SU1069433A1 - Strain streptonuces lavendofolial 12/3a producer of alkacynomycins a and b - Google Patents

Abstract

Strain Streptcimyces lavendofolial 12/ZA, No. 1670 (Collection of microorganism cultures of the All-Russian Research Institute of Agronomy) is a producer of aclacinomycins A and B.

Description

Od 00

The invention relates to the microbiological industry, in particular to the production of antitumor antibiotics aclacinomicins A and B. The strain Streptomyces galilaeus MA 1A4M1 is known as a producer of aclacinomicins A and B1. However, the known strain does not have high antibiotic-forming activity - 46 mg / l aclacinomicin A - and 23 mg / l aclacinomicin B. The aim of the invention is to increase the antibiotic-forming activity. This goal is achieved using the strain Streptomyces lavendofolial 12 / 3A. The strain was isolated from Central Asia. Deposited in the culture collection of the All-Russian Research Institute of Archaeology under No. 1670. The strain Str.lavendofolial 12 / 3A is characterized by the following features. Cultural and morphological characteristics. Forms abundant aerial mycelium of lilac-pink color. Sporebearers are spiral, spores with a smooth surface, 0.7(X)-1.4 μm in size. Substrate mycelium is from beige-pink to dirty-lilac. A brown-brown to brown-pink-violet pigment is released into the medium. The culture was grown at 28°C for 28 days, Czapek Agar. Aerial mycelium is well developed, lilac-pink. Substrate mycelium is beige-pink-lilac. Soluble pigment is brown-pinkish. Gause Agar 1. Aerial mycelium - abundant growth, pale pinkish. Substrate mycelium is brown-dark-violet. Soluble pigment is brownish-pinkish. Medium 1 Krasilnikova. Substrate mycelium is beige-gray-violet. Soluble pigment is brown-pink-violet. Starch agar. Aerial mycelium - moderate growth, pink. Substrate mycelium is beige-pink. Soluble pigment is beige-pink. Glucose-aspartic. 3 Substrate mycelium is sandy-brownish to brown. Soluble pigment is sandy-brown. Waxman agar. Aerial mycelium - abundant growth, pink-gray. Substrate mycelium is from brown-chestnut-brown to dirty-violet). Soluble pigment is brown-brown /B2/. Yeast agar with soluble starch. Aerial mycelium - abundant growth, pink-gray. Substrate mycelium is brown. Soluble pigment is sandy-brown to brown. Glucose-yeast agar. Aerial mycelium - growth is abundant, whitish. Substrate mycelium is dark brown. Soluble pigment is brown. Oat agar. Aerial mycelium - growth is good, pink-gray. Substrate mycelium is brown-brown /B2/. Soluble pigment is light brown /B7/. Malt agar. Aerial mycelium - growth is abundant, pink-gray. Substrate mycelium is brown-chestnut. Soluble pigment is brown-brown. Gause 2 agar. Aerial mycelium - growth is satisfactory, light gray. Substrate mycelium is brown. Soluble pigment is brown. Aerial mycelium - weak growth. Substrate mycelium - beige-sand. Soluble pigment absent. Tyrosine agar. Aerial mycelium - abundant growth, lilac-pink. Substrate mycelium - plum-black. Soluble pigment - umber. Tressner agar with iron citrate. Aerial mycelium - good growth, lilac-pink. Substrate mycelium - dark umber. Soluble pigment dark umber. Potato slices.

Aerial mycelium - good growth, from white to pink.

Substrate mycelium is brown. Soluble pigment is brown.

Physiological signs.

Aerobic, optimum growth 24-30C. Does not grow. Dilutes gelatin moderately, peptonizes milk with browning, hydrolyzes starch moderately. Forms melanoids (Tresner medium with ferric citrate, agar with tyrosine |.

The culture of Str.lavendofolral strain I 2/ZA makes good use of the following carbon sources: D-glucose, O-xylose, c-arabinose, D-fructose, D-galactose, 17-inositol. It uses c-rhamnose poorly or does not use it, and does not use sucrose, raffinose, or D-mannitol.

The culture of Str.lavendofolial strain 12/3-A is preserved on slanted agar media under a layer of vaseline oil at +5°C for 6 months.

Antagonistic signs.

The culture of Str.lavendofolial strain 12/3-A when cultivated in a liquid nutrient medium in flasks on shakers on the third day produces aclacinomycin A in an amount of 56-60 mg/l. which has an antimicrobial effect. Data on antimicrobial activity are given in the table.

Antimicrobial spectrum of aclacinomycin A isolated from the culture of Str.lavendofolial strain 12/3-A in comparison with the prototype.

250,0 250,0

aerugenosa

15.56 7.8

15,5615,56

g sensitivity was determined by the method of two-fold serial dilutions in m-peptone agar pH 7.0 with a microbial load of 10 cells/ml using a replicator strain after 20 hours of incubation at 37°C.

Aclacinomycin A also has cytotoxic activity and antitumor action.

. Biochemical properties.

Milk peptonizes with browning, starch hydrolyzes moderately. Dilutes gelatin moderately. Assimilates 1 / B-glucose, D-fructose, L-arabinose, β-galactose, 3-inositol; does not assimilate sucrose, L-rhamnose, β-fructose, raffinose, β-mannitol. Forms melanoids. Example I. Spore material of the culture of Str.lavendofolial strain 12/3-A is grown on oat agar at 28 °C for 12 days until abundant aerial mycelium appears. To obtain a mother nutrient, the agar block is seeded into 750 ml Erlenmeyer flasks in liquid B-Y medium without copper sulfate and foam. Growing is carried out on a shaker (250 rpm) for 2 days at . The mother culture is added to the seed inoculant (I) in an amount of 5%. Cultivation is carried out on B-2 medium without copper sulfate at 28°C, 200 rpm and aeration of 10 l/min for 24 hours. The vegetative inoculum is used in an amount of 5% of the volume of the fermentation medium. Biosynthesis is carried out in a 100 l fermenter containing 60 l of B-2 medium for 3 days at a temperature of 27°C, 200 rpm and aeration of 60 l/min. The content of aclacinomycin A and aclacinomycin B in the fermentation liquid is determined using the following method: 1. Determination of aclacinomycin A and aclacinomycin B in the mycelium. After the end of fermentation, mycelium is obtained from 5.0 MPa of the culture liquid by centrifugation. at 2000 rpm for 10 min. Antibiotics are extracted from the resulting mycelium for 2 h with 4.0 ml of acetone with stirring every 15-20 min. Acetone is distilled off from 1.0 MP extract in a vacuum at a temperature below and re-extraction of 1.0 chloroform is carried out. The concentrated chloroform extract is used for chromatographic separation of the antibiotic complex. For this purpose, the entire chloroform extract is applied to a plate with an unfixed layer of sorbent. capillary, stepping back 2 cm to the left and from the lower edge of the plate, in the form of a line 1-1.5 cm long. A solution of a standard sample of aclacinomycin A and a solution of aclacinomycin B are applied to the same plate. The plate is placed in a chromatographic chamber containing then chloroform-ethyl acetatemethanol - 7; 2. 1. When the solvent front reaches the edge of the plate, it is taken out and dried in air for 20 min. Yellow zones, corresponding in mobility to aclacinomycin A and aclacinomycin B, are eluted separately with 4 ml of methanol at pH 6 for spectrophotometry at 430 nm. The amount of aclacinomycins A and B is determined based on the specific extinction of the antibiotics (E for aclacinomycins A and B is 158.6 and 159.2, respectively, j in mg per 1 l of culture fluid according to the formula: for aclacinomycin A C., 202 D for aclacinomycin B C., mg / A 2. Determination of aclacinomycin A and aclacinomycin B in native solution. Native solution is obtained centrifugation from 5.0 ml of culture fluid. Extraction is carried out with ethyl acetate at the rate of 5 ml of ethyl acetate per 5 MP of native solution. The ethyl acetate extract in an amount of 2 ml is evaporated in a vacuum, re-extraction is carried out with chloroform. Further procedures are the same as in the case of determining aclacinomycins A and B in the mycelium. The amount of aclacinomycins A and B is determined based on the specific extinction of antibiotics according to the formula: for aclacinomycin AC (mg / l) 126.6 D for aclacinomycin BC (mg / l1 126.0 D On the 3rd day of fermentation on medium B-2, culture 12/3-A forms about 56 mg/l aclacinomycin A and 4-8 mg/l aclacinomycin B. To isolate aclacinomycin A and aclacinomycin B, the culture liquid (60 l) is filtered on a Nutsche filter at pH 4.5-5.0. Antibiotics are extracted from the native solution with ethyl acetate in a ratio of 10:1. The organic layer is separated and evaporated to the aqueous residue in a vacuum of a circulating evaporator at a temperature not exceeding 40°C. Wet mycelium (5 kg) is extracted twice with acetone in 10 l portions. The mycelium is separated by filtration and discarded. Acetone extracts are combined and evaporated. Aqueous residues obtained after distillation of ethyl acetate and acetone are combined and from the resulting concentrate (5 l/ after alkalization) 10% NaOH to .8, antibiotics are extracted three times with a double volume of toluene {10x3 l|, the toluene extracts are combined, and the aqueous residue is discarded, the toluene extract (30 l) is evaporated in vacuum at a temperature not exceeding 40 ° C to 0.1 of the original volume (3.0 l). Antibiotics are extracted from the resulting concentrate with a 0.5-fold volume of 0.01 N HCl. The emulsion formed during extraction is broken by centrifugation or separation. Antibiotics are extracted from the aqueous solution with a 0.5-fold volume of chloroform. The extraction time is 20-30 min with constant stirring. The resulting emulsion is broken on a separator. The aqueous layer is discarded, the chloroform layer (1.5 l) is dried 4 h - over anhydrous Na2SO4 at the rate of 0 g of salt per 100 ml of extract. The chloroform extract is separated by filtration from sodium sulfate and evaporated in a vacuum on a rotary evaporator to 0.1 volume, 10-12 volumes of dry hexane are added to the residue and to complete the precipitation process the mixture is left for 30 min. The precipitated antibiotic is separated by filtration on a Buchner funnel with a porous glass filter No. 4 and dried. The filtrate is evaporated to dryness on a rotary evaporator at a temperature not exceeding 40C. The residue is diluted with hexane, hexane is separated from the precipitate by filtration. Both precipitates are combined and transferred for chromatographic purification. 2.5 g of the crude drug is obtained. Chromatographic separation aclacinomycins. The column (8x95 cm) is filled with 5.5 of a suspension consisting of 1.8 kg of aqueous silicic acid (250-400 μm) and 5.5 l of chloroform, in which 0.5% triethylamine in acetic acid is pre-dissolved. After filling the column with the suspension, it is washed with 3 l of chloroform. The crude preparation (10 g), obtained in the previous stage and dissolved in 100 mN of chloroform, is layered on the column. For resorption of antibiotics, the column is connected to the elution system. To separate the antibiotics, a 0.38-step gradient elution is carried out. For this purpose, the following are passed through the column: 1) 4 l of carbon tetrachloride; 2) 8 l of the system consisting of carbon tetrachloride (CC1 and isopropanol 25:1; 3) 8.5 l of a system consisting of CCl4-isopropanol 20:1; 4) 20.5 l of a system consisting of CCl4-isopropanol 10:1. Aclacinomycin B is removed from the column with a system of CCl4-isopropanol 20:1, and aclacinomycin A with CCl4-isopropanol 10:1. Eluates of 5-7 l containing aclacinomycin A and aclacinomycin B are separately extracted with a 0.2-fold volume of 0.01 N HCl solution. Antibiotics are extracted from the aqueous layer with a 0.2-fold volume of chloroform. Chloroform extracts of aclacinomycin A and aclacinomycin B are dried separately with anhydrous Na2SO at the rate of 2 g per 100 ml of the solution, the drying agent is separated by filtration, and the filtrate is evaporated in a vacuum at 40 ° C to 0.1 volume. A 10-fold volume of dry hexane is added to this residue. The mixture is kept for 30 min, the precipitate is separated on a porous glass filter No. 4 and dried to an air-dry state. 0.7 g of aclacinomycin A and 0.1 g of aclacinomycin B are obtained from 2.5 g of the crude drug. Example 2. An agar block of a 12-day culture of Str.lavendofolial strain 12/3-A, grown on oat agar, is inoculated into seed flasks with B-2 medium without copper sulfate. Cultivation is carried out on a shaker at 250 rpm at 28 C for 48 h. The grown seed material is used in an amount of 5-10% of the volume of the fermentation medium. Fermentation is carried out in Erlenmeyer flasks on B-3 medium of the following composition, %: soy flour 1.5, potato starch 3, chalk 1.4, sodium chloride 0.3, copper sulfate 0.007. Flasks with fermentation medium are cultivated under the same conditions for 72 h. The activity of the culture liquid is 60 mg / l for aclaciomycin A and 10 mg / l for aclaciomycin B.

91069433

The use of the invention allows to increase the activity of the antibio-product.