Recombinant expression of a glucose oxidase from a cladosporium strain

• US 5,879,921 A
• Filed: 11/07/1996
• Issued: 03/09/1999
• Est. Priority Date: 11/07/1996
• Status: Expired due to Fees

• Current Assignee: Novozymes
• Original Assignee: Novo Nordisk A/S, NOVO Nordisk Biotech Incorporated (Novo Nordisk A/S)
• Inventors: Cherry, Joel R., Berka, Randy M., Halkier, Torben
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Mayhew, Bradley S.
• Application Number:
  US08/746,257
• Time in Patent Office: 852 Days
• Field of Search: 435/190, 435/320.1, 435/6, 435/252.3, 435/254.11, 435/254.7, 536/23.2, 536/24.3
• US Class Current: 435/190
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)

Abstract

The invention is directed to alkaline glucose oxidases comprising novel peptide sequences. Furthermore, the invention relates to methods for producing and using said glucose oxidases.

Claims

1. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a polypeptide having glucose oxidase activity with more than about 75% maximum activity between about pH 5 to about pH 8, determined at 30°

C. with D-glucose as substrate, wherein the nucleic acid sequence is selected from the group consisting of;

(a) a nucleic acid sequence, obtained from a Cladosporium strain, encoding a polypeptide having an amino acid sequence which has at least 70% identity with the amino acid sequence of SEQ ID NO;

1;

(b) a nucleic acid sequence, obtained from a Cladosporium strain, which hybridizes under medium stringency conditions with one or more of (i) the nucleic acid sequences of SEQ ID NO;

2 and SEQ ID NO;

3, and (ii) either of their complementary strands, wherein the medium stringency conditions are defined by prehybridization and hybridization at 42°

C. in 5×

SSPE, 0.3% SDS, 200 μ

g/ml sheared and denatured salmon sperm DNA, and 35% formamide, and wash conditions at 60°

C. for 30 minutes in 0.2×

SSC, 0.1% SDS;

(c) a nucleic acid sequence which encodes SEQ ID NO;

1; and

(d) a subsequence of (a), (b), or (c), wherein the subsequence encodes a polypeptide having glucose oxidase activity with more than about 75% maximum activity between about pH 5 to about pH 8, determined at 30°

C. with D-glucose as substrate.

2. The isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence, obtained from a Cladosporium strain, encodes a polypeptide having an amino acid sequence which has at least 70% identity with the amino acid sequence of SEQ ID NO:

1.

3. The isolated nucleic acid fragment of claim 2, wherein the nucleic acid sequence encodes a polypeptide having an amino acid sequence which has at least 80% identity with the amino acid sequence of SEQ ID NO:

1.

4. The isolated nucleic acid fragment of claim 3, wherein the nucleic acid sequence encodes a polypeptide having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

5. The isolated nucleic acid fragment of claim 4, wherein the nucleic acid sequence encodes a polypeptide having an amino acid sequence which has at least 95% identity with the amino acid sequence of SEQ ID NO:

1.

6. The isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO:

1;

or a subsequence thereof which encodes a polypeptide having glucose oxidase activity with more than about 75% maximum activity between about pH 5 to about pH 8, determined at 30°

C. with D-glucose as substrate.

7. The isolated nucleic acid fragment of claim 6, wherein the nucleic acid sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO:

1.

8. The isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence encodes a polypeptide having amino acids 24 to 613 of SEQ ID NO:

1.

9. The isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence is SEQ ID NO:

2 or SEQ ID NO;

3.

10. The Isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence comprises nucleotides 419 to 2189 of SEQ ID NO:

2.

11. The isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence, obtained from a Cladosporium strain, hybridizes under medium stringency conditions to one or more of (i) the nucleic acid sequences of SEQ ID NO:

2 and SEQ ID NO;

3 and (ii) either of their complementary strands, wherein the medium stringency conditions are defined by prehybridization and hybridization at 42°

C. in 5×

SSPE, 0.3% SDS, 200 μ

g/ml sheared and denatured salmon sperm DNA, and 35% formamide, and wash conditions at 60°

C. for 30 minutes in 0.2×

SSC, 0.1% SDS.

12. The isolated nucleic acid fragment of claim 11, wherein the Cladosporium strain is a Cladosporium oxysporum strain.

13. The isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence is contained in the plasmid pGOX4A which is contained in Escherichia coli NRRL B-21628 and plasmid pGOX6A which is contained in Escherichia coli NRRL B-21629.

14. A nucleic acid construct comprising the isolated nucleic acid fragment of claim 1, wherein the nucleic acid sequence is operably linked to one or more control sequences which direct the production of the polypeptide in a suitable expression host.

15. A recombinant expression vector comprising the nucleic acid construct of claim 14.

16. The vector of claim 15, further comprising a selectable marker.

17. A recombinant host cell comprising the nucleic acid construct of claim 14.

18. The recombinant host cell of claim 17, wherein the nucleic acid construct comprises SEQ ID NO:

2 or SEQ ID NO;

3;

or a subsequence thereof which encodes a polypeptide having glucose oxidase activity with more than about 75% maximum activity between about pH 5 to about pH 8, determined a 30°

C. with D-glucose as substrate.

19. The recombinant host cell of claim 17, wherein the nucleic acid construct comprises nucleotides 419 to 2189 of SEQ ID NO:

2.

20. The recombinant host cell of claim 17, wherein the nucleic acid construct is contained on a vector.

21. The recombinant host cell of claim 17, wherein the nucleic acid construct upon introduction into the cell integrates into the host cell genome.

22. The recombinant host cell of claim 17, wherein the host cell is a yeast or filamentous fungal cell.

23. A method for producing a polypeptide having glucose oxidase activity comprising (a) cultivating the host cell of claim 17 under conditions suitable for the production of the polypeptide;

and (b) recovering the polypeptide.

24. The method of claim 23, wherein the polypeptide having glucose activity is encoded by SEQ ID NO:

2 or SEQ ID NO;

3;

or a subsequence thereof which encodes a polypeptide having glucose oxidase activity with more than about 75% maximum activity between about pH 5 to about pH 8, determined at 30°

C. with D-glucose as substrate.

25. The method of claim 23, wherein the polypeptlde having glucose activity is encoded by nucleic acid sequence comprising nucleotides 419 to 2189 of SEQ ID NO:

2.