CS252994B1 - Trigonopsis variabilis CCY 15-1-2 strain - Google Patents

Abstract

The solution relates to the strain Trigonopsis variabilis CCY 15-1-2, which is a mutant of the strain Trigonopsis variabilis CCY 15-1-1 and which is characterized by the inductive synthesis of D-amino acid: 06-oxidoreductase deaminating EC 1-4-3-3, catalyzing the oxidative deamination of compounds of general formula Z (especially cephalosporin C) to compounds of general formula 1 (especially 7-[3-(4-carboxybutanamido)cephalosporanic acid). The cells of the production strain can be used in native or immobilized form for the transformation of especially cephalosporin C to form 7-acylamino compounds, which are a suitable substrate for the enzymatic preparation of 7-amino-cephalosporanic acid.

Description

252994

BACKGROUND OF THE INVENTION Trigono-psis variabilis CCY 15-1-2 microorganism strain with D-aminoacid activity: O1-4-3-3 deaminating oz-oxidoreductase refers to strain Tri-gonopsis variabilis CCY 15-1-2, applicable to for the enzymatic preparation of 7-acyl-amino compounds, especially for the preparation of 7-β- (4-carboxybutanamido) cephalosporinic acid, hereinafter GLT-7ACK.

Compounds of Formula 1

CH 2 X wherein X is -H, -OH, -OCOCH 3, or a n-kleophilic group, and R 1 is (CH 2) 3 — CHNH, - (CH 2) 3 —CO — COOH, may be prepared from compounds of Formula 2

COOH

wherein R 2 is CH 2 Cl 3 -CH NH 2 - COOH, by the action of enzymes catalyzing the oxidative de-amination of compounds of Formula 2. Either the isolated enzyme or whole cells of some microorganisms can be used for the naenzyme reaction.

Direct hydrolysis of compounds of Formula 2 wherein X is -OCOCH 3 and R is - (CH 2) 3 CHNH 2 COOH ie 7-aminocephalosporane (7ACK) cephalosporin C is possible, but significant complex and prepractic use is little effective and often accompanied by the main product A number of different acylase-producing microorganisms useful for converting CFS-C to 7ACK-Aspergillus, Penicillium, Alteraria, Gibercella Aspergillus, Glioclaudi-um (JP 77) are disclosed. 143,289, Brit. Patent No. 1566830, US Pat. No. 3,239,394, JP 77,038,092), the maximum conversion expected to be 7ACK is 15-18%. In this situation, the highly-desirable six-step enzyme transformation of 7-.alpha.-cylamino of compounds of Formula 2 appears to be where R is - (CH 2) 3 -CNHN 2 COOH to 7-acylamino compounds of the formula I wherein R is - (CH 2) 3 - COOH. pergilus, Neuro-spores, Aerobacter, Candida, Trigonopsis, Fusarium, Penicillium describe Japanese pat. No. 80 23,966; 75,038,092; 1 272 769. Chemical conversion of CFS-C to compounds of Formula 1 is described by jap.pat. No. 78,114,871 or 77,777,078. Formula 1 can be transformed with 7-amino-cephalosporanic acid by enzymatic hydrolysis using enzymes or cells of Pseudomonas ovalis, Commamonas, Arthobacter, Bacillus and others from Japan. pat. Nos. 53 940 93, 76 70 884., 81 85 298., 52 128 293 and others.

The present invention relates to a Trigonopsis variabilis CCY 15-1-2 strain which is a mutant of the original Trigonopsis variabilis strain CCY15-1-1 and which is characterized by inductive synthesis of D-aminoacid: Oz-oxidoreductase, deaminating EC 1-4-3- 3 (D-aminoacid oxidase, DAAox), catalysing the conversion of CFS-C (a compound of formula 2 to compounds of formula 1, or catalyzing the D-amino acid moiety to form the corresponding keto acids. The strain was obtained from the original T. variabilisCCY 15-1-1 collection strain by physical factor mutagenesis, and the strain of the stem cells obtained from the slanted agar was plated on Sabubud ' s Petri dishes and subjected to mutagenesis. UV light from a distance of 50 cm for 1 min, a monocolonium isolate having high DAAox enzyme activity was selected.

The strain is kept on a slanted agar soil according to Saburaud, and an expanded onset of glucose (2%), a maize extract of 100% (2%) and an inducer (0.25%), which may be DL-methionine, D- valine, pH soil 5.0. The cultivation in both the inoculation and production stages is 72 hours.

Biochemical characteristics of the strain: 1) Fermentation of galactose-J-maltose-lactose-sucrose-glucose + fructose -j-sorbose -j-xylose-dulcite-arabinose-ribose-mannose-{- 2) lyzedecarboxylase - 3) catalase + 4) gelation of gelatin - 5) reduction of citrates - 6 µ (S-lactamase -

The amount of DDA oxidase in the cells was monitored indirectly based on the 0-xidase deamination balance of cephalosporin C to produce glutaryl-7ACK by the high pressure method.

Claims (1)

Liquid chromatography or thin layer chromatography. The use of the Trigonopsis variabilis strain GLT-7ACK preparation is documented in the following practical example without limiting its use. Example 1 Trigonopsis variabilis strain CCY 15-1-2 is inoculated into 60 ml of a 2% glucose feed, 2% 100% maize extract and 0.25% inducer (D-valine), pH 5.0 in 500 ml culture flasks. After 72 hours of cultivation on a rotary shaker at 27 ° C, 5 ml of inoculum is inoculated into the same composition as the inoculum, only D-valine is replaced by D or DL methionine at a concentration of 0, 3 ° / o. Cultivation of the same conditions as in the inoculation step is terminated after 72 hours and the cells are separated by centrifugation at 4000 rpm, with cooling (-5 ° C) for 20 minutes. The specific activity of the cells thus prepared is determined on the basis of a balance of oxidative deamination of the cephalosporin C (compound of formula 2) at a concentration of 10 mg / ml in 0.1 M phosphate buffer pH 8.0 at 34 ° C over time 2-hour conversion to give GLT-7ACK (Compound of Formula 1), the cell activity is 15 nmol min -1 mg-1 of dry matter, compared to the flood strain of T. variabilis CCY 15- 1-1 under the same conditions, the specific activity of T.variabilis CCY 15-1-2 strain is 30% higher. 4-3-3, catalyzing the oxidative deamination of a compound of the general formula wherein X is -H, -OH, -OCOCH 3 or a n-kleophilic group, and R 1 is - (CH 2) 2 -CHNH 2 -COOH. to give the corresponding 7-acylamino compounds.