RU2420568C2 - Strain and biosynthesis method of producing antibiotic mitomycin - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Streptomyces caespitosus 3810/OE strain is a mitomycin C producer prepared from a stock strain recovered from soil by processing with chemical mutagens followed by stage selection, and deposited in the Collection of Cultures of State Institution G.F.Gauze Research Institute for Investigation of New Antibiotics of the Russian Academy of Medical Science, No. INA 01082. For producing mitomycin C, the strain is grown on a sowing medium of the following compoisiton (%): glucose - 0.6-1.4, starch - 0.6-1.4, soya flour - 2-5, corn steep - 0.6-1.0, KH₂PO₄ - 0.01-0.03, chalk - 0.05-0.12, (NH₄)₂SO₄ - 0.06-0.14, main water, pH before sterilisation 6.8-7.2 and then is transferred to an enzyme medium of the following composition (%): starch - 0.6-1.4, sucrose - 1-3, soya flour - 2-4, corn steep - 0.6-1.0, CoCl₂•6H₂O - 0.007-0.012, KH₂PO₄ - 0.02, chalk - 0.05-0.12, (NH₄)₂SO₄ - 0/06-0.04, FeSO₄•7H₂O - 0.01-0.02, MgSO₄•7H₂O - 0.01-0.03, NaCl - 0.2-0.6, main water, pH before sterilisation 6.8-7.2. The strain is grown in an aeration environment for 100-120 h, then mycelium is removed by centrifugation or filtration, and a supernatant containing an end product is extracted by mixed acetonitrile and methyl or ethyl alcohol. A level of strain produced mitomycin C is 85 to 100 mcg/ml.

EFFECT: improved method of producing antibiotic mitomycin.

2 cl, 1 ex

Description

The present invention relates to a strain producing an antibiotic mitomycin C and a method for producing mitomycin C by biosynthesis based on the use of this strain.

State of the art

Mitomycin C belongs to the group of antibiotics formed by actinomycetes, with antibacterial and antitumor activity. The mitomycin antibiotic group was first described 50 years ago (Hata T., Sano Y., Sugawara R., Matsumae A., Kanamori H., Shima T., Hoshi T. Mitomycin, a new antibiotic from Streptomyces. Int. J. Antibiotics , 1956, v. 9, p. 141). Almost immediately it was found that mitomycins have not only antibacterial, but also antitumor effects (Kanamori N., Shima T., Morita Ch., Hata T. Studies on antitumor activity of mitomycin. J. Antibiot., 1957, V.10, p.120). Mitomycins include a number of natural antibiotics, in the structure of which there are the following chemical groups: aziridine, carbamate, quinone and pyrrole core (Wakaki S., Maruto N., Tomioka K., Shimizu G., Kato E., Kamada H., Kudo S ., Fujimoto Y. Isolation of new fractions of antitumor mitomycins. Antibiot. Chemother., 1958, v. 8, p. 228; Webb JS, Cosulich DB, Mowat JH, Patrick HB, Broschard RW, Meyer WE, Williams RP, Wolf CF, Fulmor W., Pidacks Ch., Lancaster JE The structure of mitomycins A, B and C and porfiromycin. Part IJ Am. Chem. Soc. 1962, v. 84, p. 3185; Webb JS, Cosulich DB, Mowat JH , Patrick HB, Broschard RW, Meyer WE, Williams RP, Wolf CF, Fillmor W., Pidacks Ch., Lancaster JE The structure of mitomycins A, B, and C and porfiromycin. Part II. J. Am. Chem. Soc. 1962, vol. 84, p. 3187; Steve ns C. L., Taylor K. G., Munk M. E., Marshall W. S., Noll K., Shah G. D., Shah L. G., Uzu K. Chemistry and structure of mitomycin C. J. Med. Chem. 1965, n. 8, p. 1).

The most applicable in medicine for the treatment of various forms of oncological diseases is mitomycin C (Beretta G. Mitomycin C, clinical and experimental chemotherapy. Review, XVII ed., 2004, edizioni Minevra Medica, Turin, 2004). It is known that mitomycin C is obtained by biosynthetic methods using strains representing two genera of streptomycetes: Streptomyces and Streptoverticillium. The disadvantages of the known methods for producing mitomycin C is the formation by strains of producers of a complex of closely related antibiotics, and therefore the isolation and purification of mitomycin C is difficult. This application describes a method for producing mitomycin C, which consists in using a one-component producer of mitomycin C of strain Streptomyces caespitosus 3810 / OE deposited in the Microorganism Collection of the State Institution Research Institute for the Search for New Antibiotics named after GF Gauze of the Russian Academy of Medical Sciences (GU NIINA RAMS) under the number INA 01082, and nutrient media developed for it.

Description of the present invention

Strain 3810 / OE is the original strain obtained from the original strain ("wild"), isolated from the soil and assigned to the species Streptomyces caespitosus. The original strain was subjected to treatment with chemical mutagens, followed by stepwise selection.

To maintain the culture, Gauze modified agar medium No. 2 was used with the following composition (%): glucose - 1, peptone - 0.5, tryptone - 0.3, sodium chloride - 0.5, agar - 2; pH 7.2-7.4 (does not require adjustment). Colonies in spore screenings corresponding to the main active variant are at least 95% and on the 8-10th day of growth at 28 ° C have the following symptoms: substrate mycelium is greenish-brown, aerial spore-forming mycelium is developed moderately or well, gray in color, dark brown pigment. The culture is subcultured no more than three times, after which the spore suspension is sieved and colonies with the morphological characteristics described above are selected. To ensure a high level of mitomycin C biosynthesis, the following inoculum and fermentation media were developed.

Sowing medium (%): glucose - 0.6-1.4, starch - 0.6-1.4, soy flour - 2-5, corn extract - 0.6-1.0, KN ₂ PO ₄ - 0 , 01-0.03, chalk - 0.05-0.12, (NH ₄ ) ₂ SO ₄ - 0.06-0.14; tap water. The pH before sterilization should be adjusted to a value of 6.8-7.2.

Fermentation medium (%): starch - 0.6-1.4, sucrose - 1-3, soy flour - 2-4, corn extract - 0.6-1.0, CoCl ₂ · 6H ₂ O - 0.007-0.012 , KH ₂ PO ₄ - 0.02, chalk - 0.05-0.12, (NH ₄ ) ₂ SO ₄ - 0.06-0.14, FeSO ₄ · 7H ₂ O - 0.01-0.02 MgSO ₄ · 7H ₂ O — 0.01-0.03; NaCl — 0.2-0.6; tap water. The pH before sterilization should be adjusted to a value of 6.8-7.2.

The level of biosynthesis is at least 85 μg / ml (85-100) subject to the conditions described below for biosynthesis in the inoculum and fermentation culture media developed for this producer strain.

Fermentation

Inoculation of flasks with inoculum is made with a piece of agar medium with a surface culture growth of approximately 2 cm ² . Seeds are grown for 2 days at 28 ° C. 5% by volume of seed is added to the fermentation flasks. 5 days growth at 28-31 ° С. Rocking chair suspended 220 rpm.

Isolation of mitomycin C

After fermentation is completed, the mycelium is filtered and discarded, and a double volume of the precipitating mixture (a mixture of acetonitrile and methanol or ethanol in a ratio of 1: 1) is added to the filtrate of the culture liquid, kept at 0 ° C for 1 h until a precipitate forms, then centrifuged at 3000 rpm / min 20 min and the precipitate is discarded.

Determination of mitomycin C biosynthesis by high performance liquid chromatography (HPLC)

Analytical HPLC was carried out on a Shimadzu LC10 vp chromatograph using Diasfer 110-C18 columns (4.0 × 250 mm, grain size 5 μm) of JSC BioChemMak, Russia. The volume of the injector loop is 20 μl. The treated supernatant obtained above is introduced into the loop of the chromatograph. Detection is carried out at a wavelength of 356 nm. The eluting system contains a 0.01 M solution of H ₃ PO ₄ pH 2.6 and acetonitrile. With gradient elution, at a flow rate of 1.0 ml / min and room temperature, the concentration of acetonitrile changes as follows:

Time (min) 0 5 fifteen eighteen twenty thirty Acetonitrile (%) 10 fifteen twenty 70 10 10

The exit time of mitomycin C under these conditions is 10.9 minutes.

Calculation of the content of mitomycin C.

A standard solution in methanol with a concentration of 1 mg / ml was prepared from the dosage form of mitomycin C (Kyowa) (taking into account that in addition to 2 mg of the active substance, a filler is present in the vial). A standard antibiotic solution (final concentration — C _st ) is added to the culture fluid not containing mitomycin C, extracted as described above, chromatographed, and the area under the curve of the standard sample of the culture fluid (AUC _st ) is determined. Return on extraction is 87 ± 2.3%. The content of mitomycin C in the culture fluid is calculated by the formula:

A _{mg / ml} = AUC _pr · K _cross ; To _rec = (AUC _pr × C _st ): AUC _st ,

where A _{mg / l} is the concentration of mitomycin C in the culture fluid;

AUC _st - the area under the curve when chromatographic standard sample of the culture fluid;

AUC _pr - the area under the curve during chromatography of the analyzed sample;

With _article - the concentration of mitomycin C in a standard sample of culture fluid (in mg / l);

To _recount - a coefficient for converting the concentrations of mitomycin C in mg / l.

Under the chromatographic conditions described above, and With _article = 100 mg / l, the value of K _rec = _0.0508 .

The measurement accuracy is A _{mg / L} ± 2.5 mg / L.

The productivity level of mitomycin C strain No. 3810 / OE is from 85 to 100 μg / ml.

Further, the present invention is illustrated by an example, which illustrates it and does not limit the scope described in the claims.

Example

Monthly agar culture of strain 3810 / OE is sown in an amount of 2 cm ² with growing mycelium in a 750 ml Erlenmeyer flask containing 100 ml of inoculum of the following composition (%): glucose - 1, starch -1, soy flour - 4, corn extract - 0.9, KN ₂ PO ₄ - 0.02, chalk - 0.9, (NH ₄ ) ₂ SO ₄ - 0.1; tap water. The pH is adjusted to 7.0 before sterilization. The flask is incubated for 48 hours on a rocking chair with 200 rpm at 28 ° C. The resulting seed is transferred in an amount of 5 ml to an 750 ml Erlenmeyer flask containing 100 ml of a fermentation medium of the following composition (%): starch - 1, sucrose - 2, soy flour - 3, corn extract - 0.9, CoCl ₂ · 6H ₂ O - 0.01, KH ₂ PO ₄ - 0.02, chalk - 0.9, (NH ₄ ) ₂ SO ₄ - 0.1, FeSO ₄ · 7H ₂ O - 0.015, MgSO ₄ · 7H ₂ O - 0.02, NaCl - 0.4; tap water. The pH is adjusted to 7.0 before sterilization. The flask is kept on a shaker at 200 rpm at 3 ° C for 114 hours. After fermentation is completed, the mycelium is filtered and discarded, and 200 ml of the precipitating mixture of acetonitrile and methanol are added to the filtrate of the culture liquid in a ratio of 1: 1, kept in the cold (at 0 ° C) for 1 h and centrifuged at 3000 rpm for 20 min to remove the precipitate formed. 20 μl of the obtained supernatant was introduced into a loop of a Shimadzu LC10 vp chromatograph (Diasfer 110-C18 column, 4.0 × 250 mm, 5 μm granulation). The determination is carried out at a wavelength of 356 nm. The level of productivity is 92 μg of mitomycin C in 1 ml of culture fluid.

Claims (2)

1. Strain - producer of mitomycin C Streptomyces caespitosus 3810 / OE, deposited in the Collection of microorganism cultures of the State institution Research Institute for the search for new antibiotics named after GF Gauze of the Russian Academy of Medical Sciences (GU NIINA RAMS) under the number INA 01082. 2. The method of obtaining mitomycin C, which uses the strain producing mitomycin C according to claim 1, which is grown for 40-52 hours under aeration conditions on the seed medium of the following composition,%: glucose 0.6-1.4, starch 0.6 -1.4, soy flour 2-5, corn extract 0.6-1.0, KH ₂ PO ₄ 0.01-0.03, chalk 0.05-0.12, (NH ₄ ) ₂ SO ₄ 0 , 06-0.14, tap water, pH before sterilization of 6.8-7.2, and transferred to a fermentation medium of the following composition,%: starch 0.6-1.4, sucrose 1-3, soy flour 2-4 , corn extract 0.6-1.0, CoCl ₂ · 6H ₂ O 0.007-0.012, KH ₂ PO ₄ 0.02, chalk 0.05-0.12, (NH ₄ ) ₂ SO ₄ 0.06-0 , 14, FeSO ₄ · 7H ₂ O 0.01-0.02, MgSO ₄ · 7H ₂ O 0.01-0.03, NaCl 0.2-0.6, water water water, pH before sterilization, 6.8-7.2, on which it is grown under aeration conditions for 100-120 hours, after which the mycelium is removed by centrifugation or filtration, and the supernatant is extracted with a mixture of acetonitrile and methyl or ethyl alcohol.