RU2081175C1 - Strain of bacillus subtilis - a producer of riboflavin (variants) - Google Patents

Abstract

FIELD: microbiology industry. SUBSTANCE: new mutant strains of Bacillus subtilis exhibiting resistance to purines were obtained by genetic-selection methods. Each of the obtained strain has additional traits: adenine auxotrophy (2159) and reversion to prototrophicity (2160). Yield of riboflavin at these strains culturing in flasks and fermenter - 7.2 and to 12 g/l, respectively. Strains were cultured on media containing sugar or glucose, or syrup, or molasses as carbon source. EFFECT: increased yield of riboflavin. 2 cl, 1 tbl

Description

 The invention relates to the microbiological industry, specifically to the microbiological synthesis of riboflavin.

Riboflavin (vitamin B ₂ ) is used in animal husbandry, animal husbandry, and poultry farming (pigs, chickens, fur animals) as a feed additive. Crystalline riboflavin is used in the food and pharmaceutical industries.

In world practice, to obtain riboflavin, a mushroom producer is used by the microbiological method. Known strain Eremothecium ashbyii "VNIIgenetika 1906" [1] which is a medium with soy and corn extract hydrologo accumulates over 96 hours of growth at 20 ^o C of 1.9 g / l of riboflavin. The main disadvantage of the mushroom producer is the long duration of the fermentation process.

Known strains of Bacillus subtilis with overproduction of riboflavin [2] and characterized by an unregulated biosynthesis of purines and riboflavin, as well as inversion and amplification of the riboflavin operon on the Bacillus subtilis chromosome. When using complex nutrient media containing glucose, maltose, yeast extract, corn extract, potassium phosphates, glutamate and sodium citrate, Mg ⁺⁺ , Ca ⁺⁺ , Mn ⁺⁺ , Fe ⁺⁺⁺ salts, succinic acid, etc. as well introducing carbohydrate feed into the fermenter using a computer while maintaining a certain level of dissolved oxygen (15 ± 5) in the culture fluid, these strains are able to accumulate 13-14 g / l of riboflavin in 48 hours and 15 g / l in 56 hours. The disadvantage of this process is the fact that high performance is achieved only with the use of roads large and scarce components of the nutrient medium and complex technology.

 A microbiological method for producing riboflavin based on Bacillus subtilis strains of the All-Russian Research Institute of Genetics 304 and 304a [3] obtained using genetic and selection methods, including obtaining mutants resistant to the riboflavin-rosoflavin analogue, combining mutations in the operator and regulatory regions using genetic transformation, and stepwise selection has been developed using mutagens. The disadvantage of these strains is the relatively low level of riboflavin synthesis, reaching 1.0 g / L.

The USSR author's certificate N 1429568 [4] describes the Bacillus subtilis strain "VNIIgenetics 24 / rMX45", created on the basis of the Bacillus subtilis strain "VNIIgenetics 304a". This strain, considered as a prototype, was obtained by genetic selection and genetic engineering methods, including the preparation of regulatory and other mutants, the construction of a hybrid plasmid carrying the Bacillus subtilis riboflavin operon, the introduction of this plasmid into the Bacillus subtilis recipient strain, and the expression of the riboflavin operon host cell plasmids. When growing the strain in deep conditions at a temperature of 37-42 ^o C and aeration on a medium containing glucose or sucrose, green molasses, molasses, sources of mineral nitrogen, urea or ammonium salts, ammonia water, mineral salts and growth factors, for example , dry biomass of yeast, BVK, yeast extract, peptone, casein hydrolyzate in the culture fluid accumulates 6.0 g / l of riboflavin for 48-50 hours of growth.

 In the case of applying the method for producing riboflavin described in USSR author's certificate N 1561513 [5], an increase in the conversion rate of the carbohydrate source to riboflavin and a decrease in the amount of impurities in the culture fluid are achieved. The method includes cultivating producer strains of the species Bacillus subtilis under deep conditions with stirring and aeration on a fermentation medium containing carbon sources, nitrogen, growth substances and mineral salts, while the carbon source is supplied to the fermentation medium at a constant speed of 1-3 g / l hour.

 The disadvantage of the prototype strain VNIIgenetics 24 / rMX45 is the relatively low biosynthetic activity in flasks and in fermenters using the method described above.

 The aim of the invention is to obtain a highly active strain producing vitamin B in bacteria of the species Bacillus subtilis.

This goal is achieved by obtaining new strains of Bacillus subtilis Bioreactor-1 A3, collection number 2159 and Bioreactor-1 ST12, collection number 2160 in the Collection of microorganism cultures of the State Scientific Center for Antibiotics (SSCA), which after 72 hours of growth at a temperature of 37-43 ^o С under stirring and aeration, up to 7.2 g / l of riboflavin is accumulated during cultivation in flasks. In a laboratory fermenter, when the process is fed with water according to the method [5], up to 12 g / l of riboflavin is accumulated in the culture fluid in 50-60 hours.

 New strains were obtained using genetic selection methods from the original Bacillus subtilis strain VNIIgenetika 24 / rMX 45 and carry mutations with changes in purine biosynthesis. Mutants were selected according to the following characteristics: resistance to 8-azaguanine and 6-mercaptopurine analogs to purines; adenine auxotrophy (AZ) and reversal to prototrophy (ST12). As a result, strains of Bacillus subtilis Biorector-1 AZ (2159) and Bioreactor-1 ST12 (2160) were obtained, the productivity of which reaches 7.2 g / l in flasks and 12 g / l in fermenters, which is 20-90% more in comparison with the prototype Bacillus subtilis VNIIgenetics 24 / pMX45.

Obtaining riboflavin using strains of Bacillus subtilis 2159 and 2160 is as follows: One and a half or two day old culture grown on an IPA jamb is transferred to flasks with inoculum containing molasses or sucrose, yeast biomass and mineral salts. The seed is grown for 12-20 hours at 37-40 ^° C under aeration conditions and in an amount of 1-5% is transferred to the main fermentation medium. You can inoculate the fermentation medium, bypassing the stage of the inoculum by flushing from the jamb (the titer of the cells in the fermentation medium after inoculation is 10 _{5. The} fermentation medium contains glucose, or molasses, or sucrose, or molasses, dried yeast biomass as growth factors, or yeast extract or peptone or casein hydrolyzate, inorganic nitrogen sources, e.g., urea, ammonium salts, ammonia water, mineral salts -. magnesium, manganese, iron, etc. The fermentation is carried out at a temperature of 37-43 ^o with stirring and aeration. After 48-72 hours of fermentation bioreactor strains A3-1 (2159) and the bioreactor ST12 (2160) in a medium accumulates 5-7,2 g / l of riboflavin in flasks and 8-12 g / l in laboratory fermenter.

 The invention is illustrated by the following examples.

Example 1. A 2-day culture of strain 2159 grown in an incubator at 37 ^{° C.} on MPA agar medium containing 20 μg / ml of erythromycin was transferred by loop to 750 ml Erlenmeyer flasks containing 25 ml of fermentation medium. The composition of the fermentation medium, parts by weight sucrose 10, yeast biomass 2, MgSO ₄ 0.05; urea 0.6. The fermentation is carried out on a shaker 240 rpm at 40 ^o C for 72 hours. The concentration of riboflavin in the culture fluid is 5 g / L.

Example 2. A two-day culture of strain 2160, grown in a thermostat at a temperature of 37 ^o With agar medium MPA containing 20 μg / ml of erythromycin, loop transferred to Erlenmeyer flasks containing 25 ml of fermentation medium. The composition of the medium and the fermentation conditions, as in example 1. The concentration of riboflavin in the culture fluid is 7.2 g / L.

Example 3. A two-day culture of strain 2159 grown in an incubator at a temperature of 37 ^° C on agarized agarized MPA medium with 20 μg / ml erythromycin loop is transferred to a seed medium spilled in 50 ml Erlenmeyer flasks (750 ml). The composition of the seed medium, wt. sucrose 2, yeast biomass 1, magnesium sulfate 0.05, urea 0.3. After 16 hours of cultivation in flasks on a shaker at 37 ^° C, the fermentation medium is seeded in a 1.2 L Marubishi laboratory fermenter (initial working volume 0.7 L). The composition of the fermentation medium, wt. yeast biomass (BVK) 2, magnesium sulfate 0.05, antifoam (niogen) 0.1. After sowing, fermentation is carried out by aeration (1: 1), stirring (700-1000 rpm), a temperature of 41 ± 1 ^o C and a constant supply of makeup in the form of a molasses solution (concentration of about 50%) at a speed of about 2 g / l hour. After 60 hours of cultivation, 12 g / l of riboflavin accumulates in the medium.

 Example 4. A two-day culture of strain 2160 grown under conditions as in Example 3 on a seed medium is transferred to a fermentation medium, as in Example 3. After 50 hours of cultivation, 12 g / l of riboflavin accumulates in the medium.

 The proposed strains 2159 and 2160 on media similar to the prototype provide a level of riboflavin synthesis of 20-90% more in comparison with the prototype strain VNIIgenetika 24 / rMX45.

Claims (2)

 1. Strain Bacillus subtilis SSCA 2159 producer of riboflavin.  2. Strain Bacillus subtilis GNCA 2160 producer of riboflavin.

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