CS245153B1 - Strain of acremonium chrysogenum microorganism - Google Patents

Abstract

Production strain of Acremonium chrysogenura / Thirum et Sukap / W. Gams CCM F-729 produces acid under aerobic conditions 7 / D-5-amino-5-carboxy-n-valeramido / 3-acetoxymethyl-3-cephem-4-carboxylic acid called. cephalosporin C of formula I HOOC-CH-CH 2/3-CO-NH NH2 COOH ch2ococh3 / 1 / Cephalosporin C is used for preparation semisynthetic cephalosporin antibiotics. Cephalosporin C is prepared submerged fermentation in the presence of sulfur sources. Most industrial production operations DL-methionine dependent dependent auxotrophic mutants. Tribe Acremonium chrysogenum CCM F-729 is not dependent on DL-methionine, produces cephalosporin C to economically advantageous sulfur sources such as NajS ^ O.551 ^ 0.

Description

Cephalosporin C is used for the preparation of semi-synthetic cephalosporin antibiotics.

Cephalosporin C is prepared by submerged fermentation in the presence of sulfur sources. Most industrial production strains have DL-methionine dependent auxotrophic mutants.

Acremonium chrysogenum CCM F-729 strain is not DL-methionine-dependent, producing cephalosporin C on economically advantageous sulfur sources such as Na 2 SO 4.

The present invention relates to a novel industrial microorganism producing 7- (D-5-amino-5-carboxy-n-valeramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid of the general formula I, so-called. cephalosporin C / CFS-C /

HOOC-CH ICHjh-CO-NH

I 'nh ₂ (ch ₂ ococh 3/1 ₎

COOH

Cephalosporin C is used as a starting material in the preparation of semisynthetic cephalosporin antibiotics. The production strain was obtained in a screening program aimed at isolating DL-methionine-independent strains.

The starting strain was a Cephalosporium acremonium WIS OP 163 culture, the maximum production of cephalosporin C reached on sodium thiosulfate as a sulfur source was 200-400 µg / ml. Cephalosporin antibiotics derived from 7-aminocephalosporic acid are produced predominantly using the Cephalosporium acremonium strain (after reclassification by Gams W. in 1971 Acremonium chrysogenum).

The genus Cephalosporium is systematically included in the family Moniliceae in the class Fungi imperfecti / Deuteromycetes /. In the genus Cephalosporium, spores are formed only terminally on non-branched conidiophores, almost regularly seated on the main mycelium. Cephalosporium consists of septated hepthoes, septa dividing protoplasm into smaller sections.

Konidionosl are individual, strong basophilic. Known is reproduction by asexual mitosis and cellular elongation. Asexual spores are unicellular, single-core conedias, produced on hýfach. Both mycelium and perideal cells contain one haploid nucleus. They retain these properties even in submerged cultures.

Cephalosporium as a eukariotic organism has a membrane-bounded nucleus with a chromosomal structure and an oytoplasm with a relatively complex membrane structure (mitochondria, endoplasmic reticulum). In particular, extracellular accumulation of the antibiotic caused by excretion through a concentration gradient is significant.

Four types of cells are present in submerged culture: septic hyphae, conidia, germinating conidia and arthospores.

The morphology varies depending on the sulfur source used. In DL-methionine fermentation, arthospores are predominant, with the existence of maximum antibiotic formation associated with them. Fiber forms predominate in submerged culture using inorganic sulfur sources; the fiber thickness is less than that of DL-methionine.

The strain described was obtained as follows

The native strain of Caphalosporium acremonium WIS OP-163 was treated with the chemical mutagen N-nitrosomethylbiuret. The best isolate, designated 8c VULM, produced 1,800 to 2,000 µg / ml cephalosporin C in submerged culture.

The isolate 8c VULM was exposed to 0V radiation in the next phase. The obtained isolates were evaluated by a rapid screening method on agarized nutrient broth rollers with different sulfur sources. Rollers with raised columns on the surface were tested on an agar plate with the test microorganism Alcaligenes faecalis ATCC 8750, specific for cephalosporin C.

The isolate with the largest diameter of the Inhibition Zone on agarized sodium thiosulfate was further tested in a flask fermentation and in laboratory fermenters. The isolate had the working designation of 188 VULM, the activity of cephalosporin C in the fermentation pod filtrates was determined in the 168th culture hour by the spectrophotometric method / Claridge C.A., Vaughan R.W .: Antimicrob. Agents Chemother., 1969, 131 (and HPLC method). The genealogical scheme of the strain is designated as scheme no. 1. VULM Xzolate 188 is stored in Cs. collection of UJEP microorganism in Brno under the number CCM F-729.

Chemical mutagen degradation.

Kmeft OP 163 WIS was deposited in the form of a lyophilized can or briefly on sloping agar / sporulation platform labeled NB-II; the composition given below. An aqueous suspension at a concentration of 10 10 to 10® conidia / ml was prepared from slanted agar. The suspension was filtered through a cotton plug. The N-nitrosomethylbiuret mutagen was added at a concentration of 0.4 i to the conidia suspension.

The mutangen was in contact with the culture for one hour on a shaker (4Hz) at 27 ° C. After exposure, dilutions of 10 to ² to 10 conidia / ml were prepared from the mutangen-culture mixture. The diluted suspensions were inoculated with NB-II agar pad Petri dishes. As a control, the spore suspension served without the addition of mutangen.

Mutangen operations were performed without daylight access. Collaboration on Petri dishes lasted 11 days at 27 ° C. Individual colonies were transferred to NB-II sloping agar and assayed for production activity on flasks.

Effect of physical mutangen:

A conidia suspension was prepared from the slant agar culture by washing the surface with 10 ml of sterile water. The suspension was filtered through a cotton filter; Working dilutions of 10 to 10 conidia / ml were prepared from the stock suspension and seeded with Petri dishes (NB-II). The source of radiation was a germicidal OV-lamp (Philips TUV 30W, wavelength 253.7 nm contributing to a total spectral energy of 93.5%).

The conid-coated agar surface was exposed for 1 minute from a distance of 30 cm. After irradiation, Petri dishes were placed in a thermostate where the culture sporulated at 27 ° C for 11 days. Spores were transferred from the grown colonies by needle technique to the surface of the agar-roller blocks.

After 5 days, the surface of the blocks was covered with culture. The blocks were placed on agar-plate glass containing 25% Alcaligenes faecalis ATCC 8750 culture. Incubation of the blocks lasted 18 hours at 37 ° C. Evaluation of the isolates was based on measurement of the inhibition zones around the blocks. The best isolate was inoculated on a Petri dish from a colony on a sloping agar NB-II and tested in both laboratory and quarter-fermentations.

Agar platform NB-II hmot./obj. Basto-beef extract 0.3 Bacto-peptone 0.5 glucose 1.0 FeSO ₄ .7H ₂ O 0,005 _MgSO4 7H ₂ O 0,005 DL-methionine 0,005 L-cysteine 0,005 Difco agar 2.5 pH pH before sterilization -6.8.

245153 4

Preparation of vegetative inoculum:

A 500 ml flask filled with a 60 ml inoculation pod consisting of:

sucrose 1.5% w / v

corn liquor 0.5 ammonium acetate 0.5 soybean oil 0.2 pH before. by sterilization it is adjusted to 7.0.

An inoculated agar or barley grain culture is used for vaccination. canned barley. Preparation of the vegetative inoculum takes 72 hours at 27 ° C on a shaker (4 Hz). Vegetative inoculum is used to inoculate the production stages, dispensed in 500 ml flasks of 40 ml, composed of:

corn starch peanut flour sucrose glucose CaCO ₃ ^Na 2 ^S 2 ° 3 - ^5H 2 ° borax cough oil

5.0% w / v 7.0 0.5 1.25 0.5 0.9 0.05 2.8

For vaccination, 8% v / v is used. vegetative inoculum. The culture has the same conditions as the preparation of the vegetative inoculum, the cultivation length is 168 hours. A comparison of the production activity of the Cephalosporium acremonium WIS OP-163 strain with the 8c VULM and 188 VULM isolates is given in Table 1. The results obtained in the flask fermentation are compared:

Table 1

experiment number tribe prod. activity (γg / ml) CFS-C 1 WIS OP-163 350 8c VULM 1 860 2 8c VULM 1 930 188 VULM 4 000 3 188 VULM 4 000 9 VULM 1 680

Strain used in fermentation using DL-methionine as the sulfur source.

In Laboratory Glass Dancers / Ex. Workshop of the Czechoslovak Academy of Sciences /, volume 2 liters, working volume 1 liter, under conditions: aeration of 0,75 volume of air / volume of podium, mixing 10 Hz, temperature up to 24 cultivation hours 28 ° C, then by the end of fermentation 23 ° C activity 5,200 µg / ml in 168 hours of culture.

Tribe morphology:

The morphology of the strain Acremonium chysogenum CCM F-729 can be described in the following way: In monocolonium sowing, the cream-colored colonies form a beige-colored colonies with a diameter of 5 to 7 mm in the center. The colony surface is wrinkled sline, the edges are radially milled, the center of the colony is elevated by about 3 them, forming an irregular crater. Septic vines have a depth of 1.5 to 2.0 µm. Mycelium is light colored. Conidia are elongated ellipso-shaped, retaining the depth of the original vulture, their length is 5 to 6 pm (often covered with mucus) The strain is preserved as a can on clay-sand mixtures as a lyophilized can as a sloping agar under NB-II and can on loose substrate, which is shelled barley. The strain is stabilized during passage of the sloping agar-vegetative inoculum-loose substrate-vegetative inoculum as well as the passage of the sloping agar-loose substrate-loose substrate-vegetative inoculum. All materials are stored at 5 ° C, sloping agar and canned barley for 4 months.

Genealogical scheme of strain Acremonium chrysogenum CCM F-729

Cephalosporium acremonium WIS OP-163

N-nitrosomethylbiuret. Isolate 8 c VULM

OV light

Isolate 188 VULM »CCM F-729 /

Scheme no. 1

Claims (1)

Strain of Acremonium chrysogenum / Thirum et Sukap / W. Gams CCM F-729 producing 7- (D-5-amino-5-carboxy-n-valeramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid. cephalosporin C of formula 1.