1. A method for producing a heparosan compound having an isomerized hexuronic acid residue, said method comprising 5 producing the heparosan compound having the isomerized hexuronic acid residue from a heparosan compound in the presence of a protein selected from the group of consisting of the following (A) to (F): (A) a protein comprising the amino acid sequence of SEQ 10 ID No: 2; (B) a protein which comprises an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID No:2 and has a D-glucuronyl C5-epimerase activity; (C) a protein which comprises an amino acid sequence 15 having one or several deleted, substituted, added or inserted amino acid residues in the amino acid sequence of SEQ ID No:2 and has a D-glucuronyl C5-epimerase activity; (D) a protein comprising the amino acid sequence of SEQ ID NO: 5; 20 (E) a protein which comprises an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID No:5 and has a D-glucuronyl C5-epimerase activity; and (F) a protein which comprises an amino acid sequence 25 having one or several deleted, substituted, added or inserted amino acid residues in the amino acid sequence of SEQ ID No:5 and has a D-glucuronyl C5-epimerase activity.

2. The method according to claim 1, 30 wherein the protein (B) is (B1) a protein which comprises an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID No:2 and has a D-glucuronyl C5-epimerase activity, and 65 wherein the protein (E) is (E') a protein which comprises an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID No:5 and has a D-glucuronyl C5-epimerase activity.

3. The method according to claim 1 or 2, wherein said protein is a protein selected from the group consisting of the following (El) to (Fl): 10 (El) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:56, 59, 62, 65 and 68; 15 (E2) a protein which comprises an amino acid sequence having 95% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity; and (Fl) a protein which comprises an amino acid sequence having 1 to 25 deleted, substituted, added or inserted amino acid residues in an amino acid sequence selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity.

4. The method according to any one of claims 1 to 3, wherein said protein is derived from zebrafish. 25 5. The method according to any one of claims 1 to 4, wherein the heparosan compound having the isomerized hexuronic acid residue is produced in the presence of a transformed microorganism producing said protein or an extract thereof. 30

6. The method according to claim 5, wherein said transformed microorganism is a host cell comprising an expression unit comprising a polynucleotide selected from 66 the group consisting of the following (a) to (j) and a promoter operably linked thereto: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID N0:1; 5 (b) a polynucleotide which comprises a nucleotide sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO:l and encodes a protein having a D-glucuronyl C5-epimerase activity; (c) a polynucleotide which hybridizes with a 10 polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:l under the stringent conditions and encodes a protein having a D-glucuronyl C5-epimerase activity; (d) a polynucleotide comprising the nucleotide sequence 15 of SEQ ID NO: 3; (e) a polynucleotide which comprises a nucleotide sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO:3 and encodes a protein having a D- glucuronyl C5-epimerase activity; 20 (f) a polynucleotide which hybridizes with a polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:3 under the stringent conditions and encodes a protein having a D-glucuronyl C5-epimerase activity; 25 (g) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4; (h) a polynucleotide which comprises a nucleotide sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO:4 and encodes a protein having a D- 30 glucuronyl C5-epimerase activity; (i) a polynucleotide which hybridizes with a polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:4 67 under the stringent conditions and encodes a protein having a D-glucuronyl C5-epimerase activity; and (j) a degenerate mutant of a polynucleotide selected from the group consisting of (a) to (i). 5

7. The method according to claim 6, wherein the polynucleotide (b) is (b') a polynucleotide which comprises a nucleotide sequence having 90% or more homology to the nucleotide sequence of SEQ ID 10 NO:l and encodes a protein having a D-glucuronyl C5-epimerase activity, wherein the polynucleotide (e) is (e1) a polynucleotide which comprises a nucleotide sequence having 90% or more homology to the nucleotide sequence of SEQ ID 15 NO:3 and encodes a protein having a D-glucuronyl C5-epimerase activity, and wherein the polynucleotide (h) is (h') a polynucleotide which comprises a nucleotide sequence having 90% or more homology to the nucleotide sequence of SEQ ID 20 NO:4 and encodes a protein having a D-glucuronyl C5-epimerase activity.

8. The method according to claim 6 or 7, wherein said polynucleotide is a polynucleotide selected from the group 25 consisting of the following (el) to (jl): (el) a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:55, 58, 61, 64 and 67; (e2) a polynucleotide which comprises a nucleotide 30 sequence having 95% or more homology to a nucleotide sequence selected from the group consisting of SEQ ID NOS:58, 61, 64 and 67 and encodes a protein having a D-glucuronyl C5-epimerase activity; and 68 (jl) a degenerate mutant of the polynucleotide (el) or (e2) .

9. The method according to any one of claims 6 to 8, 5 wherein said transformed microorganism is a bacterium belonging to genus Escherichia.

10. The method according to claim 9, wherein said transformed microorganism is Escherichia coli. 10

11. The method according to any one of claims 1 to 10, wherein said heparosan compound is an N-sulfated heparosan.

12. The method according to any one of claims 1 to 11, 15 wherein said heparosan compound is a low-molecularized heparosan.

13. A method for producing a heparan sulfate, said method comprising subjecting a heparosan to a 20 treatment comprising C5-epimerization of a hexuronic acid residue, 2-O-sulfation of a hexuronic acid residue, N-deacetylation of an a-D-glucosamine residue, N-sulfation of an a-D-glucosamine residue, 3-O-sulfation of an a-D-glucosamine residue, and 6-O-sulfation of an a-D-25 glucosamine residue to produce the heparan sulfate, wherein the C5-epimerization is performed in the presence of a protein selected from the following (A) to (F) : (A) a protein comprising the amino acid sequence of SEQ 30 ID NO:2; (B) a protein which comprises an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:2 and has a D-glucuronyl C5-epimerase activity; 69 (C) a protein which comprises an amino acid sequence having one or several deleted, substituted, added or inserted amino acid residues in the amino acid sequence of SEQ ID NO:2 and has a D-glucuronyl C5-epimerase activity; 5 (D) a protein comprising the amino acid sequence of SEQ ID N0:5; (E) a protein which comprises an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:5 and has a D-glucuronyl C5-epimerase activity; 10 and (F) a protein which comprises an amino acid sequence having one or several deleted, substituted, added or inserted amino acid residues in the amino acid sequence of SEQ ID NO:5 and has a D-glucuronyl C5-epimerase activity. 15

14. The method according to claim 13, wherein the protein (B) is (B') a protein which comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:2 and has 20 a D-glucuronyl C5-epimerase activity, and wherein the protein (E) is (E') a protein which comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:5 and has a D-glucuronyl C5-epimerase activity. 25

15. The method according to claim 13 or 14, wherein said protein is selected from the group consisting of the following (El) to (Fl): (El) a protein comprising an amino acid sequence selected 30 from the group consisting of SEQ ID NOS:56, 59, 62, 65 and 68; (E2) a protein which comprises an amino acid sequence having 95% or more homology to an amino acid sequence 70 selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68, and has a D-glucuronyl C5-epimerase activity; and (Fl) a protein which comprises an amino acid sequence having 1 to 25 deleted, substituted, added or inserted 5 amino acid residues in an amino acid sequence selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity.

16. The method according to any one of claims 13 to 15, 10 wherein the treatment further comprises decomposition of a heparosan into small molecules.

17. A protein selected from the group consisting of the following (El) to (Fl): 15 (El) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:56, 59, 62, 65 and 68; (E2) a protein which comprises an amino acid sequence having 95% or more homology to an amino acid sequence 20 selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity; and (Fl) a protein which comprises an amino acid sequence having 1 to 25 deleted, substituted, added or inserted amino acid residues in an amino acid sequence selected from 25 the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity. 71

WE CLAIM:
1. A method for producing a heparosan compound having an
isomerized hexuronic acid residue, said method comprising
5 producing the heparosan compound having the isomerized hexuronic acid residue from a heparosan compound in the presence of a protein selected from the group of consisting of the following (A) to (F):
(A) a protein comprising the amino acid sequence of SEQ
10 ID No: 2;
(B) a protein which comprises an amino acid sequence
having 80% or more homology to the amino acid sequence of
SEQ ID No:2 and has a D-glucuronyl C5-epimerase activity;
(C) a protein which comprises an amino acid sequence
15 having one or several deleted, substituted, added or
inserted amino acid residues in the amino acid sequence of SEQ ID No:2 and has a D-glucuronyl C5-epimerase activity;
(D) a protein comprising the amino acid sequence of SEQ
ID NO: 5;
20 (E) a protein which comprises an amino acid sequence
having 80% or more homology to the amino acid sequence of SEQ ID No:5 and has a D-glucuronyl C5-epimerase activity; and
(F) a protein which comprises an amino acid sequence
25 having one or several deleted, substituted, added or
inserted amino acid residues in the amino acid sequence of SEQ ID No:5 and has a D-glucuronyl C5-epimerase activity.
2. The method according to claim 1,
30 wherein the protein (B) is (B1) a protein which comprises an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID No:2 and has a D-glucuronyl C5-epimerase activity, and
65

wherein the protein (E) is (E') a protein which comprises an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID No:5 and has a D-glucuronyl C5-epimerase activity.
3. The method according to claim 1 or 2, wherein said
protein is a protein selected from the group consisting of
the following (El) to (Fl):
10
(El) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:56, 59, 62, 65 and 68;
15
(E2) a protein which comprises an amino acid sequence having 95% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity; and
(Fl) a protein which comprises an amino acid sequence having 1 to 25 deleted, substituted, added or inserted amino acid residues in an amino acid sequence selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity.
4. The method according to any one of claims 1 to 3,
wherein said protein is derived from zebrafish.
25 5. The method according to any one of claims 1 to 4, wherein the heparosan compound having the isomerized hexuronic acid residue is produced in the presence of a transformed microorganism producing said protein or an extract thereof.
30
6. The method according to claim 5, wherein said transformed microorganism is a host cell comprising an expression unit comprising a polynucleotide selected from
66

the group consisting of the following (a) to (j) and a promoter operably linked thereto:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID N0:1; 5 (b) a polynucleotide which comprises a nucleotide
sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO:l and encodes a protein having a D-glucuronyl C5-epimerase activity;
(c) a polynucleotide which hybridizes with a
10 polynucleotide consisting of the nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO:l under the stringent conditions and encodes a protein having a D-glucuronyl C5-epimerase activity;
(d) a polynucleotide comprising the nucleotide sequence
15 of SEQ ID NO: 3;
(e) a polynucleotide which comprises a nucleotide
sequence having 80% or more homology to the nucleotide
sequence of SEQ ID NO:3 and encodes a protein having a D-
glucuronyl C5-epimerase activity;
20 (f) a polynucleotide which hybridizes with a
polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:3 under the stringent conditions and encodes a protein having a D-glucuronyl C5-epimerase activity;
25 (g) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4;
(h) a polynucleotide which comprises a nucleotide sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO:4 and encodes a protein having a D-
30 glucuronyl C5-epimerase activity;
(i) a polynucleotide which hybridizes with a polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:4
67

under the stringent conditions and encodes a protein having a D-glucuronyl C5-epimerase activity; and
(j) a degenerate mutant of a polynucleotide selected from the group consisting of (a) to (i). 5
7. The method according to claim 6,
wherein the polynucleotide (b) is (b') a
polynucleotide which comprises a nucleotide sequence having 90% or more homology to the nucleotide sequence of SEQ ID
10 NO:l and encodes a protein having a D-glucuronyl C5-epimerase activity,
wherein the polynucleotide (e) is (e1) a polynucleotide which comprises a nucleotide sequence having 90% or more homology to the nucleotide sequence of SEQ ID
15 NO:3 and encodes a protein having a D-glucuronyl C5-epimerase activity, and
wherein the polynucleotide (h) is (h') a polynucleotide which comprises a nucleotide sequence having 90% or more homology to the nucleotide sequence of SEQ ID
20 NO:4 and encodes a protein having a D-glucuronyl C5-epimerase activity.
8. The method according to claim 6 or 7, wherein said
polynucleotide is a polynucleotide selected from the group
25 consisting of the following (el) to (jl):
(el) a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:55, 58, 61, 64 and 67;
(e2) a polynucleotide which comprises a nucleotide 30 sequence having 95% or more homology to a nucleotide sequence selected from the group consisting of SEQ ID NOS:58, 61, 64 and 67 and encodes a protein having a D-glucuronyl C5-epimerase activity; and
68

(jl) a degenerate mutant of the polynucleotide (el) or (e2) .
9. The method according to any one of claims 6 to 8,
5 wherein said transformed microorganism is a bacterium
belonging to genus Escherichia.
10. The method according to claim 9, wherein said
transformed microorganism is Escherichia coli.
10
11. The method according to any one of claims 1 to 10,
wherein said heparosan compound is an N-sulfated heparosan.
12. The method according to any one of claims 1 to 11,
15 wherein said heparosan compound is a low-molecularized
heparosan.
13. A method for producing a heparan sulfate,
said method comprising subjecting a heparosan to a 20 treatment comprising C5-epimerization of a hexuronic acid residue, 2-O-sulfation of a hexuronic acid residue, N-deacetylation of an a-D-glucosamine residue, N-sulfation of an a-D-glucosamine residue, 3-O-sulfation of an a-D-glucosamine residue, and 6-O-sulfation of an a-D-25 glucosamine residue to produce the heparan sulfate,
wherein the C5-epimerization is performed in the presence of a protein selected from the following (A) to (F) :
(A) a protein comprising the amino acid sequence of SEQ
30 ID NO:2;
(B) a protein which comprises an amino acid sequence
having 80% or more identity to the amino acid sequence of
SEQ ID NO:2 and has a D-glucuronyl C5-epimerase activity;
69

(C) a protein which comprises an amino acid sequence having one or several deleted, substituted, added or inserted amino acid residues in the amino acid sequence of SEQ ID NO:2 and has a D-glucuronyl C5-epimerase activity; 5 (D) a protein comprising the amino acid sequence of SEQ ID N0:5;
(E) a protein which comprises an amino acid sequence
having 80% or more identity to the amino acid sequence of
SEQ ID NO:5 and has a D-glucuronyl C5-epimerase activity;
10 and
(F) a protein which comprises an amino acid sequence
having one or several deleted, substituted, added or
inserted amino acid residues in the amino acid sequence of
SEQ ID NO:5 and has a D-glucuronyl C5-epimerase activity.
15
14. The method according to claim 13,
wherein the protein (B) is (B') a protein which comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:2 and has 20 a D-glucuronyl C5-epimerase activity, and
wherein the protein (E) is (E') a protein which comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:5 and has a D-glucuronyl C5-epimerase activity. 25
15. The method according to claim 13 or 14, wherein said
protein is selected from the group consisting of the
following (El) to (Fl):
(El) a protein comprising an amino acid sequence selected 30 from the group consisting of SEQ ID NOS:56, 59, 62, 65 and 68;
(E2) a protein which comprises an amino acid sequence having 95% or more homology to an amino acid sequence
70

selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68, and has a D-glucuronyl C5-epimerase activity; and
(Fl) a protein which comprises an amino acid sequence having 1 to 25 deleted, substituted, added or inserted 5 amino acid residues in an amino acid sequence selected from the group consisting of SEQ ID NOS:59, 62, 65 and 68 and has a D-glucuronyl C5-epimerase activity.
16. The method according to any one of claims 13 to 15,
10 wherein the treatment further comprises decomposition of a
heparosan into small molecules.
17. A protein selected from the group consisting of the
following (El) to (Fl):
15 (El) a protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:56, 59, 62, 65 and
68;
(E2) a protein which comprises an amino acid sequence
having 95% or more homology to an amino acid sequence 20 selected from the group consisting of SEQ ID NOS:59, 62, 65
and 68 and has a D-glucuronyl C5-epimerase activity; and (Fl) a protein which comprises an amino acid sequence
having 1 to 25 deleted, substituted, added or inserted
amino acid residues in an amino acid sequence selected from 25 the group consisting of SEQ ID NOS:59, 62, 65 and 68 and
has a D-glucuronyl C5-epimerase activity.
71