# US 5,705,373 A · Production of 2-keto-L-gulonic acid using pseudogluconobacter saccharoketogenes with recycling

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- Number: US 5,705,373 A
- Document ID: US5705373A
- Status: Granted
- Kind: A
- Application number: 08/682,258
- Filed: 1996-07-16
- Granted: 1998-01-06
- Earliest claimed: 1993-12-24
- Assignees: Takeda Chemical Industries, Ltd.
- Inventors: Takamasa Yamaguchi; Kenkichi Yoneto; Giichi Tanaka
- Web page: https://patentgrep.com/patent/US5705373A

## CPC

- C12P 7/60: 2-Ketogulonic acid
- Y02P 20/582: Recycling of unreacted starting or intermediate materials
- 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)}
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

A method for producing 2-keto-L-gulonic acid (hereinafter referred to as 2KGA) which comprises (1) culturing a microorganism capable of producing 2KGA from L-sorbose, in a liquid culture medium containing a substrate to produce 2KGA and (2) recovering the produced 2KGA, while recovering the microorganism in the culture broth, (3) inoculating the recovered microorganism to a new liquid culture medium for the following culture and (4) repeating said culture and recovering at least once. The present invention provides an industrially advantageous, versatile method for producing 2KGA that shortens the overall culture time, and reduces the amounts of starting medium materials and culture waste.

## Claims

### Claim 1

A method for producing 2-keto-L-gulonic acid which comprises
(1) culturing a Pseudogluconobacter saccharoketogenes microorganism capable of producing 2-keto-L-gulonic acid from L-sorbose without immobilization, and with another microorganism capable of supplying a growth factor to the Pseudogluconobacter saccharoketogenes, in a liquid culture medium containing L-sorbose as a substrate and producing 2-keto-L-gulonic acid,
(2) recovering the produced 2-keto-L-gulonic acid, while recovering the microorganism in the culture broth,
(3) inoculating the recovered microorganism to a new sterilized liquid culture medium comprising L-sorbose, sucrose, ammonium sulfate, and optionally urea, and at least one cell reactivity retention aid selected from the group consisting of yeast extract, dry yeast and corn steep liquor, and,
(4) repeating the steps (2) and (3) at least once.

### Claim 2 (depends on 1)

The method of claim 1, wherein the microorganism capable of producing 2-keto-L-gulonic acid is selected from the group consisting of Pseudogluconobacter saccharoketogenes K591s (FERM BP-1130), Pseudogluconobacter saccharoketogenes TH14-86 (FERM BP-1128), Pseudogluconobacter saccharoketogenes 12-5 (FERM BP-1129), Pseudogluconobacter saccharoketogenes 12-15 (FERM BP-1132), Pseudogluconobacter saccharoketogenes 12-4 (FERM BP-1131), and Pseudogluconobacter saccharoketogenes 22-3 (FERM BP-1133).

### Claim 3 (depends on 1)

The method of claim 1, wherein the microorganism capable of supplying a growth factor to the microorganism capable of producing 2-keto-L-gulonic acid from L-sorbose is selected from the group consisting of Bacillus cereus IFO 3131, Bacillus licheniformis IFO 12201, Bacillus megaterium IFO 12108, Bacillus pumilus IFO 12090, Bacillus amyloliquefaciens IFO 3022, Bacillus subtilis IFO 13719, Bacillus circulans IFO 3967, Pseudomonas trifolii IFO 12056, Pseudomonas maltophilia IFO 12692, Proteus inconstans IFO 12930, Enterobacter cloacae IFO 3320, Erwinia herbicola IFO 12686, Xanthomonas pisi IFO 13556, Xanthomonas citri IFO 3835 and Flavobacterium menigosepticum IFO 12535.

### Claim 4 (depends on 1)

The method of claim 1, wherein the recovery of the microorganism of the step (2) is carried out by using a hollow fiber membrane filter, a ceramic membrane filter or a centrifuge.

### Claim 5 (depends on 1)

The method of claim 1, wherein the recovery of the microorganism of the step (2) is carried out at 5.degree. to 40.degree. C.