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

Abstract

Strain Streptcimyces lavendofolial 12 / ZA, No. 1670 (Collections of Microorganism Cultures of VNIIA) - producing aclacinomycin A and B.

Description

Od 00

This invention relates to the microbial industry, in particular, to the production of anticancer antibiotics aclacinomycin A and B. The known strain Streptomyces galilaeus MA 1A4M1 is the producer of aclacym A and B l 2. However, the known strain does not have a high antibiotic activity of 46 mg / l aclacinomycin on A-and 23 mg / l of aclacinomycin, B. The aim of the invention is to increase the activity of antibiotics. This goal is achieved by using a strain of Streptomyces lavendofolial 12 / 3A. from the soil image Strain is distinct from Central Asia. It is deposited in the collection of cultures VNIIA under number 1670. Strain Str.lavendofolial 12 / ZA is characterized by the following features. Cultural and morphological signs. Forms abundant aerial liceum lilac-pink color. Sporonosa. spiral, spores with a smooth surface of 0.7 (X) -1.4 microns in size. Substrate mycelium from beige-pink to dirty-lilac color. On Wednesday, brown-brown to brown-pink-violet pigment is released. . The culture was grown at 28 ° C for 28 days, Agar čapek. Aerial mycelium - well developed lilac-pink. Substrate mycelium - beige-roses lilac. Soluble pigment brownish-pink. Agar Gauze 1. Aerial mycelium - growth abundant, pale pink. Substrate mycelium - brown-gr zio-violet. Soluble pigment brownish-pink. Wednesday 1 Krasilnikova. Substrate mycelium - beige-gr but-purple. Soluble pigment brown-pink-purple. Starch agar. Aerial mycelium - moderate growth, pink. Substrate mycelium beige-pink. Soluble beige pigment pigment .. Gluco3oasparagin. 3 Substrate mycelium is sandy brown to brown. Sand pigment soluble pigment. Agar Waxman. Aerial mycelium - growth abundant, pink-gray. Substrate mycelium - from brown-chestnut-brown to very warm-violet). Soluble pigment brown-brown / B2 /. Yeast agar with soluble starch. Aerial mycelium - growth abundant, pink-gray. Substrate mycelium - brown. Soluble pigment - sandy brown to brown. Agar glucose yeast. Aerial mycelium - growth abundant, whitish. Substrate mycelium is dark brown. Soluble pigment brown. Oat agar. Aerial mycelium - good growth, pink and gray. Substrate mycelium - brown-brown / B2 /. Soluble pigment light brown / B7 /. The agar is malt. Aerial mycelium - growth abundant, pink-gray. Substrate mycelium brownish brown. Soluble pigment brown-brown. Agar Gauze 2. Aerial mycelium - satisfactory growth, light gray, Substrate mycelium - brown. The soluble pigment is brown. Aerial mycelium - growth is weak. Substrate mycelium - beige and sand. Soluble pigment is missing. Agar with tyrosine. Aerial mycelium - growth abundant, lilac-pinkish. Substrate mycelium - black plum. Soluble pigment umber. Agar Tresner with citric acid iron. Aerial mycelium - a good growth, lilac-pink. Substrate mycelium - dark bum. Dark pigment soluble pigment. . Sliced potatoes.

Aerial mycelium - good growth, from white to pink.

Substrate mycelium - brown. Soluble pigment brown.

Physiological signs.

Aerobe, optimum growth 24-30C. When not growing. Gelatin thins moderately, milk peptones with browning, starch hydrolyses em moderately. Melanoids form (Tresner medium with iron citrate, tyrosine agar |.

Culture Str.lavendofolral strain I 2 / 3A well uses the following carbon sources: D-glucose, O-xylose, suuarabinose, D-fructose, D-galactose, 17-inositol. Slightly used or not used with α-phrenose, does not use sucrose, raffinose and D-mannitol.

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

Antagonistic signs.

Culture Str.lavendofolial strain 12/3-A when cultivated in a liquid nutrient medium in flasks on rocking chairs on the third day forms aclacinomycin A in the amount of 56-60 mg / l. which has antimicrobial action. Data on antimicrobial activity are given in the table.

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

250.0 250.0

aerugenosa

15.56 7.8

15.5615.56

 The sensitivity was determined by the method of double serial dilutions in mi-peptone agar pH 7.0 with a microbial load of 10 cells / ml using a strain replicator after 20 h of incubation at 37 ° C.

Aklacinomycin A also has cytotoxic activity and antitumor activity.

. Biochemical properties.

Milk peptonized with starch rind hydrolyzes moderately. Gelatin thins moderately. Absorbs 1 / B-glucose, D-chsschzu, L-arabinose p-galactose, 3-inositol; does not absorb sucrose, L-rhamnose, P-fructose-y, raffinose, p-mannitol. Melanoids forms. Example I. The spore culture material Str.lavendofolial strain 12/3-A is infested on oat agar at 28 ° C for 12 days before the appearance of abundant air mycelium. To obtain a uterine inoculum, an agar block is seeded into Erlen Meier flasks with a capacity of 750 mp in liquid medium. do VI without copper sulphate and foam foam. Growing is carried out on a rocking chair (250 rpm) for 2 days at. In the inoculum inoculum (U is introduced in a uterine culture in the amount of 5%. The cultivation is carried out on medium B-2 without copper sulphate at 28 ° C at 200 rpm and aeration 10 l / min for 24 hours. The vegetative inoculum is used in the amount of 5% of the volume of the fermentation medium, Biosynthesis is carried out in a 100 liter 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 ak, H lacinomycin B in the fermentation liquid is determined by the following method: 1. Determination of aclacinomycin A and aclacinomycin B in m After the fermentation, mycelium is obtained from 5.0 MP, the culture fluid is centrifuged at 2000 rpm for 10 minutes, from the obtained mycelium antibiotics are extracted for 2 hours with 4.0 ml of acetone with stirring in 15-20 minutes. 1.0 MP extract was used to vacuum the acetone under vacuum at a temperature below and reextract with 1.0 hl of the roforme.The concentrated chloroform extract was used for the chromatographic separation of the antibiotic complex. For this purpose, the plate with a loose sorbent.capillary jam, retreating to the left and 2 cm from the bottom edge of the plate, applies the entire chloroform extract in the form of a line, 1-1.5 cm long. A standard sample of aclacinomycin A is applied to the same plate and a solution of aclacinomycin B. The plate is placed in a chromatographic chamber, containing chloroform-ethyl acetate-methanol - 7; 2 one . When the front of the solvent reaches the edge of the plate, it is removed and dried in air for 20 minutes. The yellow zones, corresponding to the mobility of aclacinomycin A and aclacinomycin B, are eluted separately by 4 mp of methanol at pH 6 for spectrophotometry at 430 nm. The number of aclacinomycin A and B is determined on the basis of the specific extinction of antibiotics (E for aclacinomycin A and B are 158.6 and 159.2, respectively, jb mg per 1 liter of culture fluid according to the formula: for aclacinomycin A C., 202D for aclacinomycin B C, MG / A 2. Determination of aclacinomycin A and aclacinomycin B in a native solution. A native solution is obtained by centrifugation from 5.0 ml of culture liquid. Extraction is carried out with ethyl acetate at a rate of 5 MP of a native solution of 5 ml of ethyl acetate. Ethyl acetate extract in an amount of 2 ml per steam, vacuum, reextraction of chloroform wire. Further procedures are the same as for the determination of aclacinomycin A and B in mycelium. The amount of aclacinomycin A and B is determined based on the specific the extinction of antibiotics according to the formula: for aclacinomycin AC (mg / l) 126.6D for aclacinomycin BC (mg / l 126, OD On the 3rd day of fermentation on medium B-2, the 12/3-A culture forms about 56 mg / l of aclacinomycin A and 4-8 mg / l of aclacinomycin B. To isolate aclacinomycin A and aclacinomycin B, the culture fluid (60 l) is filtered on a suction pipe. Eltre at pH 4.5-5.0. From a native solution, antibiotics are extracted with ethyl acetate in a ratio of 10: 1. The organic layer is separated and the aqueous residue is evaporated in a vacuum circulating evaporator at a temperature not exceeding 40 ° C. Moist mycelium (5 kg) is extracted twice with 10 liters portions of acetone. Mycelium is separated by filtration and discarded. The acetone extracts are combined and evaporated. The aqueous residues obtained after the distillation of etipacetate and acetone are combined and the resulting concentrate (5 l / after alkalinization with 10% NaOH to 8), antibiotics are extracted three times with double the volume of toluene {10x3 l |, the toluene extracts are combined, and the aqueous the residue is discarded, the toluene extract (30 l) is evaporated in vacuo at a temperature not higher than 40 ° C to 0.1 of the original volume (3.0 l) .The antibiotics are extracted from the obtained concentrate with a 0.5-fold volume of 0.01 nNS. during extraction, the emulsion is broken by centrifugation or separation Antibiotics are extracted from the aqueous solution with 0.5-fold volume of chloroform. Extraction time is 20-30 minutes with constant stirring. The resulting emulsion is broken in a separator. The aqueous layer is discarded, the chloroform layer (1.5 l is dried for 4 hours over anhydrous Na2S04 at the rate of 0 g of salt per 100 ml of extract. The chloroform extract is filtered by filtration from sodium sulfate and evaporated in a vacuum on a rotary evaporator to 0 volume, 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 precipitated antibiotic was separated by filtration funnel Byuh inequality porous glass filter and dried № 4. The filtrate is evaporated to dryness on a rotary evaporator at a temperature not exceeding 40 s. the residue is stanted with hexane; hexane is separated from the precipitate by filtration. Both sediments are combined and transferred to a chromatographic purification. 2.5 g of raw preparation are obtained. Chromatographic separation of aclacinomycin. The column (8x95 cm) is filled with 5.5 suspensions consisting of 1.8 kg of aqueous silicic acid (250-400 µm and 5.5 liters of chloroform in which 0.5% triethng | amine is first dissolved in acetic acid). acid. After the column is filled with a suspension, it is washed with 3 l of chloroform .. A raw preparation (10 g) obtained at the previous stage and dissolved in 100 ml of chloroform is layered on the column. After re-absorption of antibiotics, the column is connected to the system for elution. separation of antibiotics, wire 0 38 step gradient elution. For this purpose, through the column pass through r 1 4l chetyrehzshoristogo carbon, 2) 8n system consisting of carbon tetrachloride (CC1 and isopropanol 25: 1; 3) 8.5 L of the system consisting of SSC-isopropanol 20: 1; 4) 20.5 liters of a 10: 1 system consisting of CC14-isopropanol. Aklacinomycin B is removed from the cannonix by the CC14 system isopropanol 20: 1, and aclacinomycin A - by SCC — isopr-opanol 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, a solution of HCI. From the aqueous layer, antibiotics are extracted with a 0.2-fold volume of chloroform. Chloroform extracts of aclacinomycin A and acclacinomyc B are dried separately with anhydrous Na2SO at the rate of 2 g per 100 ml of solution, the drying agent is separated by filtration, and the filtrate is evaporated in vacuo at 40 ° C to O, 1 volume. To this residue was added a 10-fold volume of dry hexane. The mixture was incubated for 30 minutes, the precipitate was 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 preparation. Example 2. Agar block 12 daily culture Str.lavendofolial strain 12/3-A grown on oats agar, seeded flasks with medium B-2 without copper sulfate. The cultivation is carried out on a shaker at 250 rpm at 28 ° C for 48 hours. 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 medium B-3 of the following composition,%: soybean flour 1.5, starch kartufelny 3, chalk 1.4, sodium chloride 0.3, sulfuric acid copper 0.007. The flasks with the fermentation medium are cultured under the same conditions for 72 hours. The culture liquid activity is 60 mg / l for aclaciamiomycin A and 10 mg / l for aclacinomycin B.

91069433

Use of the invention of posvocomycin B (from 23 µg / ml to a lit. increase the activity of antibiotic µg / ml; simplify the process of cleavage of aklacinomycin A; emit and purify the target and increase the degree of formation of acclamation.