RU2391396C2 - Streptomyces roseolus VKPM S-1082 STRAIN - LINCOMYCIN PRODUCER AND METHOD OF PRODUCING LINCOMYCIN - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in medical industry during production of antibacterial antibiotic lincomycin hydrochloride which is efficient in treating infections caused by gram-positive agents, including methicillin with stable and certain anaerobic bacteria. The method involves culturing a Streptomyces roseolus VKPM S-1082 strain, which is a lincomycin producer, on a culture medium with given composition, extraction of lincomycin with organic solvents from the native solution, extraction of lincomycin with subsequent isolation. The obtained extract is concentrated and crystallised to obtain lincomycin in hydrochloride form.

EFFECT: invention increases output of lincomycin.

2 cl, 1 tbl, 2 ex

Description

The invention relates to the medical industry and relates to a new strain-producer of antibacterial antibiotic lincomycin and a method for its preparation.

The antibiotic lincomycin is effective in the treatment of diseases caused by gram-positive microflora and which cannot be treated with other antibiotics. Lincomycin is especially effective in the treatment of osteomyelitis, since the antibiotic selectively accumulates in bone tissue. Lincomycin is effective against methicillin-resistant strains of staphylococci and is a valuable drug for the treatment of severe traumatic and purulent-septic patients. Lincomycin is also a raw material for the preparation of a therapeutically more active semi-synthetic drug clindamycin.

Known strain No. 1146 Actinomyces roseolus - producer of the antibiotic lincomycin [1]. The disadvantage of this strain is phagosensitivity and low antibiotic levels in the culture fluid.

The essence of the invention lies in the fact that a new strain-producer of lincomycin is obtained as a result of the multistage mutagenic effect of methyl nitrosoguanidine, ultraviolet and gamma rays ⁶⁰ Co on the initial strain, followed by selection of mutants on media containing streptomycin, rifamycin and bleomycin as selective agents.

The resulting new strain was deposited in the culture collection of FSUE GosNIIgenetika under the number S-1082.

Strain 1082 has an increased productivity of lincomycin (4800 μg in 1 ml of culture fluid) and is characterized by the following cultural, morphological and physiological characteristics.

Cultural, morphological and physiological characteristics of the strain VKPM S-1082. Temperature 28 ° C; growing time (7-21) days. Spore seed from a ten-day culture of the strain grown on mineral agar No. 1 Gauze. The color is determined according to the table of Bondartsev and Prauser (table 1).

Morphological signs. Spore-carriers straight, spore chains straight, spores oval with a smooth surface.

Physiological and biochemical properties. Aerobe. It grows at (24-37) ° С; optimum growth of 28 ° C, and in the ranges of pH values (6.0-9.0) with optimum pH of 7.0; gelatin dilutes. Milk peptones. Sucrose inverts. Starch hydrolyzes. Fiber does not grow. Restores nitrates to nitrites. It does not form melanoid pigments when growing on Tresner's medium with peptone and citric acid iron. Attitude to carbohydrates: makes good use of glucose, arabinose, xylose, inositol; fructose, lactose, galactose, sucrose, rhamnose; poorly uses mannitol and raffinose.

Storage. The strain is stored in the form of spores on sterile soil with silica sand or as a mature culture on agar medium under liquid paraffin at (5-7) ° С.

Antagonistic properties. When tested by the Gauch glucose nitrate agar method No. 2, the strain inhibits the growth of gram-positive bacteria Stapylococcus aureus INA 00985 (= 209 P), Bacillus subtilis ATCC 6533, Bacillus mycoides 537, Micrococcus luteus ATCC 9341. There were no specific virulent phages fermentation does not occur. Resistant to virulent phage of the strain.

Strain S-1082 is illustrated by the following example.

Example. To obtain spore material, a culture of Streptomyces roseolus S-1082 is grown for (7-10) days at 37 ° C on agar medium No. 2 of Gouse of the following composition,%:

Tripton 0.3 Peptone 0.5 Glucose 1,0 Sodium Chloride 0.5 Agar 3.0 Water Tap pH before sterilization 7.0-7.2

The medium is sterilized for 30 minutes at a pressure of 0.6 atm at 120 ° C.

To obtain seed mycelium, culture spores are sown on Wednesday of the following composition,%:

Starch 1,0 Pea flour 1,5 Ammonium sulfate 0.3 Calcium carbonate 6.5 Sodium Chloride 0.5 Water Tap pH before sterilization 7.0-7.2

The seed is grown at 28 ° C for 48 hours in 750 ml Erlenmeyer flasks; the amount of medium in the flasks is 150 ml. Rocking rotation speed (200-240) rpm The amount of seed for inoculation is 5%.

Fermentation is carried out on a shaker at 28 ° C on medium (50 ml in Erlenmeyer flasks with a capacity of 750 ml) of the following composition,%:

Sucrose 5,0 Corn extract 1,0 Soya flour 2.0 K ₂ HPO ₄ 0.1 NaCl 0.3 CaCO ₃ 0.5 Water Tap pH before sterilization 7.2

The antibiotic activity of the culture fluid is determined by agar diffusion using a Bacillus subtilis 6633 test microorganism.

The content of lincomycin in the producer culture fluid at the end of fermentation (after 144 hours) is 4800 μg / ml.

The advantage of the claimed strain in comparison with the prototype is phage resistance and more than 8 times increased productivity, namely, the content of lincomycin in the culture fluid of the strain of the prototype is 558 μg / ml, and in the claimed strain - 4800 μg / ml.

In order to isolate lincomycin, it is extracted with n-butanol from the native solution (treated with oxalic acid and filtered culture fluid of the producer strain Streptomyces roseolus S-1082) with pH = 10.0, followed by transfer of lincomycin to an aqueous solution of hydrochloric acid and re-extraction with n-butanol . Butanol extract with the addition of 2 N. a solution of hydrogen chloride in acetone at pH = 2.0 is concentrated and lincomycin is crystallized in the form of a hydrochloride.

Lincomycin hydrochloride, obtained on the basis of the biosynthesis of a new mutant strain of Streptomyces roseolus S-1082, is a white powder, readily soluble in water, methanol, to a limited extent in ethanol and insoluble in other organic solvents. According to the physicochemical properties, antibacterial activity and toxicity, it fully complies with the requirements of VFS. Gross formula: C ₁₈ H ₃₄ N ₂ O ₆ S · HCl. Like mass 443.04 amu

Like base mass (MALDI-TOF-MS method) = 407 (MH) ⁺

IR spectrum (KBr), λ _max , cm ^-1 : 3440, 3330, 3280, 3040, 3015, 2940, 2910, 2860, 1672, 1657, 1566, 1475, 1459, 1445, 1409, 1358, 1263, 1230, 1207, 1143, 1107, 1092, 1078, 1044, 996, 963, 906, 871, 802, 738, 718;

The yield of the finished product - lincomycin hydrochloride - is 42% of the content of lincomycin in the producer culture fluid.

Thus, a new mutant strain of Streptomyces roseolus S-1082 was created, which differs from the known antibiotic formation ability, which is more than 8 times the activity of the prototype strain No. 1146 Actinomyces roseolus. The antibiotic activity of the new strain is 4800 μg / ml. The use of a new strain of Streptomyces roseolus S-1082 as a producer for lincomycin biosynthesis allows to increase the yield of the target product upon isolation of lincomycin from the culture fluid by extraction with n-butanol, followed by transfer to the aqueous phase, secondary extraction with n-butanol and crystallization in the form of hydrochloride.

LITERATURE

1. SU, USSR copyright certificate No. 287743 A1 of 1975

Table 1. Cultural and morphological characteristics of the strain Streptomyces roseolus S-1082 Agar medium Aerial mycelium growth Mycelium color Soluble pigment Air Substrate No. 1 Gause +++ White (D-1) Colorless No Oatmeal +++ White (D-1) Colorless No Capek +++ White (D-1) Colorless Yellowish Glucose Aspartic +++ From white (D-1) to yellowish (G-1) Yellow (W-6) Yellowish (F-6) Lindenbein (glycerol nitrate) ++ White (D-1) Colorless Yellowish (F-6) Glucose-nitrate No. 2 Gauze + White (D-1) Colorless no Tresner (with peptone and citric acid iron) Melanoid pigments do not form * / +++ - good; ++ - moderate; + - weak.

Claims (2)

1. The strain Streptomyces roseolus VKPM S-1082 - producer of lincomycin. 2. A method of producing lincomycin, comprising cultivating a strain of Streptomyces roseolus VKPM S-1082, extracting lincomycin with organic solvents from a native solution, reextracting lincomycin with its subsequent isolation, concentration and crystallization in the form of hydrochloride.