RU2084520C1 - Strain of bacterium brevibacterium flavum - a producer of l-glutamine (variants) - Google Patents

Abstract

FIELD: biotechnology, amino acids production. SUBSTANCE: invention relates to new strains of bacterium Brevibacterium flavum VKPM B-3757 and Brevibacterium flavum VKPM B-6642 producing L-glutamine at significant amount. Mutant strains showing resistance to sulfaguanidine and azaserine were obtained by selection way. Culturing these strains on media containing sucrose or food sugar as carbon source ensures to obtain L-glutamine at concentration up to 35.5 g/l. Invention describes cultural-morphological and physiological-biochemical properties of strains also. EFFECT: new strains indicated above, increased yield of L-glutamine. 2 cl, 1 tbl

Description

 The invention relates to the microbiological industry and relates to a method for producing l-glutamine by growing strains of microorganisms producing it on nutrient media containing carbohydrates. L-glutamine is used in the medical, chemical and cosmetic industries, as well as as a dietary supplement.

 One of the known microbiological methods for producing glutamine is based on the use of strains of glutamic acid producers that produce L-glutamine under special cultivation conditions [1] The highest level of glutamine accumulation (46.5 g / l) was achieved in Microccocus glutamicus ATCC 14752 strain on medium with 15% glucose in 96 hours of cultivation. The disadvantages of the method are complex nutrient media and prolonged fermentation.

 To obtain L-glutamine, mutants of several types of glutamate-producing corynebacteria, in particular Brevibacterum flavum, Corinebacterum glutamicum, resistant to sulfamides, are also used. The strain B. flavum FERM P-1684, resistant to sulfaguanidine, produced after 48 h of cultivation on a medium with glucose 33 g / l glutamine, and on a medium with acetate as a carbon source 42 g / l [2] This strain is the closest analogue of the strains proposed in the present invention.

In these methods for producing L-glutamine, nutrient media contain glucose, ethanol, acetate as well as vitamins: biotin, thiamine and nutritional supplements in the composition of the soy flour hydrolyzate as a carbon source. The possibility of using cheaper components as a carbon source: sucrose or food sugar, and as a source of vitamins is not shown
corn extract.

 Our task is to obtain L-glutamine in high concentrations on simple nutrient media containing sucrose or food sugar as a carbon source, and corn extract as a source of vitamins. The task is achieved by creating new mutant strains of Brevibacterum flavum VNIIgenetika 50-72 and VNIIgenetika 115, which are deposited in the All-Russian collection of industrial microorganisms and have registration numbers VKPM V-3757 and V-6642, respectively.

 The new strains were obtained from the wild-type strain B. flavum ATCC 14067 as a result of several stages of selection using N-methyl-N-nitro-N-nitrosoguanidine as a mutagenic factor. At the first stage, the cells of the wild-type strain ATCC 14067 after treatment with nitrosoguanidine (250 μg / ml, 30 min) were plated on a minimal agar medium containing 0.5 mg / ml sulfaguanidine (SG). The selected mutant, resistant to sulfaguanidine and producing glutamine in a medium with sucrose, was used as the parent strain for the second stage of selection using a higher concentration of SG-1.5 mg / ml. After checking the obtained mutants for the ability to accumulate L-glutamine, the All-Russian Research Institute of Genetics strain 50-72 (VKPM B-3757) was selected, producing up to 25 g / l after 70 h of cultivation.

 At the next stage of selection for this strain, a mutation of resistance to the glutamine analogue of azaserin, which was not previously used in the selection of producers of this amino acid, was introduced. For this, the cells of the strain VNIIgenetika 50-72 were treated with a mutagen, as described above, and plated on a minimal medium containing 0.3 mg / ml azaserin (AS). Among the mutants resistant to azaserin, the VNIIgenetika-115 strain was selected. (VKPM B-6642), producing up to 35.5 g / l L-glutamine in 70 hours

 New strains, except for relation to azaserin, do not have significant differences in cultural-morphological and physiological-biochemical characteristics and therefore are further described together.

 Cultural and morphological characters. The cells are oval, sizes 1.0-2.0 x 0.5-1.0 microns, motionless, gram-positive, do not form spores.

After 3-5 days of growth at 30 ^o C on MPA or Hottinger agar, colonies with a diameter of 3-4 mm are creamy, the surface is smooth, the shape is convex, the edge is even, the structure is uniform, pasty. When sowing by injection, growth in the depth of the agar is weak, on the surface is moderate.

After 3-5 days of growth of 30 ^o C on Glover's glucose-mineral medium containing biotin and thiamine, colonies of 1-2 mm, smooth, uniform structure, pasty consistency, smooth surface, the color of the colonies is white. Stroke growth after two days on this medium is moderate.

 Physiological and biochemical characteristics.

 Aerobe. It does not dilute gelatin.

 It grows well on glucose, sucrose, maltose, fructose, mannose, ethanol, acetic acid. Does not grow on xylose, arabinose, lactose. Absorbs nitrogen in the form of ammonium and urea salts. Does not absorb nitrate nitrogen.

It grows at a temperature of 24-34 ^o C. The optimum temperature is 30 ^o C.

 It grows on media with a pH of 6 to 8.5. The optimal pH value is 6.8-7.2.

 For growth in a minimal environment, it needs biotin, thiamine.

 Resistance to antimetabolites. Strain B-3757 is resistant to sulfaguanidine, and strain B-6642 is resistant to sulfaguanidine and azaserin.

The strains are stored at a temperature of 4 ^o C on mowing agarized medium Hottinger for 3 months or in a freeze-dried state in ampoules for 1 year.

 Obtaining L-glutamine using strains of B. flavum BB3757 and B-6642 is as follows.

The strain culture is grown on a complete agarized medium, then plated in flasks with a seed medium for growing seed. After seeding, the flasks are mounted on a circular shaker with 220-250 rpm at 30 ^o C. After incubation, the finished seed is introduced into flasks or fermenters with a fermentation medium containing sources of carbon, nitrogen, vitamins and mineral salts. All components are sterilized under pressure. Fermentation is carried out in flasks on a rocking chair or in a fermenter equipped with a system of mixing, aeration and temperature control.

 The concentration of L-glutamine in the culture fluid at the end of fermentation reaches 35.5 g / L. The preparation of crystalline L-glutamine from a fermentation solution is obtained by known methods.

 Example 1

Culture B-6642 is grown for 24 hours at 30 ^° C. on meat peptone agar. A loop of 50 ml of inoculum inoculated into the flasks is seeded. 750 ml. The composition of the inoculum (%): sucrose-4.0, ammonium sulfate-2.5, monosubstituted potassium phosphate 0.05, magnesium sulfate-seven-water-0.03, corn extract-1.0, chalk-1.0, pH of the medium -7.4. The flasks are incubated on a circular shaker (250 rpm) at 30 ^o C for 20 hours. Ready-flown inoculum in an amount of 5% by volume of the fermentation medium is inoculated with flasks. 750 ml, containing 30 ml of a fermentation medium of the following composition (%): sucrose -12.0, ammonium sulfate (sterilized separately as a 50% solution and added to the medium before inoculation) -7.0, monosubstituted potassium phosphate-0, 25, magnesium sulphate heptahydrate-0.04, corn extract-0.5, thiamine-0.00035, chalk-4.0, pH-7.2. Fermentation is carried out on a circular shaker (250 rpm) for 70 hours. Towards the end of fermentation, the concentration of L-glutamine is 33.2 g / L.

 Example 2

 Inoculum is prepared according to example 1, but edible sugar is used instead of sucrose in the inoculum. Fermentation is also carried out as in example 1, but instead of sucrose, food sugar is used in the composition of the fermentation medium and thiamine is not added. After 70 hours, the concentration of L-glutamine is 35.5 g / L.

 Example 3

The seed is prepared according to example 2. Seed in an amount of 5% by volume is seeded with a 3 L laboratory fermenter containing 1.5 L of the fermentation medium described in Example 2. The fermentation is carried out at 30 ^° C., the stirrer rotates at 800 rpm air supply of 0.5 volume per volume of medium in min. After 70 hours, the concentration of L-glutamine reaches 34.3 g / L.

 Example 4

The culture of the strain B. flavum B-3757 is grown according to example 1, after which the seed medium of the following composition (%) is seeded: molasses-8.0, ammonium chloride-0.5, ammonium sulfate-0.5, monosubstituted potassium phosphate-0, 05, magnesium sulphate heptahydrate-0.03, corn extract-0.3, chalk-1.0, pH-7.4. The cultivation of seed is carried out according to example 1. Seed in an amount of 5% by volume of the volume of the fermentation medium is seeded with a laboratory fermenter with a capacity of 0.6 l, containing 0.4 l of the fermentation medium described in example 1. Fermentation is carried out at 30 ^o C, speed stirrer rotation 800 rpm, air supply 1 volume per volume of medium in min. After 70 hours, the concentration of L-glutamine reaches 25.0 g / L.

Claims (2)

 1. The bacterial strain Brevibacterium Flavum VKPM B-3757-producer of L-glutamine.  2. The bacterial strain Brevibacterium flavum VKPM B-6642 producer of L - glutamine.

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