RU2033426C1 - Strain micromonospora purpurea var violacea - a producer of antibiotic gentamycin, and a method of gentamycin producing - Google Patents

Abstract

FIELD: biotechnology, antibiotics. SUBSTANCE: vegetative inoculum of culture strain-producer Micromonospora purpurea var. violacea deposited in Collection ВНИИА at number 1931 is grown for two stages. At the first stage aqueous nutrient medium of the following composition is used, %: potato starch 2.4-2.6; pea flour 1.0-1.2; corn extract 0.6-0.8; chalk 0.4-0.5; at the second stage aqueous nutrient medium of the following composition is used, % : potato starch 2.5-3.5; glucose 0.15-0.3; soybean flour 1.5-2.5; ammonium sulfate 0.25-0.32; sodium chloride 0.25-0.35; chalk 0.4-0.6; cobalt chloride hexahydrate 0.00006-0.00016. Strain fermentation is carried out on the nutrient medium of the following composition, %: potato starch 4.5-5.2; glucose 0.15-0.3; soybean flour 3.2-4.0; ammonium sulfate 0.15-0.3; sodium chloride 0.25-0.35; chalk 0.5-0.8; cobalt chloride hexahydrate 0.00008-0.0002, and water - the rest. Gentamycin concentration in cultural fluid reaches 2100 mcg/ml. EFFECT: increased yield of antibiotic. 3 cl

Description

 The invention relates to biotechnology and can be used to obtain gentamicin.

 Gentacimin is a broad-spectrum antibiotic, one of the valuable properties of which is its high activity against blue-green pus sticks.

 It is known to use the strain Micromonospora purpurea TCC 31849 to obtain gentamicin, the activity of the strain is 400-500 μg / ml (established by calculation) [1] The noted level of activity is insufficient to obtain gentamicin industrially.

 The aim of the invention is to increase the yield of gentamicin in the process of fermentation to obtain this therapeutically valuable antibiotic.

 The goal is achieved by obtaining a new highly productive strain of Micromonospora purpurea var. violacea 5309 / 5AB, which is obtained from a strain of Mocromonospora purpurea var. violacea VNIIA 7R using mutagenic factors and selective background.

 Strain 5309 / 5AB is characterized by the following features.

 Cultural and morphological characters.

 Disputes are single (0.6-0.83). They are sitting on the spore-bearing leg. Aerial mycelium is absent.

 Agar Gauze-1. Growth is good. Substrate mycelium dark purple black (g1-A1) (Color is given on the color scale A. Bondartseva Edition of the USSR Academy of Sciences, M. 1954). There is no soluble pigment.

 G-1 agar with pea flour and starch. Growth is good. The substrate mycelium is gray-dark violet dirty brown-violet (p2-m3). There is no soluble pigment.

 Oat agar. Growth is good. Substrate mycelium umbra black (o7-a1). There is no soluble pigment.

 Bennett's agar. Growth is good. The substrate mycelium is gray-dark-violet rusty (n2-g1). There is no soluble pigment.

 Corn agar N 6. Growth is good. Substrate mycelium gray-dark purple pale terracotta (n2-zh4); There is no soluble pigment.

 Agar Gauze-2. Growth is good. The substrate mycelium is from pale terracotta brownish-red to dark purple (w4-b1-g1). There is no soluble pigment.

 Tyrosine agar. Growth is very weak. The substrate mycelium is pale grayish (a6). There is no soluble pigment.

 Emerson agar. No growth.

 Physiological and biochemical characteristics.

Aerobe. Optimum growth of 32-37 ° ^C. The milk is not peptonised not coagulate. Gelatin does not thin. Starch hydrolyzes.

 Relation to carbon sources. Absorbs starch, glucose, sucrose, maltose, arabinose, xylose; weak galactose, fructose; very weak raffinose, lactose; does not absorb mannitol, inositol, rhamnose.

 Cultivation on corn agar medium No. 6 with 200 μg / ml streptomycin sulfate. No growth.

 Cultivation on G-1 medium with pea flour and starch with 6000-8000 μg / ml sisomycin sulfate. Growth is satisfactory.

 Antagonistic properties. Producer of the antibiotic gentamicin, active against most gram-positive and gram-negative bacteria, which has high activity against the stick of blue-green pus.

 On a medium of the following composition, potato starch 5.0; soybean flour 3.0; ammonium sulfate 0.35; sodium chloride 0.3; chalk 0.7; cobalt hexavalent chloride 0.00008; tap water, natural pH, in Erlenmeyer flasks with a capacity of 0.75 L from 0.05 L of medium, the average activity of strain 5309 / 5AB for 120 hours is 1407 μg / ml, maximum 1700 μg / ml, average activity of strain for 144 hours (full fermentation cycle) is 1600 μg / ml, maximum 1928 μg / ml.

 A known method of producing gentamicin using a producer strain of Micromonospora purpurea ATCC 31849 [1] the Method consists in inoculating spore material of the strain Micromonospora purpurea ATCC 31849 inoculation medium containing meat extract 0.3; tryptone 0.5; dextrose 0.1; soluble starch 2.4; yeast extract 0.5; chalk 0.4; water, followed by fermentation on a medium containing glucose 2; soy flour 0.75; Hypericum flowers 0.4; chalk 0.4; cobalt hexavalent chloride 0.0001; antifoam 0.01; water. Fermentation is carried out under aeration for 140 hours. After isolation and purification from 10 l of the culture fluid, 1.98 g of the target product is obtained (taking into account losses on the allocation, the activity of the culture fluid is 400-500 μg / ml).

 The disadvantages of this method are the low yield of the target product and the use of meat extract as a raw material for the nutrient medium.

 The aim of the invention is to increase the level of biosynthesis of gentamicin.

 This is achieved by using the biosynthesis of a new, more productive strain of Micromonospora purpure var. violacea 5309 / 5AB, new nutrient media, as well as the cultivation of vegetative seed in 2 steps.

 The method is as follows.

Growing vegetative inoculum of strain 5309 / 5AB (I stage uterine flasks). In this case, the spores of the strain are introduced into Erlenmeyer flasks with a capacity of 0.5 l from 0.05 l of medium of the following composition, potato starch 2.4-2.6; pea flour 1.0-1.2; corn extract (tech. weight) 0.6-0.8; chalk 0.4-0.5; water the rest, pH is natural. The flasks are shaken on a rocking chair from 230-240 rpm for 40-42 hours at 33 ^about C.

Grow vegetative seed (II stage, working flasks). At the same time, the vegetative inoculum grown on the 1st stage is introduced into Erlenmeyer flasks with a capacity of 0.75 L from 0.05 L of medium of the following composition, potato starch 2.4-3.5; glucose 0.15-0.3; soy flour 1.5-2.5; ammonium sulfate 0.25-0.32; sodium chloride 0.25-0.35; chalk 0.4-0.6; cobalt six-water 0.00006-0.00016, the rest is water, pH is natural. The flasks were shaken on a shaker with 230-240 rev / min for 40-44 hours at 33 ° ^C. Using the vegetative inoculum is grown to stage II, flasks were seeded with 0.75 l 0.045 liter fermentation medium of the following composition, potato starch 4 5-5.2; glucose 0.15-0.3; Soya flour 3.2-4.0; ammonium sulfate 0.15-0.3; sodium chloride 0.24-0.35; chalk 0.5-0.8; hexavalent cobalt chloride 0.00008-0.0002; water the rest, pH is natural. The fermentation was conducted at 33 ° ^C. The flasks were shaken on a shaker with 230-240 rev / min, the duration of the fermentation 144 h. The method provides a culture liquid with the concentration of gentamicin in the end of fermentation in 2100 ug / ml.

 A feature of the method is the use of a new strain N 5309 / 5AB and modified compositions of the inoculum and fermentation media, which allows to increase the level of biosynthesis of gentamicin.

PRI me R 1. Spore material of the culture Micromonospora purpurea var. violancea 5309 / 5AB sown in Erlenmeyer flasks (uterine flasks with a capacity of 0.75 l from 0.05 l of nutrient medium of the following composition, potato starch 2.4; pea flour 1.0; corn extract (tech. weight) 0.6; chalk 0.4, tap water, natural pH flasks were shaken on a shaker with 230-240 rev / min at 33 ^{° C} for 42-44 hours vegetative inoculum from the flask is transferred to a masterbatch flasks with 0.75 l of 0.05.. l of medium of the following composition, potato starch 2.5; glucose 0.15; soy flour 1.5; ammonium sulfate 0.25; sodium chloride 0.25; chalk 0.4; cobalt hexahydric chloride 0,000 06; water else natural pH flasks were shaken on shaker at 33 C ^for 40-44 h from inoculum flasks were transferred to a working capacity of 0.75 liter flask with 0.05 L of fermentation medium with the following composition, potato starch, 5.0..; glucose 2.0; soybean flour 3.0; ammonium sulfate 0.3; chalk 0.7; sodium chloride 0.3; cobalt chloride, hexahydrate 0.00008; tap water; pH natural. 10% of the seed from the volume of the fermentation medium is introduced . Biosynthesis carried out at 33 ^{° C} on a shaker with 230-240 rev / min. The duration of the biosynthesis process is 144 hours. The activity per 120 hours of fermentation is 1700 μg / ml. Activity for 144 h 1920 μg / ml.

PRI me R 2. The cultivation of vegetative material (N 1) on the medium of the following composition, potato starch 2.6; pea flour 1.2; corn extract 0.8; chalk 0.5, water the rest, natural pH. The flasks were shaken on shaker at 33 ^C for 40-42 h from the inoculum flasks masterbatch in an amount of 10% by volume of the medium in the working transmit flask into the flask seed medium composition, potato starch 2.5.; glucose 0.15; soya flour 1,5; ammonium sulfate 0.25; sodium chloride 0.25; chalk 0.4; cobalt hexahydrate chloride 0.00006; water the rest, pH is natural. The flasks are shaken for 40-44 hours. The seed from the working flask is transferred to a flask with a capacity of 0.75 L from 0.045 L of fermentation medium of the following composition, potato starch 4.5; glucose 0.15; soy flour 3.2; (NH ₄ ) ₂ SO ₄ 0.15; sodium chloride 0.25; chalk 0.5; cobalt hexavalent chloride 0,0008; tap water, natural pH, inoculation of a fermentation medium 10% of seed from the volume of medium. The flasks are incubated on a shaker from 230-240 rpm for 144 hours. The activity for 120 hours of fermentation is 1700 μg / ml, for 144 hours 1980 μg / ml.

 PRI me R 3. The cultivation of vegetative material in the fallopian flasks according to example 1. Seed from the fallopian flask is transferred in an amount of 10% to a working flask with a capacity of 0.75 l from 0.05 l of seed medium with the following ratio of components, potato starch 3.0; glucose 0.2; soy flour 2.0; ammonium sulfate 0.3; sodium chloride 0.3; chalk 0.5; cobalt hexahydrate chloride 0.00008, tap water, natural pH. The cultivation conditions of example 2. Seed from the working flask in an amount of 10% is transferred to a flask with a capacity of 0.75 l from 0.45 l of fermentation medium with the following ratio of components, potato starch 5.0; glucose 0.2; soy flour 3.5; ammonium sulfate 0.2; sodium chloride 0.3; chalk 0.7; cobalt chloride 0.00016, tap water, natural pH.

 The cultivation conditions of example 2. The activity for 120 hours of fermentation is 1750 μg / ml, 144 2100 μg / ml.

 PRI me R 4. The cultivation of vegetative inoculum in the fallopian flasks according to example 1. Seed from the fallopian flask in the amount of 10% is transferred to a working flask with a capacity of 0.75 l from 0.05 l of inoculum with the following ratio of components, starch potato 3.5; glucose 0.3; soy flour 2.5; ammonium sulfate 0.32; sodium chloride 0.35; chalk 0.6; cobalt chloride 0.00016; tap water, natural pH. The cultivation conditions of example 2. Seed from a working flask in an amount of 10% is transferred to a flask with a capacity of 0.75 l from 0.45 l of fermentation medium with the following ratio of components, potato starch 5.2; glucose 0.3; soy flour 4.0; ammonium sulfate 0.3; sodium chloride 0.35; chalk 0.8; cobalt hexavalent chloride 0.0002; tap water, natural pH. The cultivation conditions of example 2. The activity for 120 hours of fermentation is 1745 μg / ml, 144 2050 μg / ml.

Claims (2)

 1. Strain Micromonospora purpurea var. violacea VNIIA N 1931 - producer of the antibiotic gentamicin.  2. A method of producing gentamicin by growing a vegetative inoculum of the producer of the culture Micromonospora purpurea in an aqueous nutrient medium containing sources of carbon, nitrogen and mineral salts, followed by inoculation of a fermentation medium containing sources of carbon, nitrogen and mineral salts, characterized in that, in order to increase the concentration of the target product in the culture fluid, a Micromonospora purpurea var. strain is used as a producer. violacea VNIIA N 1931, the cultivation of the vegetative inoculum is carried out in two stages, the first stage using an aqueous nutrient medium containing potato starch 2.4 - 2.6, pea flour 1.0 1.2, corn extract 0.60, 8, chalk 0.4 0.5, in the second stage, an aqueous nutrient medium containing potato starch 2.5 3.5, glucose 0.15 0.3, soy flour 1.5 2.5, ammonium sulfate 0.25 - 0.32, sodium chloride 0.25 0.35, chalk 0.4 0.6, cobalt chloride, six-water 0.00006 0.00016, and fermentation is carried out on the following composition, potato starch 4.5 5.2, glucose 0 , 15 0.3, soy flour hydrochloride 3.2 4.0, ammonium sulfate 0.15 0.3, sodium chloride 0.25 0.35, chalk 0.5 - 0.8, cobalt chloride, six-water 0.00008 0.0002, the rest is water.