RU2261273C2 - Method for preparing riboflavin, strains bacillus subtilis as producers of riboflavin (variants) - Google Patents

Abstract

FIELD: biotechnology, microbiology, vitamins.

SUBSTANCE: method relates to a method for preparing riboflavin by culturing the microorganism Bacillus subtilis as a producer in the nutrient medium containing rib-operon from Bacillus amyloliquefaciens, or microorganism able for utilization of glycerophosphate as a single carbon source, or eliciting the resistance against inhibition of growth by glyoxylate, and extraction of riboflavin prepared. Invention uses the following strains as producers of riboflavin: B. subtilis GM51/pMX45, B. subtilis GM41/pMX45, B. subtilis GM44/pMX45. Invention provides preparing riboflavin of the high degree of effectiveness.

EFFECT: improved preparing method.

5 cl, 4 ex

Description

Technical field

The present invention relates to the microbiological industry, in particular, to a method for producing riboflavin. More specifically, the present invention relates to a method for producing riboflavin using bacteria belonging to the genus Bacillus.

State of the art

Riboflavin (Vitamin B ₂ ) is an essential compound for higher animals, including humans. Riboflavin deficiency leads to various types of diseases, such as baldness, inflammation of the skin and other injuries, conjunctivitis, blurred vision, stunted growth and others.

Therefore, riboflavin is used for the preparation of commercial vitamin preparations used for vitamin deficiency, as well as as a dietary supplement. In addition, riboflavin is used as a food coloring, for example, in mayonnaise, ice cream and other products.

Riboflavin is obtained by both chemical and microbiological methods. In chemical methods, riboflavin is usually obtained as the end product of a complex multi-stage process that uses a rather expensive starting component, such as D-ribose.

The production of riboflavin by fermentation of fungi such as Ashbya gossypii or Eremothecium ashbyii (The Merck Index, Windholz et al., Eds. Merck & Co., page 1183, 1983) is described. Yeast such as Candida or Saccharomyces (WO 93/03183, JP63098399A2, US Pat. 4,794,081) are also suitable for riboflavin production. In addition, fungi from the Ashbya genus with increased isocytratliase activity (ICL) having the ability to produce and accumulate riboflavin have been described (US Pat. 5,976,844).

Mutants of Bacillus subtilis are described, obtained by selection on purine analogues, azaguanine and azaxanthin, producing significant amounts of riboflavin (U.S. Pat. No. 3,900,368). In general, treatment with purine and riboflavin analogues allows one to obtain mutants with increased riboflavin synthesis, since these mutations allow the microorganism to displace analogs through increased riboflavin production due to the competitive mechanism (Matsui et al., Agric. Biol. Chem. 46: 2003, 1982) .

It was shown that mutant strains of Bacillus subtilis, with activity of releasing phosphoric acid from 5'-guanilic acid no higher than 0.081 μmol / min / (mg protein), are capable of producing riboflavin (EP 0531708 B1).

Mutant strains of Bacillus subtilis, selected for resistance to roseoflavin and 8-azaguanine and containing a mutation in the ribC gene, produce riboflavin (USSR patent 908092). Such strains are strains of Bacillus subtilis VNIIGenetika-304 and 304a. Next, a strain of Bacillus subtilis 304 / pMX45 was obtained, containing a plasmid with a rib operon from Bacillus subtilis, which has increased ability to produce riboflavin (up to 3.5 g / l) (French patent 2546907).

A strain of Bacillus subtilis 62 / pMX30ribO186 is described, producing up to 12.5 g / l of riboflavin within 42 hours of fermentation (RF patent 2081906). The strain Bacillus subtilis 62 / pMX30ribO186 was obtained from the strain Bacillus subtilis RK6121 as a mutant resistant to 8-azaguanine, methionine sulfoxide, diacetyl and psicofuranine and additionally containing a plasmid with the mutant (ribO mutation) ribus subt operon.

Bacterial strains obtained by introducing riboflavin biosynthesis genes from Bacillus sublilis into the chromosome of this bacterium also produce riboflavin (US Pat. 5,837,528, 5,925,538 and 6,322,995). The best known strain is the strain RB50 :: [pRF69] ₆₀ (Ade ⁺ ) carrying a transcriptionally modified riboflavin operon containing two SPO1-15 promoters, producing 13.0-14.0 g / l riboflavin for 48 hours and 15 g / l riboflavin a for 56 hours (US Pat. 5,925,538) when grown on standard commercial media and standard commercial conditions.

Although riboflavin production has been significantly improved by eliminating the above microorganisms or improving its production process, it is necessary to develop more efficient riboflavin production processes in order to meet the growing demand for this vitamin.

Description of the invention

The aim of the present invention is to increase the productivity of riboflavin by strains producing riboflavin and to provide a method for producing riboflavin using such strains.

To increase the production of riboflavin, the authors of the present invention constructed a strain containing a deregulated rib operon from Bacillus amyloliquefaciens. Then, a mutant strain was obtained with the ability to utilize glycerophosphate and resistant to growth inhibition by glyoxylate.

Thus, the present invention was accomplished.

The present invention provides a bacterium belonging to the genus Bacillus and having the ability to produce riboflavin. In particular, the present invention provides a bacterium with increased ability to produce riboflavin, due to the presence of a deregulated rib operon from Bacillus amyloliquefaciens, the ability to utilize glycerophosphate and / or a mutation that confers glyoxylate resistance.

The present invention also provides a method for producing riboflavin by a fermentation method, comprising the steps of growing the above bacteria in a culture medium and isolating riboflavin from the culture fluid.

The present invention is as follows:

1. The bacterium Bacillus subtilis, with the ability to produce riboflavin, containing a heterologous rib operon in the chromosome of the bacterium, in which the specified rib operon is a rib operon from Bacillus amyloliquefaciens.

2. The bacterium Bacillus subtilis in accordance with 1, in which the specified rib operon from Bacillus amyloliquefaciens is deregulated.

3. The bacterium Bacillus subtilis. with the ability to produce riboflavin and the ability to utilize glycerophosphate.

4. The bacterium Bacillus subtilis, with the ability to produce riboflavin and resistance to growth inhibition by glyoxylate.

5. The bacterium Bacillus subtilis, with the ability to produce riboflavin and having one or more of the following characteristics:

- contains rib operon from Bacillus amyloliquefaciens in the chromosome of this bacterium;

- has the ability to utilize glycerophosphate and

- It is resistant to growth inhibition by glyoxylate.

6. A method of producing riboflavin, comprising the stage of growing bacteria in accordance with 1-5 in a nutrient medium and isolating riboflavin from the culture fluid.

In more detail, the present invention will be described below.

(1) A bacterium according to the present invention.

The bacterium of the present invention is the bacterium Bacillus sublilis, which is capable of producing riboflavin, in which the production of riboflavin is increased by introducing the heterologous rib operon from Bacillus amyloliquefaciens into the bacterial chromosome.

As used herein, the term “bacterium with the ability to produce riboflavin" means a bacterium capable of producing and accumulating in the nutrient medium riboflavin in an amount greater than the natural or parental strain of B. subtilis, such as strain of B. subtilis 168, and preferably means that said microorganism is capable of producing and accumulating in a nutrient medium not less than 0.5 g / l, more preferably not less than 1.0 g / l of riboflavin.

The term "bacterium Bacillus subtilis" means a bacterium that is classified as Bacillus subtilis in accordance with the classification known to a person skilled in the field of microbiology. And the term "bacterium Bacillus amyloliquefaciens" means a bacterium that belongs to the species Bacillus amyloliquefaciens in accordance with the classification known to a specialist in the field of microbiology.

The term “rib operon” means a chromosome fragment containing genes encoding proteins essential for the production of riboflavin, rib operon in bacteria belonging to the genus Bacillus, contains the following genes: ribO gene, encoding a regulatory element, ribG gene, encoding deaminase / reductase, gene ribB encoding riboflavin synthase (α-subunit), ribA gene encoding GTP cyclohydrolase / 3,4-dihydroxy-2-butanone-4-phosphate synthase, ribH gene encoding limazine synthetase, and ribT gene encoding a protein with unknown function (Morozov and et al., Mol. Genet. Mik. Virusol., 7:42, 1948). The nucleotide sequences of the ribG, ribB, ribA, ribH, and ribH genes from Bacillus subtilis are presented in the GenBank database under the numbers gi: 16079385, gi: 16079384, gi: 16079383, gi: 16079382 and gi: 16079381, respectively (nucleotides 2429492 to 2430577, from 2428834 to 2429481, from 2427623 to 2428819, from 2427126 to 2427590 and from 2426639 to 2427013 in the sequence NC_000964.1).

The term “heterologous operon” as used herein means that said operon has been isolated from a chromosome of an organism other than Bacillus sublilis. A bacterium containing a heterologous operon in the chromosome can be obtained by standard methods of recombinant DNA, transformation, transfection.

The term “deregulated” means that the level of production of riboflavin is higher than the level of production observed in bacteria with a natural regulatory system for the synthesis of riboflavin (namely, a natural bacterium). Examples of bacteria having a deregulated rib operon are bacteria that are resistant to various purine analogs and their antagonists, or riboflavin analogs.

The deregulation of the regulatory system for riboflavin synthesis can be carried out by changing the regulatory region of the operon, replacing the regulatory region with another strong region with a constitutive expression level, inactivating the gene encoding the repressor, obtaining mutations in the repressor protein, etc. This deregulation can be carried out by traditional methods, such as mutagenic treatment using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination and / or insertional deletion mutagenesis.

The term “glycerophosphate recycling ability” means the ability to metabolize glycerophosphate more efficiently than the parent strain, for example, the ability of the B. subtilis strain used in the present invention to grow faster than the parent strain when these strains are grown on a medium containing glycerophosphate as the sole carbon source. More specifically, this means that the strain of B. sublilis has the ability to utilize glycerophosphate if this strain grows faster than the parent strain, when these strains are grown under suitable conditions on a medium containing glycerophosphate as the sole carbon source, for example, liquid medium containing 0.1% glycerophosphate. More specifically, we can say that the strain B. subtilis has the ability to utilize glycerophosphate if this strain colonizes within 2 days at 37 ° C when growing this strain on an agar medium containing glycerophosphate as the sole carbon source, for example, on a medium containing 0.1% glycerophosphate and agar, under suitable conditions. The term “suitable conditions” refers to temperature, pH, access to air, or the optional presence of essential nutrients and other conditions necessary for growing a strain of B. subtilis.

Glyconeogenesis enzymes are known to have an activity level insufficient for the recycling of glycolysis products, such as glycerophosphates. It is also known that B. subtilis strains grow very poorly on media containing glycerophosphate as the sole carbon source. Based on this, it was suggested that the isolation of mutants capable of growing on a medium containing glycerophosphate as the sole carbon source will allow mutants to be obtained with an increased level of activity of enzymes involved in glyconeogenesis and, as a result, increase the production of riboflavin during the fermentation process by sucrose.

The term "bacterium is resistant to growth inhibition by glyoxylate" means a bacterium obtained from the parent strain by modifying the genetic properties so that the strain becomes capable of growth on a nutrient medium containing glyoxylate. Specifically, glyoxylate resistance means the ability of a bacterium to grow on a minimal medium containing glyoxylate at a concentration at which the parent strain of said bacterium cannot grow, or the ability of a bacterium to grow faster on a nutrient medium containing glyoxylate than a natural or parent strain of said bacterium. For example, a bacterium that is able to form colonies when grown for 3-5 days at 34 ° C on plates with an agar medium containing 0.5 mg / ml or more, preferably 1.0 mg / ml or more glyoxylate, is resistant to glyoxylate.

Glyoxylate is a very important intermediate compound of the glyoxylate shunt, necessary for growth on carbon sources such as acetate or fatty acids, since this pathway allows direct conversion of acetyl-CoA to intermediate metabolic products. The indicated pathway includes three enzymes. Two of them, isocitratliase and malatesynthase A, convert some intermediates of the tricarboxylic acid cycle from isocitrate to malate. It was shown that, unlike E. coli, the activity of the glyoxylate shunt enzymes in B. subtilis is very low. In addition, glyoxylate inhibits the growth of natural B. subtilis cells at a concentration of 1 mg / ml. To increase the activity of glyoxylate shunt enzymes, it was decided to obtain glyoxylate resistant mutants.

The bacterium of the present invention can be obtained from a bacterium already possessing the ability to produce riboflavin by imparting stability and / or the ability to utilize glycerophosphate. In addition, the bacterium according to the present invention can be obtained by giving the ability to produce riboflavin bacteria, already possessing specific resistance and / or the ability to utilize glycerophosphate.

Thus, any known strain of the bacterium belonging to the genus Bacillus with the ability to produce riboflavin can be subjected to one or more of the mutagenic treatments described above, and then the obtained mutant strains can be checked for compliance with the above requirements of the present invention, including glycerophosphate utilization and glyoxylate resistance, and therefore suitable for use in the present invention. The obtained strains are checked by growing in a nutrient medium, and strains with the ability to produce riboflavin with a yield greater than the parent strain are selected and used in the present invention.

As parent strains that can be improved to produce bacteria according to the present invention, bacterial strains belonging to the genus Bacillus can be used, riboflavin producers such as Bacillus subtilis strains VNIIGenetika-304 and 304a (USSR patent 908092), Bacillus strain subtilis 304 / pMX45 (CMPM B-2694) (French patent 2546907), Bacillus subtilis 62 / pMX30riO186 strain (VKPM B-6797) (RF patent 2081906), Bacillus subtilis RB50 strain :: [pRF69] ₆₀ (Ade ⁺ ) (patent USA 5,925,538), a strain of Bacillus subtilis FERM BP-3855 and FERM BP-3855 (European patent EP 0531708B1) and the like.

(2) Method for producing riboflavin

The method according to the present invention is a method for producing riboflavin, comprising the steps of growing a bacterium according to the present invention in a culture medium and isolating riboflavin from the culture fluid.

In the method according to the present invention, the cultivation of a bacterium belonging to the genus Bacillus, the isolation and purification of riboflavin from the culture fluid can be carried out in a manner similar to traditional methods for producing riboflavin by fermentation using a bacterium.

The nutrient medium for the production of riboflavin can be either synthetic or natural, provided that it contains sources of carbon, nitrogen, inorganic ions and other necessary organic components. As a carbon source, saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trialose and starch hydrolyzate can be used; alcohols such as glycerin, mannitol and sorbitol; organic acids such as gluconic, fumaric, citric and succinic acids, and the like. Inorganic nitrogen salts such as ammonium sulfate, ammonium chloride and ammonium phosphate can be used as a nitrogen source; organic nitrogen compounds such as soybean hydrolyzate; ammonia gas; aqueous ammonia and the like. It is desirable that vitamins such as vitamin B ₁ , other necessary compounds, for example, nucleic acids such as adenine and RNA, or yeast extract and the like, are present in suitable amounts as organic nutritional supplements. In addition, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese and similar compounds can be added, if necessary.

The cultivation is preferably carried out under aerobic conditions from 16 to 72 hours, and the temperature during the cultivation is maintained in the range from 30 to 45 ° C, the pH is in the range from 5 to 8. The pH is maintained using inorganic and organic acidic and basic compounds, as well as gaseous ammonia.

Riboflavin can be isolated from the culture fluid by any or a combination of any known methods, such as those using ion exchange chromatography and precipitation,

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1. Construction of a strain of Bacillus subtilis containing riboflavin operon from Bacillus amyloliquefaciens on the chromosome.

It is known that direct homologous recombination of the rib operon from B. amyloliquefaciens to the B. subtilis chromosome does not lead to success.

To clone the riboflavin biosynthesis genes, the chromosomal DNA of B. amyloliquefaciens A50 strain was treated with HindIII restriction enzyme. The resulting mixture of DNA fragments was ligated into the pACYC184 vector (NBL Gene Sciences Ltd., UK, GenBank / EMBL accession number X06403) pretreated with the same restriction enzyme. The resulting product was used to transform E. coli strain BSV821. Transformants capable of growth on a nutrient medium containing chloramphenicol were selected. A plasmid was isolated from such transformants and the fragment containing the rib operon was cloned into the pCB20 vector. The resulting plasmid was named pBX2. Plasmid pBX2 complemented mutations in all rib operon genes, with the exception of ribTD genes.

To remove mutants with constitutive expression of riboflavin biosynthesis genes, plasmid pBX2 was treated with hydroxylamine using a standard mutagenesis procedure. The laboratory auxotrophic strain of B. subtilis ribG850 was transformed with a mutant DNA plasmid and the ability of the obtained transformants to produce riboflavin was evaluated. Using PCR, it was found that riboflavin production was due to mutations in the ribO gene. The ribO1 mutation (replacing T with C at position +87) was identical to the known mutation in the B. subtilis operon. The ribO2 mutation turned out to be a new mutation C to T at position +149. The plasmid containing the ribO2 mutation was named pBX21. The strain B. subtilis ribG850 transformed with plasmid pBX21 produced up to 65 mg / L of riboflavin, while the same strain transformed with plasmid pBX2 did not produce riboflavin at all.

The plasmid-free Bacillus subtilis strain VNII Genetika-304 (USSR patent 908092) was used as the parent strain for constructing the Bacillus subtilis strain GM49 containing the deregulated riboflavin operon from Bacillus amyloliquefaciens on the chromosome. To integrate the deregulated riboflavin operon from Bacillus amyloliquefaciens into the chromosome of the strain Bacillus subtilis 304, a vector for integration of pEK14 (Km ^R , Ap ^R , Rib ⁺ ) was constructed. The pEK14 vector consists of E. coli plasmid pUC19, the kanamycin nucleotidyl transferase (kan) gene from plasmid pUB110 and the deregulated (ribO2 mutation) riboflavin operon Bacillus amyloliquefaciens from plasmid pBX21. Plasmid pEK14 is not capable of replication in B. subtilis.

To integrate pEK14 into the chromosome, the method of "random integration" was used.

First, the BamHI fragment of plasmid pEK14 was ligated with BamHI fragments of chromosomal DNA from B. subtilis strain 168. The plasmid containing regions homologous to the B. subtilis genome was able to integrate into the chromosome by homologous recombination. Then, the mixture of fragments obtained after ligation was used to transform the laboratory auxotrophic strain of B. subtilis rib G850. Transformants with the phenotype Km ^R , Rib ⁺ were selected. The frequency of transformation was about 100 per 1 γ DNA. 60 clones with the phenotype Km ^R , Rib ⁺ were analyzed. Most integrants were unstable or auxotrophic. They lost the Km ^R and Rib ⁺ markers during growth in a culture medium without antibiotics. One stable integrative clone, named VK53, was found. The B. subtilis strain BK53 ribG850 aab :: pEK14 (Rib _{Bam +} Km ^R ) contained the riboflavin operon from B. amyloliquefaciens (Rib _{Bam +} ) and the kanamycin resistance gene Km ^R (kan) integrated into the B. subtilis chromosome at a locus called "aab "

The integrated operon of B. amyloliquefaciens was transferred to the chromosome of the recipient plasmid-free strain of B. subtilis 304, a producer of riboflavin from B. subtilis strain BK53 ribG850 aab :: pEK14 (Pib _{Bam +} Km ^R ) by transduction using phage AR9. The kanamycin resistance gene integrated into the B. subtilis chromosome together with the riboflavin operon from B. amyloliquefaciens was used as a selection marker. The resulting strain was named GM49. The indicated strain GM49 aab :: рЕК14 (Rib _{Bam +} Km ^R ) contains two riboflavin operons in the chromosome: its own operon and the B. amyloliquefaciens operon.

The ability of the strain B. subtilis GM49 to produce riboflavin was tested in flasks with a nutrient medium, described below in the section "Fermentation conditions". The fermentation process (“batch” fermentation) was carried out in 850 ml flasks. The culture volume was 150 ml. Strain GM49 produced 3 g / L riboflavin in 72 hours.

Example 2. Construction of a strain of Bacillus subtilis containing both riboflavin operon from B. amyloliquefaciens on the chromosome and riboflavin operon from B. subtilis on the plasmid.

The strain B. subtilis GM51 / pMX45 was obtained from the plasmid-free strain B. subtilis GM49, a producer of riboflavin.

First, a strain deficient in the recombination system was obtained.

To obtain strain GM51 with an insertion mutation recE :: cat, the plasmid pBT69 was used in the recE gene. Plasmid pBT69, derived from plasmid pBT61 containing the recE gene from Bacillus subtilis (Gassel M. and Alonso JC, "Expression of the recE gene during induction of the SOS response in Bacillus subtilis recombination-deficient strains", Mol. Biology, 1989, 3 (9), 1269-1276), contained the chloramphenicol resistance gene (cat) encoding chloramphenicol acetyltransferase. Plasmid pBT69 was integrated into the recE gene at the recognition sites of the ClaI restriction enzyme (recE :: cat) present in the gene. The mutant recE :: cat gene was integrated into the chromosome of B. subtilis GM49 strain by double crossing over recombination. Thus, the strain B. subtilis GM51 was obtained (aab :: pEK14 recE :: cat (Rib _{Bam +} Km ^R Cm ^R )).

Then, the C.subtilis GM51 strain deficient in the recombination system (recE :: cat) Cm ^R was transformed with the plasmid pMX45 containing the riboflavin operon from the B. subtilis ribO335 strain. The indicated plasmid pMX45 contains a large (10 kb) DNA fragment of the chromosome B. subtilis. Plasmid pMX45 is a low-copy plasmid (1-2 copies per cell). Plasmid pMX45 is described in detail in French patent 2546907.

Strain GM51 / pMX45 produced 5.8 g / l of riboflavin for 72 hours of fermentation in flasks and 15 g / l of riboflavin for 70 hours of fermentation in 1 l of jar fermentors.

Example 3. The removal of a strain of Bacillus subtilis, capable of utilizing glycerophosphate as the sole carbon source.

The strain B. subtilis GM41 / pMX45 was obtained from the riboflavin producer B. subtilis GM51 / pMX45 as a spontaneous mutant capable of utilizing glycerophosphate (0.1%) as the sole carbon source. The strain B. subtilis GM41 / pMX45 was obtained by selection on Spitzizen's minimal nutrient medium containing glycerophosphate (0.1%) instead of glucose. The strain B. subtilis GM41 / pMX45 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 113545 Moscow, 1st Dorozhniy proezd, 1) in accordance with the Budapest Convention on September 17, 2002 under accession number VKPM B-8386.

The strain B. subtilis GM41 / pMX45 produced 8.8 g / l of riboflavin for 72 hours of fermentation in flasks and 18 g / l of riboflavin for 70 hours of fermentation in 1 l jar fermentors.

Example 4. The removal of the strain of Bacillus subtilis, resistant to glyoxylate.

The strain B. subtilis GM44 / pMX45 was obtained from strain B. subtilis GM41 / pMX45 as a spontaneous mutant resistant to glyoxylate at a concentration of 1 mg / ml. The strain B. subtilis GM44 / pMX45 was obtained by selection on Spitzizen minimal nutrient medium containing 1 mg / ml glyoxylate and glycerophosphate (0.1%) instead of glucose.

The strain B. subtilis GM44 / pMX45 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 113545 Moscow, 1st Dorozhniy proezd, 1) in accordance with the Budapest Convention on September 17, 2002 under accession number VKPM B-8387.

The strain B. subtilis GM44 / pMX45 produced 9.6 g / l of riboflavin for 72 hours of fermentation in flasks and 21 g / l of riboflavin for 70 hours of fermentation in 1 l jar fermentors.

Section: Fermentation Conditions

A seed culture (50 ml) was obtained by growing strains in flasks on a rotary shaker (200-220 rpm) for 18 hours at 37 ° C.

Sowing medium:

Sucrose 20.0 g / l Yeast autolysate 10.0 g / l MgSO ₄ × 6H ₂ O 1.0 g / l Erythromycin 10 γ / ml pH before sterilization 7.1-7.2

The production process of riboflavin was carried out on a 1 L Marubishi laboratory fermenter with a bioprocessor. The cultivation conditions were as follows:

stirring 1100 rpm, temperature 39 ° С, aeration 1: 1 (v / v), initial volume 300 ml, inoculation with inoculum 10% of the initial volume of the medium for fermentation, the pH was maintained at 7.0 ± 0.2 using 6% aqueous ammonia solution and 5% sulfuric acid. Mixtures of sugars were added during fermentation along with top dressing.

During fermentation, residual sugars (fructose, glucose, sucrose and maltose) were monitored. Usually, the feeding regimen was chosen so that the total concentration of sugars did not exceed 10 g / l.

The composition of the initial environment:

Sucrose 30.0 g / l Yeast powder 32.0 g / l Cereal extract 10.8 g / l KN ₂ PO ₄ 3.0 g / l K ₂ HPO ₄ 9.0 g / l MgSO ₄ × 7H ₂ O 0.6 g / l Urea 6.0 g / l

Top-up composition:

Molasses (type C) 227 g / l Syrup VHM 227 g / l Sucrose 419 g / l

Top dressing contains approximately 720 g / l of sugars (glucose, sucrose, maltose, etc.)

Cooking the initial environment

Nutrient yeast powder was sterilized in a boiling water bath for 40 minutes. 9.6 g of yeast powder was dissolved in 100 ml of sterilized water at 45-55 ° C. The procedure was carried out in sterile flasks. The suspension was kept in a boiling water bath for 40 minutes. All other components of the medium were sterilized in a standard way in an autoclave.

Initial Volume (AS) 300 ml Seed 30 ml Top dressing (0-120 h) about 170 ml Water (28-48 h) 90 ml Water ammonia 1: 1 about 20-30 ml Antifoam (20% solution in water) if necessary Final volume (theoretical) 620 ml (real) 550-570 ml Top dressing: 1.2 ml / h (7-24 h) 1.5 ml / h (25-72 h)

The duration of fermentation is up to 120 hours.

The amount of riboflavin produced was determined spectrophotometrically at 464 nm and confirmed by reverse phase HPLC (eluent 34% methanol).

Claims (4)

1. A method for producing riboflavin, comprising the steps of growing the bacterium Bacillus subtilis, a producer of riboflavin, and isolating riboflavin from the culture fluid, characterized in that the bacterium producing riboflavin uses the bacterium Bacillus subtilis, which has at least one of the following characteristics: contains rib operon from Bacillus amyloliquefaciens in the chromosome of this bacterium; has the ability to utilize glycerophosphate or possesses resistance to growth inhibition by glyoxylate. 2. The strain Bacillus subtilis GM51 / pMX45 containing the rib operon from Bacillus amyloliquefaciens on the rib chromosome is a producer of riboflavin. 3. The strain Bacillus subtilis GM41 / pMX45 (VKPM B-8386), with the ability to utilize glycerophosphate, is a producer of riboflavin. 4. The strain Bacillus subtilis GM44 / pMX45 (VKPM B-8387), which is resistant to growth inhibition by glyoxylate, is a producer of riboflavin.