conditions comprise maintaining the protein and the fructose substrate between pH 4 and pH10. 
     
     
         17 . A method according to  claim 12 , wherein the conditions comprise maintaining the fructose substrate concentration between 75% and 95% (WN). 
     
     
         18 . A nucleic acid molecule comprising a polynucleotide sequence which:
 i) has at least 70% sequence identity to SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3; or   ii) hybridizes under highly stringent conditions to a polynucleotide having a sequence complementary to the sequence set forth in SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3.   
     
     
         19 . A host cell comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having ketose 3-epimerase activity, wherein the polynucleotide sequence:
 i) has at least 70% sequence identity to SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3; or   ii) hybridizes under highly stringent conditions to a polynucleotide having a sequence complementary to the sequence set forth in SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3.   
     
     
         20 . A vector comprising a nucleic acid molecule comprising a polynucleotide encoding a polypeptide having ketose 3-epimerase activity, wherein the polynucleotide sequence:
 i) has at least 70% sequence identity to SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3; or   ii) hybridizes under highly stringent conditions to a polynucleotide having a sequence complementary to the sequence set forth in SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3.   
     
     
         21 . A method of producing allulose comprising the steps of:
 i) providing a vector comprising a nucleic acid molecule having a polynucleotide sequence encoding a protein having ketose 3-epimerase activity wherein the polynucleotide sequence: a) has at least 70% sequence identity to SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3; or b) hybridizes under highly stringent conditions to a polynucleotide having a sequence complementary to the sequence set forth in SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3;   ii) synthesising the protein having ketose 3-epimerase activity encoded by the polynucleotide sequence;   iii) contacting fructose with the protein having ketose 3-epimerase activity and maintaining the fructose and protein under conditions to permit the conversion of fructose to allulose; and   iv) at least partially purifying the allulose produced in step iii).