# US 5,707,833 A · Process of making cyclodextrin glucanotransferase with brevibacterium

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- Number: US 5,707,833 A
- Document ID: US5707833A
- Status: Granted
- Kind: A
- Application number: 08/471,235
- Filed: 1995-06-06
- Granted: 1998-01-13
- Earliest claimed: 1993-02-09
- Assignees: Amano Pharmaceutical Co., Ltd.
- Inventors: Shigeharu Mori; Tamio Mase; Takaichi Ohya
- Web page: https://patentgrep.com/patent/US5707833A

## CPC

- C12N 9/1074: {Cyclomaltodextrin glucanotransferase (2.4.1.19)}
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- Y10S 435/84: Brevibacterium
- 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)}
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

This invention relates to a .gamma.-cyclodextrin glucanotransferase having novel properties, to a process for the production of the .gamma.-cyclodextrin glucanotransferase which comprises culturing a strain belonging to the genus Brevibacterium capable of producing cyclodextrin glucanotransferase, thereby allowing the strain to produce the .gamma.-cyclodextrin glucanotransferase in a culture medium, and subsequently collecting the enzyme, to a process for the production of cyclodextrin which comprises allowing the cyclodextrin glucanotransferase to react with substrate dissolved in a solution, thereby effecting formation of .gamma.-cyclodextrin as a main product, and to a method for increasing .gamma.-cyclodextrin yield without accompanying yield increase of total cyclodextrin, which comprises adding ethyl alcohol to a reaction solution in which .gamma.-cyclodextrin and .beta.-cyclodextrin are formed from starch by the action of .gamma.-cyclodextrin glucanotransferase, thereby effecting repressed formation of .beta.-cyclodextrin and enhanced formation of .gamma.-cyclodextrin. Development of the process of this invention has rendered possible low cost production of .gamma.-cyclodextrin and therefore expands practical use of .gamma.-cyclodextrin to the field of food and the like.

## Claims

### Claim 1

A process for producing .gamma.-cyclodextrin glucanotransferase which comprises:
(1) culturing Brevibacterium sp. FERM BP-4537 or a mutant thereof, in an aqueous nutrient culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under conditions such that said Brevibacterium sp. FERM BP-4537 or said mutant thereof produces and secretes .gamma.-cyclodextrin glucano-transferase into said culture medium, and
(2) collecting the resulting culture medium of step (1) containing said cyclodextrin glucanotransferase, and
(3) purifying said .gamma.-cyclodextrin glucano-transferase from the collected culture medium of step (2), wherein when purified, the collected .gamma.-cyclodextrin glucanotransferase has the following properties:
(a) action and substrate specificity: acts upon a substrate selected from the group consisting of starch, dextrin and maltooligosaccharides to produce .gamma.-cyclodextrin as a primary product and .beta.-cyclodextrin as a secondary product;
(b) optimum reaction pH: about 8 to about 9;
(c) optimum reaction temperature: around 45.degree. C.;
(d) pH stability: about 6 to about 8; and
(e) temperature stability: about 85% activity remains after 30 minutes at 40.degree. C. in the absence of a calcium salt, and about 100% activity remains after 30 minutes at 45.degree. C. in the presence of 20 mM of a calcium salt.