SU298131A1 - - Google Patents

Description

METHOD OF OBTAINING KAZUGAMICIN ANTIBIOTICS

The invention relates to methods for producing antibiotics by cultivating the production fungus on a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts, in particular to methods for producing kazugamycin by using the fungi from the genus Strepiomyces as a producer.

A known method for producing kazugamycin in the culture of the fungus producer Sir. kasugaensis ATCC 15714 and ATCC 15715.

A method for the production of kazugamycin, including the cultivation of a fungus-producer of the genus Streptomyces on a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts, by a deep method, in which Str is taken as a representative of the genus Streptomyces, is proposed. kasugaspinus, and the cultivation is carried out at a temperature of from 25 to 35 ° C.

Str. The kasugaspinus was isolated from a soil sample taken at Maru, Usada-Ho in Nagano prefecture in June 1965 on a glycerol-casein-agar medium. He received the laboratory number MA 350-SZ and found the following features.

Czapek's glycerol burning is cultivated at 27 ° С: colorless or slightly yellowish culture. Abundant airy mycelium from white to slightly brown-gray

silver no-gray 3 fe, by Color Harmony Manual. (CHM) J. Soluble pigment no or slightly yellow. On glucose-asparagino-agar medium

Krainski, cultivation at 27 ° C: colorless culture. Abundant aerial mycelium from white to brown-gray (dark gray 5 ih, according to SNM). There is no soluble pigment.

On calcium block agar, grown at 27 ° C: culture from colorless to light yellow or light orange. White aerial mycelium. There is no soluble pigment or yellowish orange. Malic acid calcium around the culture is sometimes dissolved.

In a peptone solution containing 1% sodium nitrate, grown at 27 ° C: a colorless culture with a white aerial mycelium. Soluble pigment no or slightly yellow. Restores nitrates.

On a slice of potatoes, grown at 27 ° C: mycelium from colorless to light bluish gray or light gray (ashy 5 fe, according to SNM). There is no soluble pygmyite.

light gray or light brown gray. Soluble pigment no or light yellow. No hydrolysis or weak starch hydrolysis.

On food agar, grown at 37 ° C: culture from colorless to light yellowish brown, good growth. Thin and white aerial mycelium. There is no soluble pigment.

On food agar, expressing a culture from colorless to yellowish-brown with a white aerial mycelium. . There is no soluble pigment.

On the blood agar (10% of human blood) cultivation at 37 ° C: colorless culture without aerial mycelium. There is no soluble pigment. Severe hemolysis.

On the medium of coagulated serum (grown at 37 ° C): wrinkled, colorless culture. There is no airy mycelium and soluble pigment. Coagulated serum does not decompose.

On gelatinous medium (injection into solid medium), cultivation at 20 ° С: culture from colorless to one-white. Lean white aerial mycelium. There is no soluble pigment. Gelatinu liquefies.

In the dairy culture, grown at 37 ° C: culture from colorless to pale yellow or brown. There is no airy mycelium and soluble pigment. Strong coagulation and peptonization of the medium.

On tyrosine agar, grown at 27 ° C: the culture is colorless with aerial mycelium from white to light olive gray or brownish gray. There is no soluble nigment, there is no tyrosinase reaction.

On cellulose (filter paper), growing at 27 ° C: the culture is weak, but without decomposition of the paper.

With the use of carbohydrates on the Pridgema-Gottlieb base medium, cultivation at 27 ° C: mannose, inositol, starch, dextrin, lactose, glycerin, galactose, glucose, fructose and maltose - good growth. With rhamnosa, inulin, dulcite and arabinose there is no growth. Xylose is probably unsuitable. Mannitol, sorbitol, salicin, raffinose and sucrose give different results.

Summarizing the above characteristics, it can be concluded that the MA 350-C3 strain belongs to nonchromogenic type streptomycetes. There is no curl formation, but there is a spiral structure.

The surface of the dispute in contrast to Sir. villous kasugaensis. The culture is colorless to yellowish in various media, with aerial mycelium ranging from white to brownish gray. Soluble pigment pet or eat slightly yellow. Starch hydrolysis is absent or weak. Strong proteolytic effect on the environment. Positive nitrate recovery.

ricia (1948) (S.A. Waksman, The actinomycetes, vol. 2), HO differs from it in fuzzy

surface dispute. It is established that the strain

can give, besides kazugamytsnna, aureotricin and thiolutine similarly to Sir. kasugaensis.

However Str. kasugaspinus can be clear

different from Sir. kasugaensis on the surface

dispute and on other grounds.

Received 600 µg1 cmz kazugamycin on

the eighth day of incubation, when the strain isolated from the soil was grown at 27 ° C in an environment consisting of 3.0% maltose soy flour powder (product Proric from Agimoto) 0.1% K2HPO4, 0.1 MgSO4-7H2O, 0 , 3%

NaCl, 0.0007% CuS04-7H2O, 0.0002% ZnSO4-5 NaO, 0.5% peptone, 0.35% CAS02, and 0.08% soybean oil (pH 7.0), with agitation: in the machine which made 140 strokes / h with an amplitude of 8 cm.

3950 µg / cm3 of kazugamycin was obtained when the strain in monoscore breeding was grown in the same way as above, in a medium consisting of 5.0 e / o soy flour powder (product S3 - m from Agimoto)

and 4, OVo soybean oil (Oil Shiraschimi - product of the company Agimoto) (do not regulate pH). It is believed that by proper selection from a strain, a strain with a high ability to form kazugamycin can be obtained.

Example 1. Medium consisting of 4.0Vo of maltose, 3.0 ° / o of soy flour (product Proric from Agimoto), 0.1% K2HPO4, 0.1% MgS04-H2O; 0.3 NaCl, 0.0007% CuSO4-7H20,

0.001% ZnS04, 0.0008% MnCl2-7H20, 0.0002% ZnSO4-5 HaO, 0.059 / o peptone (polypepton of Daigo Sayku company), 0.35 o / o CAS02 and 0.08% soybean oil, poured over 125 ml in flasks with a capacity of 500 ml. pH

adjusted to 7.4 and autoclaved for 20 minutes at a temperature of 120 ° C. This autoclaved sterilized medium is inoculated with a pure strain using a platinum loop and cultured in a shaker machine at a temperature of

27 ° C for five days. On the fifth day, the pH reaches 7.6.

The culture fluid is filtered to separate the mass of mycelium, 1N is added to the filtrate. HC1 for precipitating insoluble

substances at pH 5.0. The filter is then filtered, and 450 ml of a clear solution are obtained, which contains 360 mg of casugamycin. The filtrate is passed through a column with a diameter of 1.2 cm, filled with acidic ion exchange resin (amberlite XE-100) of ammonia type. Kazugamycin, adsorbed resin, washed with 0.5 n. ammonia. The active eluate contains 300 mg of kazugamycin. 90 C. of the active eluate is poured into a 0.8 cm diameter column filled with activated carbon (Wako product). After flushing with 50 l of deionized water, kasugamycin is washed out with 0.5N. HC1 in the form of its hydrochloric acid salt 30.2 ml of active eluate contain 180 Mf,

an evaporator under reduced pressure and a temperature below 40 ° C. A tenfold volume of the standard is added to 5 liters of the concentrate to precipitate the casugamycin. The precipitated kasugamycin is dried, and 140 mg of crude crystals containing 132 mg of casugamycin are obtained. These crude crystals were dissolved in 8 ml of water and 12 ml of ethanol was added. After settling at room temperature, 96 mg of crystalline kazugamycin are obtained. The identity of these crystals with hydrochloric kazugamycin is confirmed by infrared spectrum, paper chromatography, thin layer chromatography, high-voltage electrophoresis, and elemental analysis.

Example 2. In flasks with a capacity of 500 ml, 80 ml are added of a medium consisting of SVo of soy flour and 4.0% of soybean oil (the pH is not adjusted), and inoculated with shtamma. Incubated at 27 ° C in a shaking mask that makes 144 hol / min with an amplitude of 8 cm. On the eighth day, the pH reaches 7.5. Get 3570 mcg / cm kazugamycin. 920 liters of cultured liquid is centrifuged to remove the micelle mass. 400 ml of ethyl ether is added to the resulting clear liquid containing 3570 µg1 ml of antibiotic.

to remove the remaining oil. The bottom layer is evaporated to remove ethyl ether, and 750 ml of liquid containing 4250 mg 1 ml of antibiotic is obtained.

This liquid is passed through a chromatographic column with a diameter of 3.2 cm with coal. After washing with water, elute 0.05 n. HC1 and collect the active fractions, which are neutralized and concentrated to 20 ml. To them

ethanol is added to precipitate the crude powder. The latter is dissolved in 20 ml of water, 35 ml of ethanol are added and left to stand overnight, after which 2.65 g of crystalline solid is obtained. It is identified with causugamycin hydrochloride by melting point, optical rotation, infrared spectrum, and other properties.

Subject invention

The method of obtaining the antibiotic kasugamycin, including the cultivation of the fungus-producer of the genus Steptomyces in aerobic conditions by the deep method on a liquid nutrient medium containing sources of carbon, nitrogen

and mineral salts, characterized in that Streptomyces kasugaspinus is taken as a producer fungus, and cultivation is carried out at a temperature of 25-35 ° C.