RU2198928C2 - Strain streptomyces nodosus 472 vniischm d-666 as producer of amphotericin b - Google Patents

Abstract

FIELD: microbiology, antibiotics. SUBSTANCE: strain Strepromyces nodosus 472 VNIISCHM D-666 is obtained by selection method by effect with mutagens N-nitrosomethylurea and N-nitroso-N-methylbiuret. Strain shows the enhanced productivity of amphotericin B that is effective antifungal antibiotic used in medicinal practice for treatment of deep and systemic mycosis. EFFECT: increased yield of amphotericin B.

Description

 The invention relates to the medical and microbiological industry and for the production of antibiotics.

 The objective of this technical solution was to obtain a new strain with higher productivity for the production of amphotericin B, which is an effective antifungal drug used in medical practice for the treatment of deep and systemic mycoses. The antibiotic also has an antiviral, antitumor effect and suppresses the vital activity of protozoa, and is also effective against various phytopathogenic fungi in plants. As a result of the work, a strain was selected that has a high ability to synthesize amphotericin B.

 Also known is strain 510 of the producer of amphotericin (materials of a scientific conference of the Leningrad Scientific Research Institute of Antibiotics, Leningrad, 1967). However, the antibiotic activity of Actinomyces nodosus 510 is 1750-2400 units / ml. The disadvantage of this technical solution is the low antibiotic activity.

 The proposed strain of Streptomyces nodosus 472 was obtained by selection from a strain of Actinomyces nodosus 143 using N-nitrosomethylurea and N-nitroso-N-methylburet as mutagens.

 The strain produces up to 8000 μg / ml of culture fluid. The strain according to the invention is stored in the All-Russian Research Institute of Agricultural Microbiology under registration number D-666 VNIISCHM in the group of crops used to control pests of agricultural plants.

 The essential features of the claimed strain of Streptomyces nodosus 472.

 The strain grows well in the presence of glucose, fructose, xylose, rhamnose. It grows weakly in the presence of arabinose, does not grow with sucrose. The strain liquefies gelatin, does not peptone milk, hydrolyzes starch, does not grow on fiber. Restores nitrates to nitrites.

 Forms a well-developed air and substrate mycelium. On the surface of the colonies forms spores cylindrical with cut edges, the spore membrane is smooth. The size of the spores is 1.2x2 microns.

 Distinctive features of the proposed strain from the known.

 Cultural and morphological characters.

 Forms spiral sporonosos with 1-3 curls.

 On a medium with corn extract and starch, the aerial mycelium is dark gray, light gray at the edges, the medium is dark sand.

 On the glycerol-aspartic agar of the colony with a diameter of 1 to 5 mm, the aerial mycelium is dark gray, along the edge of the colony gray. The substrate mycelium is dark chestnut.

 On potato agar, colonies are gray with a rim of light gray, convex with radial folds.

 On oat agar, the edge of the colony is flat or vague.

 On the organic environment of Gauze, the colonies are small, with a diameter of 1 to 3 mm smooth. Folding is absent.

 It does not grow on a medium with inositol. The inventive strain exceeds the level of biosynthesis of amphotericin B by 15-25% prototype.

 Morphological and cultural characteristics.

 Strain 472 forms a well-developed aerial and substrate mycelium. On the surface of the colonies forms spiral spore-carriers (1-3 curls), spores are cylindrical, with cut edges, the spore membrane is smooth. The size of the spores is 1.2x2.0 microns.

On a medium with corn extract and starch (21/12) on 21 days of growth at 28 ± 1 ^o C forms colonies with a diameter of 10-12 mm, convex in the center, with a concentric fold in the center, from which 3-4 radial folds depart. The edge of the colony is flat, uneven. Aerial mycelium is well developed, velvety, dark gray (a-2), light gray along the edges (a-6). The substrate mycelium is dark chestnut (o-7), the medium is dark sand (z-6).

 On starch-ammonia agar, colonies from 2 to 10 mm in size are slightly convex, with radial, sometimes concentric folds. Aerial mycelium is dark gray (a-2) or gray (a-5), along the edges of the colony pale grayish (a-6) or whitish (d-1), sometimes does not completely cover the colony, the edge of the colony is uneven. Slate substrate mycelium (i-6). The environment is pale brown (b-4).

 On glycerol-aspartic agar, colonies with a diameter of 1 to 5 mm; convex with concentric folds. The edge of the colony is flat or blurry. Aerial mycelium is dark gray (a-2), along the edges of the colony gray (a-5). The substrate mycelium is dark chestnut (o-7). There is no soluble pigment.

 On oat agar, colonies with a diameter of 4 to 8 mm are slightly convex with a crater in the center, concentrically folded. The edge of the colony is flat or blurry. Aerial mycelium is gray (a-3), darker in the center (a-5). Covers the colony completely. There is no soluble pigment.

 On potato agar, the colonies are small, with a diameter of 1 to 3 mm, convex with radial folds. Aerial gray mycelium (a-2) with a rim of light gray color (a-6). Covers the colony completely. The substrate mycelium is brown (v-7) or brown-brown (v-2). The medium is not colored.

 On the organic medium of Gauze, 2 colonies with a diameter of 1 to 3 mm, slightly convex in the center, are smooth. The edge of the colony is uneven. Aerial mycelium is often absent. The substrate mycelium is pale sand (k-3). There is no soluble pigment.

 On meat and peptone agar, the colonies are round, slightly convex in the center, and smooth. Aerial mycelium is often absent, less often covers the colony, pale sand color (k-3). There is no soluble pigment.

 Physiological signs.

 On the environment, Pridhema-Gottlieb grows well in the presence of glucose, fructose, xylose, rhamnose, mannitol, maltose, lactose. It grows weakly in the presence of arabinose. Does not grow with sucrose and inositol. The strain liquefies gelatin, does not peptone milk, hydrolyzes starch. Fiber does not grow. Restores nitrates to nitrites. It grows well on potatoes.

 The storage conditions of the strain.

Periodic reseeding after 4 months of storage at 4-6 ^o C on Wednesday 21/12.

 The composition of the medium is 21/12 (in%).

 Corn extract (by dry weight) - 0.5; soluble starch - 2.0; disubstituted soluble ammonium - 0.4; monosubstituted potassium phosphate - 0.2; magnesium sulfate - 0.025; calcium carbonate - 0.1; agar - 2.0; PH - 6.9-7.0.

 Antagonistic properties.

 It exhibits antagonistic properties against gram-positive bacteria, yeast, yeast-like and mycelial fungi.

 Conditions for the formation of amphotericin B.

For carrying out the biosynthesis process, a culture of Streptomyces nodosus strain 472 is used for inoculation of seed flasks with a capacity of 750 ml, containing 100 ml of medium having the following composition (in%): soy flour - 3.0; glucose - 2.0; chalk - 0.25; sodium chloride - 0.1; pH after sterilization is 6.9-7.1. Seeds were grown for 28-30 hours at (27 ± 1) ^o С on a rocking chair with a rotation frequency of 220-240 rpm. / min Fermentation flasks with a capacity of 750 ml containing 50 ml of medium of the following composition (in%) are seeded from seed flasks: corn extract (by technical weight) - 1.0; glucose - 1-1.2; corn flour -5.0-7.0; ammonium sulfate - 0.5-0.65, chalk - 0.5-0.8; pH after sterilization - 6.9 - 7.1. The amount of sowing is 10%. Fermentation is carried out at (28 ± 1) ^o C for 72 hours on a rocking chair with a rotation frequency of 220-240 rpm.

 The antibiotic activity in 1 ml of culture fluid is 7500-8000 μg / ml.

Claims (1)

 Strain Streptomyces nodosus 472 VNIISCHM D-666 - producer of amphotericin B.