# US 5,352,599 A · Co-enzyme-independent L-sorbosone dehydrogenase of gluconobacter oxydans: isolation, characterization, and cloning and autologus expression of the gene

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- Number: US 5,352,599 A
- Document ID: US5352599A
- Status: Granted
- Kind: A
- Application number: 07/415,208
- Filed: 1990-06-11
- Granted: 1994-10-04
- Earliest claimed: 1988-01-14
- Assignees: Hoffmann-La Roche Inc.
- Inventors: Akiko Fujiwara; Tatsuo Hoshino; Masako Shinjoh
- Web page: https://patentgrep.com/patent/US5352599A

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12P 7/60: 2-Ketogulonic acid
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}

## Abstract

A novel coenzyme independent L-sorbosone dehydrogenase originating from a microorganism belonging to the genus Gluconobacter oxydans which acts on L-sorbosone to produce 2-keto-L-gulonic acid. The enzyme has the following physico-chemical properties: a) optimum pH: about 7.0, b) optimum temperature: about 30.degree. C. to about 40.degree. C., c) molecular sturucture: consisting of one type of unit having a molecular weight of about 47,500.+-.5,000 as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, d) thermostability: stable below 30.degree. C., and e) inhibition: by Cu.sup.2+ -ions.

## Claims

### Claim 1

An isolated and purified DNA segment consisting essentially of a region encoding a Gluconobacter, coenzyme independent, L-sorbosone dehydrogenase, said dehydrogenase capable of converting L-sorbosone to 2-keto-L-gulonic acid.

### Claim 2 (depends on 1)

The isolated and purified DNA segment according to claim 1, wherein said Gluconobacter coenzyme independent L-sorbosone dehydrogenase has the following partial amino acid sequences:

### Claim 3 (depends on 1)

The isolated and purified DNA segment, DNA fragment according to claim 1, wherein said region is characterized by restriction map; depicted in FIG. 3.

### Claim 4

A recombinant Gluconobacter microorganism transformed with a recombinant DNA vector, said vector having a DNA segment consisting essentially of a region encoding a Gluconobacter, coenzyme independent, L-sorbosone dehydrogenase said dehydrogenase capable of converting L-sorbosone to 2-keto-L-gulonic acid.

### Claim 5 (depends on 4)

The recombinant microorganism of claim 4, wherein said coenzyme independent L-sorbosone dehydrogenase has the following partial amino acid sequences: ##STR5##

### Claim 6 (depends on 5)

A recombinant microorganism according to claim 5, which is a Gluconobacter selected from the group consisting of Gluconobacter oxydans OX-4/p7A6, Gluconobacter oxydans N44-1/p7A6, Gluconobacter oxydans N44-1/p7A6.DELTA.4, Gluconobacter oxydans U-13/p7A6.DELTA.4, Gluconobacter oxydans IFO 3292/p7A6.DELTA.4, Gluconobacter oxydans IFO 3293/p7A6.DELTA.4 and Gluconobacter oxydans IFO 3294/p7A6.DELTA.4.

### Claim 7 (depends on 6)

A recombinant microorganism according to claim 6, which is a transconjugant of a strain belonging to the genus Gluconobacter.

### Claim 8

A process for producing 2-keto-L-gulonic acid comprising cultivating a Gluconobacter microorganism in a medium comprising a sugar selected from the group consisting of L-sorbose, L-sorbosone and D-sorbitol, wherein said microorganism is transformed with a recombinant DNA vector, said vector having a DNA segment consisting essentially of a region encoding a Gluconobacter, coenzyme independent, L-sorbosone dehydrogenase, said dehydrogenase capable of converting L-sorbosone to 2-L-gulonic acid.

### Claim 9 (depends on 8)

The process of claim 8, wherein the sugar is L-sorbosone.

### Claim 10 (depends on 9)

A process according to claim 9, wherein said coenzyme independent L-sorbosone dehydrogenase has the following partial amino acid sequences: ##STR6##

### Claim 11 (depends on 10)

A process according to claim 10, wherein said microorganism is Gluconobacter oxydans OX-4/p7A6, Gluconobacter oxydans N44-1/p7A6, Gluconobacter oxydans N44-1/p7A6.DELTA.4, Gluconobacter oxydans U-13/p7A6.DELTA.4, Gluconobacter oxydans IFO 3292/p7A6.DELTA.4, Gluconobacter oxydans IFO 3293/p7A6.DELTA.4 or Gluconobacter oxydans IFO 3294/p7A6.DELTA.4.

### Claim 12 (depends on 8)

The process of claim 8, wherein the sugar is L-sorbose.

### Claim 13 (depends on 12)

A process according to claim 12, wherein said coenzyme independent L-sorbosone dehydrogenase has the following partial amino acid sequences: ##STR7##

### Claim 14 (depends on 13)

A process according to claim 13, wherein said microorganism is Gluconobacter oxydans OX-4/p7A6, Gluconobacter oxydans N44-1/p7A6, Gluconobacter oxydans N44-1/p7A6.DELTA.4, Gluconobacter oxydans U-13/p7A6.DELTA.4, Gluconobacter oxydans IFO 3292/p7A6.DELTA.4, Gluconobacter oxydans IFO 3293/p7A6.DELTA.4 and Gluconobacter oxydans IFO 3294/p7A6.DELTA.4.

### Claim 15 (depends on 8)

The process of claim 8, wherein the sugar is D-sorbitol.

### Claim 16 (depends on 15)

The process according to claim 15, wherein said coenzyme independent L-sorbosone dehydrogenase has the following partial amino acid sequences: ##STR8##

### Claim 17 (depends on 16)

A process according to claim 16, wherein said microorganism is Gluconobacter oxydans OX-4/p7A6, Gluconobacter oxydans N44-1/p7A6, Gluconobacter oxydans N44-1/p7A6.DELTA.4, Gluconobacter oxydans U-13/p7A6.DELTA.4, Gluconobacter oxydans IFO 3292/p7A6.DELTA.4, Gluconobacter oxydans IFO 3293/p7A6.DELTA.4 and Gluconobacter oxydans IFO 3293/p7A6.DELTA.4.

### Claim 18

A method for producing a transconjugant using a microorganism belonging to the genus Gluconobacter as a recipient, comprising contacting said recipient with a donor having a plasmid derived from pVK100 or pVK102, wherein said plasmid contains both a Mob site and cos site, and transferring the plasmid from the donor to the recipient through the use of the Tra gene function.