CELLOBIOSE 2-EPIMERASE, ITS PREPARATION AND USES

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.