RU2433169C1 - STRAIN Streptoalloteichus cremeus subsp tobramycini ALL-RUSSIAN COLLECTION OF INDUSTRIAL MICROORGANISMS Ac-1083 - PRODUCENT OF TOBRAMYCIN AND METHOD OF OBTAINING IT - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: strain Streptoalloteichus cremeus subsp.tobramycini 8150 All-Russian collection of industrial microorganisms Ac-1083 is obtained as a result of induced mutagenesis with further selection of variants on selective media, containing streptomycin, rifamycin and tobramycin. Method of obtaining tobramycin from culture liquid of novel strain with application of column chromatography on carboxyl cationites with step-by-step elusion with ammonia solutions, hydrolysis at 100C in (0.1-0.3) H solution of caustic soda, repeated chromatographic purification, concentration in vacuum, acidation with solution of sulfuric acid to pH 4.5 and sedimentation with metghanol in form of sulfate provides 30% output of target product from tombamycin content in culture liquid. ^ EFFECT: invention makes it possible to increase efficiency of antibiotic obtaining. ^ 2 cl, 1 tbl, 1 ex

Description

The invention relates to a new producer strain of the antibacterial antibiotic tobramycin from the aminoglycoside group.

Aminoglycoside antibiotics, having a high antibacterial activity and a wide spectrum of antibacterial action, are necessary drugs for the treatment of severe life-threatening infectious diseases. Due to its high activity against gram-negative pathogens and especially Pseudomonas aeruginosa, tobramycin, as a leader among aminoglycoside antibiotics, is widely used in medical practice.

Known strain of Streptoalloteichus cremeus var. tobramycini var. nov. 2242 - producer of aminoglycoside antibiotics of the nebramycin complex [1]. The disadvantage of this strain is the low level of tobramycin in the culture fluid (not more than 1.0 g of tobramycin in 1 liter of culture fluid).

The essence of the invention lies in the fact that a new strain producing tobramycin is obtained as a result of the multistage mutagenic effect of ultraviolet and gamma ( ⁶⁰ Co) rays and N-nitrosoethylurea on the initial strain, followed by selection of mutants on media containing streptomycin, rifamycin and tobramycin as selective agents .

The resulting new strain was deposited in the culture collection of FSUE GosNIIgenetika under the number VKPM Ac-1083.

Strain Ac-1083 has an increased productivity of tobramycin - (4.0 g in 1.0 l of culture fluid) and is characterized by the following cultural, morphological and physiological characteristics.

Cultural, morphological and physiological characteristics of strain 1083. Temperature (28-37) ° C; growing time (7-21) days. Spore seed of a ten-day culture of the strain grown on Lindenbein glycerin nitrate agar medium. The color is determined according to the table of Bondartsev and Prauser (table 1).

Morphological signs. Spore chains are spiral, spirals are extended, with 2-6 turns, the surface of the spores is smooth.

Physiological and biochemical properties. Aerobe. It grows at (24-37) ° С; optimum growth of 28 ° C and in the ranges of pH values (5.0-10.0) with optimum growth at pH 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: good use of fructose and glucose, moderately sucrose, mannitol, lactose, poorly uses inositol, rhamnose, raffinose, does not use arabinose, xylose and cellulose.

Storage. The strain is stored in the form of spores on sterile soil with silica sand or on millet grains, as well as a mature culture on agar media under liquid paraffin.

Antagonistic properties. When tested by the bar method on glucose-nitrate agar No. 2 Gauze, the strain inhibits the growth of gram-positive and gram-negative bacteria and yeast-like fungi. The strain is phage resistant. Phage in culture is absent.

The strain VKPM Ac-1083 is illustrated by the following example.

Example. To obtain spore material, a Streptoalloteichus cremeus subsp. tobramycini 1083 is grown for 8 days at 37 ° C on Lindenbein glycerin nitrate agar medium of the following composition,%:

Glycerol 3.0 K ₂ HPO ₄ 0.1 MgSO ₄ 0.05 Kcl 0.05 FeSO ₄ 0.001 NaNO ₃ 0.2 Agar 2.0 Water Tap pH before sterilization 7.0-7.2

The medium is sterilized for 30 minutes at a pressure of 1.0 atm and 120 ° C.

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

Soya flour 3.0 Glucose 3.0 NaCl 0.5 CaCO ₃ 0.5 Water Tap pH before sterilization 7.0-7.2

Seed is grown at 37 ° C for 48 h in Erlenmeyer flasks with a capacity of 750 ml, the amount of medium in flasks is 150 ml. Rocking rotation speed (200-240) rpm The amount of seed for inoculation is (5-7)%.

Fermentation is carried out on a shaker at 37 ° C on medium (100 ml in Erlenmeyer flasks with a volume of 750 ml) of the following composition,%:

Glycerol 2.0 Soya flour 2.0 NH ₄ Cl 0.3 NH ₄ NO ₃ 0.1 MgSO ₄ 0.5 CaCO ₃ 0.3 ZnSO ₄ 0.01 pH before sterilization 6.9 Water Tap

The antibiotic accumulates in the culture fluid of the producer strain.

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

The content of tobramycin in the producer culture fluid at the end of fermentation (after 144 h) is 4000 μg / ml.

The advantage of the claimed strain in comparison with the prototype is 4 times the increased productivity of the antibiotic, namely, the content of tobramycin in the culture fluid of the prototype tobramycin strain 1000 μg / ml, and in the claimed strain 4000 μg / ml.

In order to isolate tobramycin, it is sorbed from the native solution (treated with oxalic acid and filtered from the mycelium of the culture fluid) with pH = (6.7-6.9) on the KB-2 carboxyl cation exchanger in salt form, followed by chromatographic purification using the two-column method. The desorption of tobramycin is carried out by stepwise elution with solutions of (0.19-0.22) n. ammonia. The eluate is concentrated in vacuo to a concentration of (100-150) mg / ml and the antibiotic is hydrolyzed at 100 ° C in (0.1-0.3) n. sodium hydroxide solution, while carbamoyl tobramycin is converted to tobramycin. Tobramycin is separated from the reaction mixture by sorption on a KB-2 carboxyl cation exchanger followed by desorption with a dilute ammonia solution. The resulting tobramycin solution was concentrated in vacuo, a (24.0-24.2) 5th sulfuric acid solution was added to pH 4.5, and tobramycin sulfate was precipitated by the addition of an excess of methanol.

Tobramycin sulfate obtained from the biosynthesis of a new mutant strain of Streptoalloteichus cremeus subsp. tobramycini 1083, is a white powder, highly soluble in water and insoluble in organic solvents. According to their physicochemical properties ( ¹³ C-NMR data, Rf values for thin-layer chromatography and electrophoretic mobility for electrophoresis on paper in electrolytes in the range pH = (1.7-2.5), antibacterial activity and toxicity (LD ₅₀ is 80 mg / kg when administered intravenously to mice)) the tobramycin sulfate obtained is fully consistent with tobramycin described in the literature. Gross formula: C ₁₈ H ₃₇ N ₅ O ₉ H ₂ SO ₄ . Like mass = 565.5 amu Like base mass (MALDI-TOF-MS method) = 468.5 amu (MH) ⁺ . [α] ²⁰ _D + 128 ° (s 1.0, H ₂ O).

The yield of the target product is 30.0% of its content in the culture fluid.

Thus, a new mutant strain of Streptoalloteichus cremeus subsp. tobramycini VKPM Ac-1083 producer of tobramycin, characterized by a high ability to antibiotic formation, exceeding the activity of the prototype strain 2242-LB 4 times

Antibiotic activity of a new strain of Streptoalloteichus cremeus subsp. tobramycini VKPM Ac-1083 is 4000 μg / ml. Use of a new strain of Streptoalloteichus cremeus subsp. tobramycini VKPM Ac-1083 as a producer for the tobramycin biosynthesis allows to increase the yield of the target product when tobramycin is isolated from the culture fluid by sorption on carboxyl cation exchangers, followed by chromatographic purification by the two-column method by stepwise elution with solutions (0.19-0.22) n. ammonia by hydrolysis at 100 ° C in (0.1-0.3) n. caustic soda solution, repeated chromatographic purification, concentration in vacuo and precipitation in the form of sulfate, which provides a 30% yield of the target product from the content of tobramycin in the culture fluid.

LITERATURE

1. Sinyagina O. P., Lapchinskaya O. A., Lavrova-Balashova M. F., Nesterova T. P., Ponomarenko V. I. Obtaining mutants forming tobramycin in the culture of the producer of the complex of aminoglycoside antibiotics Streptomyces cremeus var. tobramycini var. nov. 2242. // Antibiotics No. 12, p. 891-894 (1980).

Table 1. Cultural and morphological characteristics of the strain Streptoalloteichus cremeus subsp. tobramycini VKPM Ac-1083 Agar medium Aerial mycelium growth ^* Mycelium color Soluble pigment Air Substrate No. 1 Gause ++ Whitish cream (d-3, b-6) Brownish-yellow (d-4) Light yellow (d-4) Oatmeal + - White to Cream (B-6) Pale brown (b-4) Pale brown (b-4) Capek - - - - Glucose Aspartic ++++ Whitish cream (a-3, b-6) Pale brown (b-4) Pale brown (b-4) Lindenbein (glycerol nitrate) ++++ Whitish (d-3) Brownish yellowish (d-2) Brownish yellowish (d-2) Glucose-nitrate No. 2 Gauze + - Whitish cream (d-3, b-6) Light yellow (d-4) Brownish (b-7) Tresner (with peptone and citric acid iron) Melanoid pigments do not form * / (+++) - good; (++) - moderate; (+) - weak; (-) - lack of growth.

Claims (2)

1. The strain Streptoalloteichus cremeus subsp. tobramycini VKPM Ac-1083 - tobramycin producer. 2. A method of producing tobramycin, comprising the cultivation of a strain of Streptoalloteichus cremeus subsp. tobramycini VKPM Ac-1083, isolation of tobramycin from the culture fluid by sorption on carboxylic cation exchangers, followed by chromatographic purification by the two-column method by stepwise elution with (0.19-0.22) n solutions. ammonia by hydrolysis at 100 ° C in (0.1-0.3) n. caustic soda solution, repeated chromatographic purification, concentration in vacuo and precipitation in the form of sulfate.