RU2054483C1 - Strain of actinomyces streptomyces avermitilis - a producer of avermectines - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: strain Streptomyces avermitilis ВНИИСХМ-54 is obtained by natural selection from the population of strain Streptomyces avermitilis-51. Strain N 54 produces two-fold more avermectines as compared with the known strain. Percent content of the most active substance avermectine B_1a is above 35%. Strain Streptomyces avermitilis ВНИИСХМ-54 is deposited in Collection of microorganism ВНИИ agriculture microbiology (Sanct-Petersburgh) at number Streptomyces avermitilis ВНИИСХМ-54. EFFECT: isolation of strain indicated above. 2 tbl

Description

 The invention relates to the production of biologically active substances by microbiological synthesis, namely the production of antiparasitic drugs based on avermectins.

 A known strain producing avermectins Streptomyces avermitilis ATCC 31372, producing an average of 500 μg / ml avermectins.

The purpose of the invention is the creation of a strain producing increased amounts of avermectins with a predominant content in the avermectin complex of avermectins of the B ₁ series (B _1a and B _1b ) that are most biologically active.

The goal is achieved by obtaining a strain of Streptomyces avermitilis VNIISKHM-54, producing an average of 1,500 μg / ml avermectins with a variability of 1200-1800 μg / ml. The proposed strain was obtained by targeted selection from a population of strain Streptomyces avermitilis-51. The selection of active variants was carried out according to two characteristics: by the ability to avermectin formation (the ability of the variants to produce the entire avermectin complex was evaluated); and by the ability to biosynthesize avermectins of series B ₁ (the ability of the variants to produce only avermectin B _1a and avermectin B _1b was evaluated). The scheme for obtaining the strain shown in the drawing.

The drawing shows the total content of avermectins (μg / ml) the percentage of avermectins of series B ₁ in the avermectin complex (%).

 The proposed strain is characterized by the following cultural, morphological and physiological characteristics.

 Micromorphological signs of the strain.

 Culture has spore-carriers in the form of regular spirals.

 Macromorphological and cultural characteristics.

On 2% agar glyukozokartofelnom at 14-21 days of growth at ²⁸ ° C, pH 6.8-7.2 formed colony diameter of 10 mm with light gray aerial mycelium and dark chestnut reverse side of the colony. The population always has up to 5% of light gray colonies with a beige reverse side. The edge of the colonies is uneven with a white rim. The surface of the colony is folded. In the center of the colony there is an elevation, sometimes with a crater. By the tenth day of growth, white velvety inclusions form on the surface of the colony.

 In the table. Figure 1 presents data on the color of air and substrate mycelium, the rate of sporulation, as well as the presence or absence of soluble pigments on various organic and synthetic agarized media.

 Information on physiological and biochemical characteristics are summarized in table. 2.

The maximum accumulation of avermectin observed on 5-7 th day at ²⁸ ° C and the oxygen dissolving velocity of 1.0-1.2 mM O ₂ / L / min.

 Antagonistic properties.

 Mycelial extracts cause the death of nematodes related to the genera: Haemonehus, Trichostronglus, Ostrertagis, Nematodimes, Coopema, Ascams, Bunostomum, Oesophagostonum, Chabertia, Triehuris, Strongylus, Trichonema, Dictyocanlusaricaricaria, Capricaria Parascaris, Ascoridia.

 Also, their effect is detrimental to ticks, aphids, lice, fleas, flies, cockroaches, dipterans and orthopterans, pest moths, carpet and wallpaper bugs.

 The proposed strain is illustrated by the following example.

Spores of strain S. avermitilis-ARRIAM-54 is grown on oatmeal agar for 10 days at 28 ± 1 ° ^C. A piece of agar medium with spores inoculated into shake flasks of 750 ml capacity containing 75 ml of medium with the following composition, glucose 4.0; soy flour 0.6; protein-vitamin concentrate 1.0; corn extract 0.3; CaCO ₃ 0.1; K ₂ NRA ₄ 0.06; tap water. Glucose is sterilized separately at 0.5 atm for 30 minutes, it is introduced into the flasks at the time of inoculation. The remaining components are sterilized at 0.8 atm for 40 minutes. The pH value before sterilization is 6.2.

The flasks were mounted on a rocker at a speed of 220 per minute and the inoculum grown at 28 ± 1 ^{° C} for 24 h then an inoculum of 5% are transmitted in flasks with fermentation medium of the following composition, glucose 7.0.; soy flour 0.6; protein-vitamin concentrate 1.0; corn extract 0.3; CaCO ₃ 0.1; K ₂ NRA ₄ 0.06; tap water. The pH value before sterilization is 6.2. Glucose is sterilized at 0.5 atm for 30 minutes, and the remaining components at 0.8 atm for 40 minutes. Glucose is introduced into the flasks at the time of inoculation of the medium.

Fermentation was carried out for 168 hours at 28 ^C. The avermectins was isolated from the mycelium as follows. First, mycelium was separated in a centrifuge at 3000 rpm for 10 minutes. To a wet cake obtained from 10 ml of culture liquid, 10 ml of chilled tap water was added, mixed thoroughly at 3000 rpm for 5 minutes. 10 ml of ethanol was added to the wet washed precipitate, and avermectins were extracted at room temperature with constant stirring for 20 minutes. After separating the mycelium at 3000 rpm for 5 min, the total content and component composition of avermectins was determined by thin layer chromatography with quantitative assessment of the metabolites in spots on the chromatogram using Opton KM-3 scanning spectrophotometer (Austria) at a wavelength of 244 nm (maximum absorption of avermectins). Chromatographic separation was carried out on Merk G-60 silica gel plates without a phosphor in a solvent system hexane: acetone: methanol (14.0: 6.0: 0.5). The concentration of avermectins was determined by the equations of the calibration curves for avermectins A ₂ and B ₁ .

The average content of avermectins in the mycelium of S. avermitilis-VNIISKHM-54 is 1500 μg / ml with a variability range of 1200-1800 μg / ml. The composition of the avermectins is as follows, group A ₁ (A _1a + A _1c ) 18.0 ± 1.85; A ₂ (A _2a + A _2c ) 26.32 ± 1.62; B ₁ (B _1a + B _1c ) 36.75 ± 1.35 and B ₂ (B _2a + B _2c ) 18.88 ± 1.64.

 The known S. avermitilis-51 strain produces an average of 700 μg / ml avermectins.

 Thus, the proposed strain producing avermectins S. avermitilis-VNIISXM-54 allows you to get avermectins twice as much as the known strain S. avermitilis-51.

Claims (1)

 The strain of actinomycete Streptomyces avermitilis VNIISKHM-54 - producer of avermectins.

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