SU1419521A3 - Method of producing antibiotic 2188 and strain of mold fungus penicillium rugulosum ferm bp-142 used for production of antibiotic 2188 - Google Patents

Abstract

PURPOSE:To obtain a novel antibiotic substance No. 2188, by cultivating a microorganism, belonging to the genus Penicillium, and having the ability to produce the antibiotic substance No. 2188 aerobically in a culture medium. CONSTITUTION:Penicillium rugulosum (FERM-P No. 142) newly separated from a soil is cultivated in a culture medium for production at 20-30 deg.C to produce the aimed novel antibiotic substance No. 2188. The resultant antibiotic substance has the following properties: Molecular weight: 193. Molecular formula: C9H7NO4. Melting point: 135-136 deg.C (decomposition). A wide range of antibacterial spectrum, e.g. the genuses Bacillus, Streptococcus, Micrococcus, Corynebacterium, Proteus, Pseudomonas, Vibrio, Salmonella, Aerobacter, Xanthomonas, etc.

Description

cm

The invention relates to the microbiological industry and can be used in the production of antibiotics.

A new antibiotic 2188 is 3- (6-isocyan-3, 7-dioxacrylo 4.1.0. Hept-4-yl propenoic acid following the formula

1419

n / soon

n

Antibiotic 2188 is obtained from the culture fluid of a strain of Panicillium rup, ulosum FERM BP-142.

The strain was isolated from soil, deposited at the Institute of Fermentation Research at the Agency of Industrial Science and Technology of the Ministry of International Trade and Industry in Japan under number 142 (FERM BP-142).

Cultural and morphological

naki. Colonies are usually velvety at 30 ° C and woolly with dull yellowish green, light green, light grayish green or green gray depending on the type of culture medium, dark green conidia, yellow or orange yellow mycelium, reverse side of the colony - yellow-orange or orange-red. At 37 ° C growth is absent. On malt extract agar, good growth On potato dextrose agar, good growth. On agar Saburo good growth. On oat flour agar, growth is good. On glucose agar with dry draws, growth is good. On a mukor-synthetic medium, growth is limited. On Capeka agar, growth is bad.

Conidiophores: grow directly from creeping or twisting hyphae and air hyphae, 2.5-3.0 X 100-300 µm, the surface of the walls is smooth or slightly coarse. Penicillus (tuft of bristles, brush): symmetric collar. Metula: 2.5-3.5x7.5- -15 μm, a bunch of 3-6 methyl. Phialida is typically lanceolate, 2.0-3.8x7.5- -13 µm, in clusters of 2-6 elements in a whorl. Conidia: elliptical, 2, 5-3x3-3,8 microns, green, smooth walls, tangled or parallel chains.

Physiological and biochemical signs. Grows at 10-35 ° C, optimum growth

ten

15

25

. 195212

at 20-30 ° C. Utilizes glucose, maltose, dextrin, starch, lactose, sucrose, glycerin as a carbon source. As a source of nitrogen, it utilizes peptone, meat extract, yeast, yeast extract, soy flour, peanut flour, cotton seed refuse, liquid corn extract, rice bran, inorganic nitrogen.

The metabolic product of the strain, antibiotic 2188, exhibits antibacterial activity against various gram-positive and gram-negative bacteria, and does not show acute toxicity to mice upon intraperitoneal administration in an amount of 100 mg / kg.

Example 1. Culture of the strain Penicilliura rugulosum FERM BP-142 from oblique agar is inoculated in 100 ml of Bennett medium (0.1% yeast extract, 0.1% beef extract, 0.2% NZ amine A-type, and 1% glucose, pH 7.2) in a 300 ml Erlenmeyer flask. 50 flasks

20

thirty

35

. 40

D5 ,,

50

culture medium is incubated at 248 ° C for 72 hours on a rotating shaker at 180 rpm.

5 liters of the obtained culture are inoculized in 100 liters of sterilized medium in a 200-liter fermenter at 24 ° C for 60 hours with aeration at a rate of 50 l / min and stirring at 200 rpm. The composition of the nutrient medium,%: soluble starch 1.5; glycerin 1.5; cotton seed waste 1.0; Soya flour 1.0; yeast extract 1.0; CrHP04 0.7; MgS04 7H, jO 0.05; 0.001; defoamer (A, CECANOL G 126, Asahi Denk Kogio KK) 0.1.

The whole culture (100 l) was passed through Shapless (type GR-U 122-1, Tomoye Indiginering Co.) and Delabal (type BPP X 309-355-60, Sweden) to remove the mycelial sediment. The clear or clear floating top layer was adjusted to pH 4 with hydrochloric acid and extracted with a fifth part by volume ethyl acetate using a continuous centrifugal extraction device (type C-1333, Hitachi Ltd.). The extract is concentrated with a condenser, and the concentrate is then subjected to back extraction with aqueous 1% sodium bicarbonate solution. Water phase

 1419521

Colonize to pH 4.0 with hydrochloric acid and extract with ethyl acetate. The extract is dried over non-sodium sulfate.

and evaporated at a temperature of less than 40 ° C under vacuum to obtain a crude (neo-C (enry)) product.

The Neochersen product is passed through a column of activated carbon placed on the top of a column (4 cm in diameter and 50 cm high) filled with silica gel (CARGO GEL Q 23, Waco Chemical Co.), and developed with a mixture of ethyl acetate and chloroform (1:19 volume) to elute the active fraction, to which n-hexane is then added. 3 g of white powder are obtained.

The resulting product has the following physico-chemical properties.

Found,%: C 56.35; H 3.49; N 7.34; About 32.82.

C, H-, KO (mol.m. 193).

M.p. 135-136 ° C (decomposition), specific rotation ot j, -128.8 (with 1.0% in ethyl acetate).

UV spectrum, T (methanop). 215 nm (t 14378).

IR (KRr): 3450; 3000; 2150; 1700; 1665; 1425; 1330; 1090; 980; 900; 875.

NMR spectrum (deuterated acetone) S, MD: 2.55 (dd, 1H. G 16, G 1.0); 3.05 (dd, 1H, G 16 D 0.5); 3.98 (d. 111, D 1.0); 4.45 (d. 1H, G 0.5); 6.11 (d. 1H, D 16); 6.71 (d. 1H, G 16).

 NMR-sectr (deuterated meld, d: 168, Uc); 165.1 (s);

Example 2. Five 300 ml Erlenmeyer flasks containing respectively 100 ml of medium with pH 7.2, which includes 1% glucose, 0.1Z yeast extract, 0.1% beef extract, and 0.2% polypepton sterilized under pressure. Spores from the culture of oblique mold agar iioculi

10 on Wednesday in flasks and cultured at 26 ° C for 70 hours with shaking at 180 rpm. The resulting culture (500 ml) is inoculated into 15 liters of sterilized medium with a pH of 7.0.

15 30-liter fermenter and cultured at 26 ° C for 60 hours with aeration purified with air at a rate of 15 liters / min and stirring at 300 rpm. Composition sterilized

20 mediums next1 1 (,%: sodium nitrate

0.3; 0.1; magnesium sulfate heptahydrate 0.05; potassium chloride 0.05; iron (II) sulfate heptahydrate 0.001; sucrose 3; yeast ex

25 path 1; defoamer (ADECANOL ZG 109) 0.1.

The resulting culture is filtered using as 30

ve filtering agent Celite 545. The filtrate is adsorbed on a column with a diameter of A cm and a height of 50 cm, filled with Liapon HP 20 resin, washed with water and washed with 60% methano-35 scrap. The eluate is condensed under vacuum at a temperature of less than 40 ° C. The concentrated material is extracted with ethyl acetate, and the extract is dried with anhydrous sodium sulfate and then concentrated

tanol) 5

141.9 (d); 125.8 (d); 66.4 (d); 66.1 (d); 40 are centered under vacuum at tempera64, 9 (s); 62.5 (s); 36.6 (t). Pour less than 40 ° C.

The product is soluble in methanol, eta- The resulting liquid material is omitted, acetone, ethyl acetate, chloroform and benzene, insoluble in hexane and petroleum ether.

Thin layer chromatography using C-picagel: only one spot at, 62 p of a mixture of chloroform-ethyl acetate-acetic acid (10: 10: 1); R, 0.44 in chloroform-ethyl acetate-gg. The column is developed with ethyl acetate-acetic acid (10: 10: 0.2); R chloroform (1:19 by volume) dn elu- 0.79 in a mixture of n-butanol-acetic acid / water (4: 1: 1).

cabins through the column (diameter 1.5 cm, height 30 cm), zap ;; enuvnuyu activated upps: -. The active fraction was concentrated in vacuo, the concentrated material was applied to a column (20 X 300 mg-O from silica gel (CARGO G C 200) and washed with chloroform.

ting the active fraction to which n-hexane is added. Get 200 mg of white powder. The resulting product exhibits the same physicochemical and biological properties as the product of example 1.

Reversible thin layer chromatography with KS-18: only one spot at, 86 in methanol-water (4: 1); , 94 in a mixture of propanol-water (4: 1).

Example 2. Five 300 ml Erlenmeyer flasks containing respectively 100 ml of medium with a pH of 7.2, which includes 1% glucose, 0.1Z yeast extract, 0.1% beef extract, and 0.2% polypepton, sterilized under pressure. Spores from the culture of oblique mold agar iioculi

on Wednesday in flasks and cultured at 26 ° C for 70 hours with shaking at a speed of 180 rpm. The resulting culture (500 ml) is inoculated into 15 liters of sterilized medium with a pH of 7.0.

A 30-liter fermenter and cultured at 26 ° C for 60 hours with aeration purified with air at a rate of 15 liters / min and stirring at 300 rpm. Composition sterilized

next media 1 1 (,%: sodium nitrate

0.3; 0.1; magnesium sulfate heptahydrate 0.05; potassium chloride 0.05; iron (II) sulfate heptahydrate 0.001; sucrose 3; yeast extract 1; defoamer (ADECANOL ZG 109) 0.1.

The resulting culture is filtered using as

ve filtering agent Celite 545. The filtrate is adsorbed on a column with a diameter of A cm and a height of 50 cm, filled with Liapon HP 20 resin, washed with water and filled with 60% methanol. The eluate is condensed under vacuum at a temperature of less than 40 ° C. The concentrated material is extracted with ethyl acetate, and the extract is dried with anhydrous sodium sulfate and then finished

The resulting liquid material is

The column is developed with a mixture of ethyl acetate-chloroform (1:19 by volume) one day.

cabins through the column (diameter 1.5 cm, height 30 cm), zap ;; enuvnuyu activated upps: -. The active fraction was concentrated in vacuo, the concentrated material was applied to a column (20 X 300 mg-O from silica gel (CARGO G C 200) and washed with chloroform.

The column is developed with a mixture of ethyl acetate-chloroform (1:19 by volume) one day.

ting the active fraction to which n-hexane is added. Get 200 mg of white powder. The resulting product exhibits the same physicochemical and biological properties as the product of example 1.

Thus, the use of the invention will make it possible to obtain a new antibiotic with a broad antibacterial spectrum of action.

Claims (2)

1. The method of half a day of antibiotic 2188 of the general formula n soon H concluded in the fact that the strain Penicillium rugulosum ferm BP-142 cul Sifted in nutrient medium containing organic sources of nitrogen and carbon, mineral salts and antifoam, under aeration and mixing conditions, extracted with a separate filtrate of the culture liquid with ethyl acetate, followed by passing the extract through a column of activated carbon, eluting with a mixture of ethyl acetate and chloroform and crystallization of the target product in the presence of n-hexane in the eluate. 2. The strain of mold fungus Penicilium rugulosura FERM BP-142, used to obtain antibiotic 2188.