Cellobiose 2-epimerase, its preparation and uses

Abstract

The present invention has objects to provide a thermostable cellobiose 2-epimerase, its preparation and uses. The present invention attains the above objects by providing a thermostable cellobiose 2-epimerase, a DNA encoding the enzyme, a recombinant DNA and transformant comprising the DNA, a process for producing the enzyme, and a process for producing isomerized saccharides using the enzyme.

1 - 14 . (canceled) 
     
     
         15 . A purified cellobiose 2-epimerase which catalyzes the following isomerization:
 (1) 2-Epimerization   epimerizing 2-hydroxyl group of D-glucose and D-galactose to convert into corresponding D-mannose and D-talose, and vice versa;   epimerizing 2-hydroxyl group of reducing end glucose in maltose, cellobiose, and lactose to convert into corresponding epimaltose, epicellobiose, and epilactose; and   epimerizing 2-hydroxyl group of reducing end glucose in maltooligosaccharide and cellooligosaccharide with a glucose polymerization degree of 3 or higher to convert into corresponding epimaltooligosccharide and epicellooligosacharide;   (2) Aldose-Ketose conversion   converting D-glucose or D-mannose into D-fructose, and vice versa;   converting D-galactose or D-talose into D-tagatose, and vice versa;   converting maltose or epimaltose into maltulose;   converting cellobiose or epicellobiose into cellobiulose; and   converting lactose or epilactose into lactulose.   
     
     
         16 . The purified cellobiose 2-epimerase of  claim 15 , which has the following physicochemical properties:
 (1) Molecular weight   44,000±5,000 daltons on SDS-gel electrophoresis;   (2) Optimum temperature   80° C. when reacted at pH 6.0 for 20 min;   (3) Optimum pH   pH 7.8 when reacted at 50° C. for 20 min;   (4) Thermal stability   Stable up to the temperature of 70° C. when incubated at pH 6.0 for 60 min;   (5) pH Stability   Stable in a range of pH 4.5 to 9.5 when incubated at 4° C. for 24 hours.   
     
     
         17 . The purified cellobiose 2-epimerase of  claim 15 , which has an amino acid sequence of SEQ ID NO:10 or an amino acid sequence where one or more but less than 10 amino acid residues in SEQ ID NO:10 are replaced, deleted, or added without altering the enzyme activity. 
     
     
         18 . An isolated DNA, which encodes the cellobiose 2-epimerase of  claim 17 . 
     
     
         19 . The isolated DNA of  claim 18 , which comprises a nucleotide sequence of SEQ ID NO:9, a nucleotide sequence where one or more but less than 30 nucleotides in SEQ ID NO:9 are deleted, replaced, or added without altering the encoded enzyme activity, or complementary nucleotide sequences thereof. 
     
     
         20 . The isolated DNA of  claim 18 , which is obtainable by replacing one or more nucleotides of SEQ ID NO:3 or a nucleotide sequence where one or more but less than 30 nucleotides in SEQ ID NO:9 are deleted, replaced, or added without altering the encoded enzyme activity, without altering the amino acid sequence encoded thereby based on the degeneracy of genetic code. 
     
     
         21 . An isolated replicable recombinant DNA, which comprises the isolated DNA of  claim 18  and an autonomously replicable vector. 
     
     
         22 . A transformed cell, which is obtainable by introducing the isolated recombinant DNA of  claim 21  into an appropriate host. 
     
     
         23 . A process for producing a cellobiose 2-epimerase, comprising the steps of:
 culturing a microorganism capable of producing the cellobiose 2-epimerase of  claim 15  in a nutrient culture medium; and   collecting the formed cellobiose 2-epimerase from the resulting culture.   
     
     
         24 . The process for producing a cellobiose 2-epimerase of  claim 23 , wherein said microorganism is a cell transformed with a replicable recombinant DNA comprising an autonomously replicable vector and a DNA encoding the cellobiose 2-epimerase, which cellobiose 2-epimerase has an amino acid sequence of SEQ ID NO:10 or an amino acid sequence where one or more but less than 10 amino acid residues in SEQ ID NO:10 are replaced, deleted, or added without altering the enzyme activity. 
     
     
         25 . A process for producing an isomerized saccharide, comprising the steps of:
 allowing the cellobiose 2-epimerase of  claim 15  to act on a saccharide selected from the group consisting of D-glucose, D-galactose, maltose, cellobiose, lactose, maltooligosaccharide with a glucose polymerization degree of 3 or higher, and cellooligosaccharide with a glucose polymerization degree of 3 or higher, for forming the corresponding isomerized saccharide selected from the group consisting of D-mannose, D-talose, epimaltose, epicellobiose, epilactose, epimaltooligosaccharide with a glucose polymerization degree of 3 or higher, and epicellooligosaccharide with a glucose polymerization degree of 3 or higher; and   collecting the formed isomerized saccharide.   
     
     
         26 . A process for producing isomerized saccharide, comprising the steps of:
 culturing a microorganism capable of producing the cellobiose 2-epimerase of  claim 15  in a medium containing a saccharide selected from the group consisting of D-glucose, D-galactose, maltose, cellobiose, lactose, maltooligosaccharide with a glucose polymerization degree of 3 or higher, and cellooligosaccharide with a glucose polymerization degree of 3 or higher; and   collecting the corresponding isomerized saccharide selected from the group consisting of D-mannose, D-talose, epimaltose, epicellobiose, epilactose, epimaltooligosaccharide with a glucose polymerization degree of 3 or higher, and epicellooligosaccharide with a glucose polymerization degree of 3 or higher, formed in the culture broth.   
     
     
         27 . A process for producing isomerized saccharide, comprising the steps of:
 allowing the cellobiose 2-epimerase of  claim 15  to act on a saccharide selected from the group consisting of D-glucose, D-galactose, maltose, cellobiose, and lactose, for forming the corresponding isomerized saccharide selected from the group consisting of D-fructose, D-tagatose, maltulose, cellobiulose, and lactulose; and   collecting the formed isomerized saccharide.   
     
     
         28 . A process for producing isomerized saccharide, comprising the steps of:
 culturing a microorganism capable of producing the cellobiose 2-epimerase of  claim 15  in a medium containing a saccharide selected from the group consisting of D-glucose, D-galactose, maltose, cellobiose, and lactose; and   collecting the corresponding isomerized saccharide selected from the group consisting of D-fructose, D-tagatose, maltulose, cellobiulose, and lactulose, formed in the culture broth.