# US 7,745,599 B1 · Hexose oxidase-encoding DNAs and methods of use thereof

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- Number: US 7,745,599 B1
- Document ID: US7745599B1
- Status: Granted
- Kind: B1
- Application number: 11/979,133
- Filed: 2007-10-31
- Granted: 2010-06-29
- Earliest claimed: 1995-06-07
- Assignees: Danisco A/S
- Inventors: Peter Stougaard; Ole Cai Hansen
- Web page: https://patentgrep.com/patent/US7745599B1

## CPC

- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method of producing hexose oxidase by recombinant DNA technology, recombinant hexose oxidase and the use of such enzyme, in particular in the manufacturing of food products such as doughs and dairy products, animal feed, pharmaceuticals, cosmetics, dental care products and in the manufacturing of lactones. Suitable sources of DNA coding for the enzyme are marine algal species including Chondrus crispus, Iridophycus flaccidum and Euthora cristata. In useful embodiments, the recombinant hexose oxidase is produced by Pichia pastoris, Saccharomyces cerevisiae or E. coli.

## Claims

### Claim 1

An isolated DNA fragment encoding a polypeptide having hexose oxidase activity, wherein said polypeptide oxidizes at least maltose and wherein said DNA fragment comprises a nucleic acid sequence isolated by hybridization with the nucleic acid sequence set forth in SEQ ID NO: 30 under stringent conditions comprising
(1) hybridization at 65° C. for at least 14 hours in a buffer containing 1×Denhardt's solution, 2×SSC, 0.1% dextran sulphate, 50 μg/ml denatured salmon sperm DNA, followed by
(2) two washes at 65° C. for 10 minutes in 2×SSC, 0.1% SDS, and followed by
(3) two washes at 65° C. for 10 minutes in 1×SSC, 0.1% SDS.

### Claim 2 (depends on 1)

The isolated DNA fragment according to claim 1 wherein said polypeptide encoded by said DNA fragment comprises the amino acid sequences
(i) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X-Asp (SEQ ID NO:6),
(ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-X-X-X-Gly-Tyr-X-Val-Ser-Ser- (SEQ ID NO:2),
(iii) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr (SEQ ID NO:4),
(iv) Tyr-Tyr-Phe-Lys (SEQ ID NO:5),
(v) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe- (SEQ ID NO:3),
(vi) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu-Asp (SEQ ID NO:7),
(vii) X-Ile-Arg-Asp-Phe-Tyr-Glu-Glu-Met (SEQ ID NO:8), and
(viii) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO:1),
in the order of (i) to (viii);
where X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, H is, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

### Claim 3 (depends on 1)

A recombinant DNA molecule comprising the isolated DNA fragment according to claim 1 operably linked to an expression signal.

### Claim 4 (depends on 1)

A host cell comprising the DNA fragment of claim 1.

### Claim 5 (depends on 4)

The host cell according to claim 4 wherein said host cell is a yeast cell.

### Claim 6 (depends on 4)

The host cell according to claim 4 wherein said host cell is a Saccharomyces cerevisiae cell or a Pichia pastoris cell.

### Claim 7 (depends on 4)

A method of manufacturing a food product wherein a host cell according to claim 4 is used wherein said method comprises the steps of adding said cell expressing hexose oxidase or the hexose oxidase produced by said cell to ingredients for said food product
(a) prior to, during or after any process step;
(b) during packaging of the finished product; and/or
(c) during storage of the finished product.

### Claim 8 (depends on 4)

A method for producing a polypeptide having hexose oxidase activity, said method comprising cultivating the host cell of claim 4 in a cultivation medium under conditions leading to the expression of the hexose oxidase active polypeptide and recovering the polypeptide from the cultivation medium and/or from the host cell; wherein said host cell comprises the DNA fragment operably linked to an expression signal.

### Claim 9 (depends on 8)

The method according to claim 8 wherein said polypeptide encoded by said DNA fragment comprises the amino acid sequences
(i) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X-Asp (SEQ ID NO:6),
(ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-X-X-X-Gly-Tyr-X-Val-Ser-Ser- (SEQ ID NO:2),
(iii) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr (SEQ ID NO:4),
(iv) Tyr-Tyr-Phe-Lys (SEQ ID NO:5),
(v) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe- (SEQ ID NO:3),
(vi) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu-Asp (SEQ ID NO:7),
(vii) X-Ile-Arg-Asp-Phe-Tyr-Glu-Glu-Met (SEQ ID NO:8), and
(viii) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO:1),
in the order of (i) to (viii);
where X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

### Claim 10 (depends on 8)

The method according to claim 8 wherein the DNA fragment is derived from a marine algal species selected from the group consisting of Chondrus crispus, Iridophycus flaccidum and Euthora cristata.

### Claim 11 (depends on 8)

The method according to claim 8 wherein said host cell is a yeast cell.

### Claim 12 (depends on 8)

The method according to claim 8 wherein said host cell is a Saccharomyces cerevisiae cell or a Pichia pastoris cell.

### Claim 13 (depends on 8)

The method according to claim 8 comprising as a further step a purification of the polypeptide preparation initially recovered from the cultivation medium and/or the host cells to obtain a preparation in which the polypeptide is in a substantially pure form.

### Claim 14 (depends on 8)

The method according to claim 8 wherein the polypeptide having hexose oxidase activity is a fusion product.