RU2392311C2 - Strain of streptoalloteichus cremeus subsp tobramycini vkpm s-1084- producer of apramicyne and method of apramicyne obtaining - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves cultivation of strain Streptoalloteichus cremeus subsp. tobramycini VKPM S-1084, producer of apramicyne. Emanation of apramicyne from culture fluid with sorption on carboxylic cationites followed by chromatographic purification with two-column method by stepwise elution with solutions (0.19-0.22)n of ammonia. The resulting eluate is concentrated in vacuum and precipitated with excess of acetone apramicyne in the form of sulfate.

EFFECT: increased output of apramicyne.

2 cl, 1 tbl, 2 ex

Description

The invention relates to a new strain-producer of the antibacterial antibiotic apramycin from the group of aminoglycosides and a method for producing apramycin sulfate.

The special importance of aminoglycosides in modern antibiotic therapy of bacterial infections is due to their high activity against gram-negative microorganisms, whose specific gravity in the etiology of purulent-septic processes is constantly increasing

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 apramycin in the culture fluid (not more than 0.8 g of apramycin in 1 liter of culture fluid), where it is formed along with other antibiotics of the aminoglycoside complex - carbamoyltobramycin, carbamoylcanamycin B in a ratio of 2: 1: 1, respectively.

The essence of the invention lies in the fact that a new strain producing apramycin is obtained as a result of the multistage mutagenic effect of ultraviolet rays and nitrosomethyl biuret on the initial strain, followed by selection of mutants on media containing streptomycin and apramycin as selective agents.

The resulting new strain was deposited in the culture collection of FSUE GosNIIgenetika under the number VKMP S-1084.

Strain 1084 has increased productivity - (4 g in 1 liter of culture fluid) apramycin, synthesizes an antibiotic in the form of a monocomponent and is characterized by the following cultural, morphological and physiological characteristics.

Cultural, morphological and physiological characteristics of strain 1084. Temperature (20-37) ° C; growing time (7-21) days. Spore seed with a ten-day culture of the strain grown on Lindenbein glycerol-nitrate agar medium. The color is determined according to the table of Bondartsev and Prauser (see table).

Cultural and morphological characteristics of the strain Streptoalloteichus cremeus subsp. tobramycini VKPM S-1084 Agar medium Aerial mycelium growth * Mycelium color Soluble pigment air substrate No. 1 Gause + Whitish cream (K-2; K-3, Coo 6a) Colorless No Oatmeal + Whitish (K-2, Coo 7a) Colorless No Capek + Whitish (D-1; Coo 7a) Colorless No Glucose Aspartic - Yellowish (G-5; Coo 4a) Yellowish (F-6) Lindenbein (glycerol nitrate) +++ Whitish cream (D-1; Coo 7a) Brownish (b-4; Coo 4a) Brownish (b-4; Coo 4a) Glucose-nitrate No. 2 Gauze +++ White (D-1; COO 7a) to light cream (bb; Coo 5a) Brownish (b-4; Coo 4a) Brownish (b-4; Coo 4a) Tresner (with peptone and citric acid iron) Melanoid pigments do not form * / (+++) - good; (++) - moderate; (+) - weak.

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: it uses fructose and inositol well, moderately rhamnose and glucose, weakly uses sucrose and lactose, does not use raffinose, arabinose, beckon, xylose and cellulose.

Storage. The strain is stored as spores on sterile soil with silica sand or on millet grains.

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.

Strain 1084 is illustrated by the following example.

Example. To obtain spore material, a Streptoalloteichus cremeus subsp. tobramycini 1084 is grown for 7 days at 37 ° C on Lindenbein glycerol nitrate agar 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 hours in 750 ml Erlenmeyer flasks; the amount of medium in flasks is 150 ml. Rocking speed 200-240 rpm. The amount of seed for inoculation of 5-7%.

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

Soya flour 1,0 Glucose 0.4 Starch 1,5 (NH ₄ ) ₂ SO ₄ 0.3 CaCO ₃ 0.3 Water Tap pH before sterilization 6.6

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

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

The advantage of the claimed strain in comparison with the prototype is the ability to biosynthesize apramycin in the form of a monocomponent and 5 times the increased productivity of the antibiotic, namely the content of apramycin in the culture fluid of the prototype strain of 800 μg / ml in the complex of aminoglycosides, and in the claimed strain of 4000 μg / ml

In order to isolate apramycin, it is sorbed from a native solution (treated with oxalic acid and filtered from the mycelium of the culture fluid) with a pH of 6.7-6.9 on KB-2 carboxyl cation exchange resin in salt form, followed by chromatographic purification using a two-column method. The desorption of apramycin is carried out by stepwise elution with solutions of (0.19-0.22) n ammonia. The eluate is concentrated in vacuo, a (24.0-25.2)% sulfuric acid solution is added to pH 6.0, and apramycin sulfate is precipitated by the addition of an excess of acetone.

Apramycin sulfate obtained on the basis of the biosynthesis of a new mutant strain of Streptoalloteichus cremeus subsp. tobramycini 1084, is a white powder (a brownish tint is allowed), it is 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 pH range (1.7-2.5), antibacterial activity (650 μg / mg) and toxicity (LD ₅₀ is 220 mg / kg when administered intravenously to mice) the obtained apramycin sulfate is fully consistent with apramycin described in the literature. Gross formula: C ₂₁ H ₄₁ N ₅ O _11. H ₂ SO _4. Mol. Mass = 637.6 a e.m.Mole base mass (MALDI-TOF-MS method) = 540 (MH) ⁺ . [α] ²⁰ _D +174 (s 1.0, H ₂ O); ¹³ C-NMR (ppm): 101.59, 96.43, 95.32, 87.74, 78.40, 76.80, 74 .17, 73.42, 71.70, 70.99, 67.99, 66.18, 62.31, 61.63, 53.06, 51.08, 50.23, 49.74, 36.51, 32.82, 32.76.

The yield of the target product 73.0% of its content in the culture fluid.

Thus, a new mutant strain of Streptoalloteichus cremeus subsp. tobramycini VKPM S-1084, which differs from the known antibiotic-forming ability of monocomponent apramycin, which is 4 times higher than the activity of the prototype strain 2242-LB.

Antibiotic activity of a new strain of Streptoalloteichus cremeus subsp. tobramycini VKPM S-1084 is 4000 μg / ml. Use of a new strain of Streptoalloteichus cremeus subsp. tobramycini VKPM S-1084 as a producer for apramycin biosynthesis allows to increase the yield of the target product when apramycin is isolated from the culture fluid by sorption on carboxylic cation exchangers, followed by chromatographic purification by the two-component method by stepwise elution with solutions of (0.19-0.22) n ammonia, concentration in vacuum and sulfate precipitation.

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, S.891-894 (1980).

Claims (2)

1. The strain Streptomyces cremeus subsp. tobramycini VKPM S-1084 - producer of apramycin. 2. A method of producing ampramycin, comprising cultivating a strain of Streptomyces cremeus subsp. tobramycini VKPM S-1084, isolation of ampramycin 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, vacuum concentration and sulfate precipitation.