RU2087535C1 - Strain of actinomyces streptomyces avermitilis - a producer of avermectines - Google Patents

Abstract

FIELD: microbiological industry, biotechnology. SUBSTANCE: invention relates to new strain of actinomyces Streptomyces avermitilis. Strain is obtained by two-stage selection from the strain S. avermitilis-198 treated with nitrosomethylurea and ultraviolet radiation. Strain is deposited in Collection VNIISXM at N 56. Under submerged condition of culturing the yield of avermectines is 300-600 mcg/ml on the 7-th day. EFFECT: increased yield of the end product, new strain indicated above.

Description

 The invention relates to the field of industrial microbiosynthesis, namely the production of avermectins with a wide spectrum of antiparasitic action. Avermectin-based preparations are used in crop production and veterinary medicine.

 The known producer strain of the avermectin complex Str. avermitilis ATCC 31272 [1] This strain is used to obtain antiparasitic substances and is characterized by activity up to 500 μg / ml.

Also known producer of avermectins Str. avermitilis VKM AC 1301, which is stored in the domestic collection of microorganisms and has an activity of up to 130 μg / ml [2]
Based on the VKM AC 1301 strain, strain 198 was obtained, which was deposited in the VNIISKhM collection under number 50 and had an activity of up to 300 μg / ml. A disadvantage of known strains of producers of avermectins is the relatively low activity.

 The aim of the invention is to expand the range of domestic strains of producers of avermectins and obtain more active cultures for the biosynthesis of the avermectin complex.

 To achieve the goal, a new strain of Streptomyces avermitilis (VNIISHM 56), an avermectin producer, is used. The strain obtained by two-stage selection from the Str strain. avermitilis 198 treated with nitrosomethylurea and ultraviolet radiation. The proposed strain is deposited in VNIISHM under the number 56.

 Cultural and morphological characters. Mycelium is nonseptic, forms a substrate and aerial mycelium of gray color, on the medium it releases pigment from brown to dark brown. The average thickness of the hyphae is 0.6 0.8 microns, spore-bearing spiral branches branching on the sides of the hyphae of the aerial mycelium. Spores are spherical, oval in size from 0.95 microns and above.

On agar media, the culture forms velvety colonies from white to dark gray. The colonies are radially folded with smooth edges, a crater is observed in the center, a soluble dark brown pigment is released into the medium. The substrate mycelium is well attached. The aerial mycelium is first white, then gray, velvety. It grows at a temperature of 20 - 40 ^o C, the optimum temperature is 28 32 ^o C, does not grow at 50 ^o C.

 The range of pH-growth of 5.0 to 9.0.

 Growth on synthetic media. On Chapek’s environment with glucose or sucrose forms small colonies from white to gray, the surface is folded. The pigment is light gray or light brown is not plentiful. Starch-ammonia agar: small, slightly folded colonies, slightly pigmented, white.

 Growth on organic media: potato agar; growth; abundant mycelium gray; reverse side of agar dark brown or brown-violet. Soluble brown pigment.

 On potato slices, growth is plentiful, aerial mycelium is gray, pigmentation is gray-brown. On wort agar, growth is average, colonies are small, gray, pigment is brown. Meat peptone agar: colonies are white and grayish with a ring, folded, slightly pigmented.

 Physiological and biochemical characteristics: aerob, dilutes gelatin, milk peptones, hydrolyzes starch. It restores nitrates, does not decompose cellulose, and tyrosinase is positive. Wednesday Tresner-Dang blackens.

 Attitude towards carbohydrates: assimilates glucose, arabinose, fructose, lactose, maltose, mannose, raffinose, rhamnose, xylose, sucrose, starch, dextrins, inositol. Does not assimilate cellulose.

 Attitude to nitrogen sources: uses both mineral (salts of nitric acid, ammonium salts) and organic nitrogen (urea, peptone, egg albumin, amino acids).

 When cultivated in deep conditions, the biosynthesis of avermectins begins with 24 hours of growth and lasts up to 10 days. depending on cultivation conditions.

 The invention is illustrated by the following examples.

 Example 1

 Lyophilized culture Str. avermitilis (VNIISKHM-56) is transferred from an ampoule to a 750 ml rocking flask with 50 ml of sterile medium of composition, glucose 4, BVK 0.6, corn extract 0.3, chalk 0.3, soy flour 1.2, water the rest. The initial pH value is 7.0.

The cultivation is carried out on a shaker with a speed of 180 rpm at a temperature of 28 ^o C. After 24 hours of cultivation, the grown mycelium is used as an inoculum for inoculation of rocking flasks with the above medium. To do this, in a 750 ml flask with 50 ml of medium add 2 ml of inoculum and cultivate at 180 rpm, 28 ^o C. Every day, 3 flasks are removed to determine avermectins in the culture fluid. The determination is carried out by high performance liquid chromatography. For this, 5 ml of culture fluid is taken and placed in a centrifuge tube. The biomass is separated by centrifugation at 8000 rpm for 15 minutes. The supernatant is drained, distilled water (5 ml) is added to the precipitate, stirred and centrifuged again. The supernatant was drained and 5 ml of ethanol was added to the precipitate to extract avermectins. The extraction is carried out with stirring for 20 minutes The biomass from the extract is separated by centrifugation. The supernatant alcohol solution of avermectins is used for chromatography. In this case, 0.003 ml of an alcoholic solution is applied to the column and the analysis is carried out in the following mode: temperature 20 ^° C., eluent flow rate 0.1 ml / min, detector at 246 nm.

где С концентрация авермектинов в культуральной жидкости, г/мл,
К калибровочный коэффициент,
ΣSi Si сумма площадей пиков авермектинов, см²,
Y объем образца, нанесенного на колонку, см³.To build a calibration graph using the brand-name drug IVOMEC with a concentration of 0.01 g / ml. Calibration solutions have a concentration of 0.001, 0.0008, 0.0006, 0.0004, 0.0002 and 0.0001 g / ml. The calculation of the total content of avermectins in the culture fluid is carried out according to the formula

where C is the concentration of avermectins in the culture fluid, g / ml,
K calibration factor,
ΣSi Si the sum of the peak areas of avermectins, cm ² ,
Y is the volume of the sample deposited on the column, cm ³ .

 The concentration of avermectins in the culture fluid on the first day of growth is 70 μg / ml. Further dynamics of daily accumulation of avermectins is 90, 120, 200, 260, 290, 300 μg / ml.

 Example 2

 All operations are carried out as in example 1, except that the cultivation of the producer is carried out with aeration of 220 rpm Moreover, the intensity of culture development is higher, and the final yield of avermectins is 7 days. growth is 600 mcg / ml.

 Example 3

 All operations are carried out as in example 1, but the producer is cultured with aeration of 280 rpm, while the yield of avermectins is 500 μg / ml.

 Example 4

 All operations are carried out as in example 2, but the following composition is used as the fermentation medium, starch 5.0, BVK 0.6, corn extract 0.3, chalk 0.3, soy flour 1.2, water up to 100% avermectins for 7 days. growth is 580 mcg / ml.

 Example 5

All operations are carried out as in example 2, but the producer is cultured at a temperature of 35 ^o C. In this case, the productivity of the strain is 300 μg / ml.

 Example 6

All operations are carried out, as in example 2, but the cultivation of the producer is carried out at a temperature of 25 ^o C. The productivity of the strain is 540 μg / ml.

 Example 7

To determine the biological activity of avermectins produced by the proposed strain, rice nematode Arhelenchoides bessei is used. The determination is carried out in parallel with the company IVOMECOM. Take a 1% alcohol solution of avermectins and IVOMEC. Dilutions from 100 to 10,000 times are prepared from each preparation. From each dilution, 100 μl was added to the microplate wells. Then, 15 20 nematodes were placed in the cells and the microplates were incubated in an incubator at 30 ^° C. After 2 hours, the status of the nematodes was analyzed. It was found that when diluted 1000 times, both drugs cause paralysis (lack of movement) of test objects.

 Thus, the presented examples demonstrate that the strain of Streptomyces avermitilis (VNIISXM-56) is highly active in terms of biosynthesis of avermectins, and the produced avermectin complex has a nematicidal effect. Therefore, the strain can be used to obtain antiparasitic drugs.

 The strain is stored in the collection of the All-Russian Research Institute of Agricultural Microbiology at number 56.

Sources of information
1. Bung RW Miller BM et. all. Avermectins new family at potenf antyhelmintic agents, producing organism and fermentation. Antimicrob. Agents Chemother. 1979, 15, p. 361 367.

 2. Chermensky D. N. Adanin V. M. and others. Avermectins: Biotechnological features of the producer strain Str. avermitilis VKMAc 1301. Applied Biochemistry and Microbiology, N 6, 1991.

Claims (1)

 The strain of actinomycete Streptomyces avermitilis VNIISCHM-56 producer of avermectins.