Method for preparing riboflavin, strains bacillus subtilis as producers of riboflavin (variants)

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

 

The technical field

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

The level of technology

Riboflavin (vitamin b2) is an essential connection for higher animals, including humans. Lack of Riboflavin leads to various types of diseases, such as alopecia, inflammation of the skin and other damage, conjunctivitis, blurred vision, delayed growth and other.

Therefore, Riboflavin used to prepare commercial vitamin preparations used in the deficiency of vitamins, and also as a food additive. In addition, Riboflavin is used as a food dye, for example, mayonnaise, ice cream and other products.

Riboflavin get both chemical and microbiological methods. In chemical methods Riboflavin usually get as an end product of a complex multistage process, in which use relatively expensive source component, such as D-ribose.

Describes the obtaining 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). Also for production and Riboflavin suitable yeast such as Candida or Saccharomyces (WO 93/03183, JP63098399A2, US Pat. 4,794,081). It also describes the mushrooms of the genus Ashbya with increased activity isocitrate (ICL), with the ability to production and accumulation of Riboflavin (US Pat. 5,976,844).

Described mutants of Bacillus subtilis obtained by selection on the analogues of purines, azaguanine and azaxanthone, producing significant amounts of Riboflavin (U.S. Pat. No. 3,900,368). In General, processing analogues of purine and Riboflavin allows to obtain mutants with increased synthesis of Riboflavin, as these mutations allow the microorganism by increased production of Riboflavin analogs to displace 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 on the release of phosphoric acid from 5'-juaneloboy acid is not higher than 0.081 mmol/min/mg protein)capable of production of Riboflavin (EP 0531708 B1).

Mutant strains of Bacillus subtilis, electrovanne resistance to roseflower and 8-azaguanine and containing a mutation in the gene ribC, produce Riboflavin (patent of the USSR 908092). Such strains are strains of Bacillus subtilis Vniigenetika-304 and a. Next was derived strain of Bacillus subtilis 304/pMX45 containing a plasmid with the rib operon of Bacillus subtilis with a high potential for the production of Riboflavin (up to 3.5 g/l) (French patent 2546907).

Described strain of Bacillus subtilis 62/MX30ribO186, producing up to 12.5 g/l of Riboflavin within 42 hours of fermentation (RF patent 2081906). The strain of Bacillus subtilis 62/pMX30ribO186 was obtained from a strain of Bacillus subtilis RK6121 as a mutant resistant to 8-azaguanine, methanesulfonamido, diacetyl and picoturbine and optionally containing a plasmid with a mutant (mutation in the gene ribO) the rib operon of Bacillus subtilis.

The bacterial strains, obtained by introducing the genes of the biosynthesis of Riboflavin from Bacillus sublilis in the chromosome of the bacteria also produce Riboflavin (US Pat. 5,837,528, 5,925,538 and 6,322,995). The best known among strains is the strain RB50::[pRF69]60(Ade+)carrying transcriptional modified ariboflavinosis operon containing two promoters SPO1-15 was produced 13.0-14.0 g/l of Riboflavin within 48 hours and 15 g/l of Riboflavin within 56 hours (US Pat. 5,925,538) when grown on standard commercial environments and standard commercial terms.

Although the production of Riboflavin was significantly improved by removing the above-mentioned microorganisms or process improvement of its receipt, it is necessary to develop more efficient processes for the production of Riboflavin in order to meet the growing needs in this vitamin.

Description of the invention

The aim of the present invention is to increase the productivity of Riboflavin strains - producers of Riboflavin and providing JV is soba obtain Riboflavin using such strains.

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

Thus was accomplished the present invention.

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 a high potential for the production of Riboflavin, due to the presence of deregulated rib operon from Bacillus amyloliquefaciens, capacity for utilization of glycerol and/or mutations giving resistance to glyoxylate, being.

The present invention also provides a method of producing Riboflavin by fermentation, comprising the stage of growth of the above bacteria in a nutrient medium and extraction of Riboflavin from the culture fluid.

The present invention is as follows:

1. The bacterium Bacillus subtilis is capable of production of Riboflavin containing heterologous rib operon in the chromosome of the indicated bacteria, which specified the rib operon is rib operon from Bacillus amyloliquefaciens.

2. The bacterium Bacillus subtilis according to 1, which is pointed to by the second rib operon from Bacillus amyloliquefaciens deregulated.

3. The bacterium Bacillus subtilis. with the ability to production of Riboflavin and capacity for utilization of glycerol.

4. The bacterium Bacillus subtilis is capable of production of Riboflavin and resistance to growth inhibition by glyoxylate, being.

5. The bacterium Bacillus subtilis is capable of production of Riboflavin and possessing one or more of the following characteristics:

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

- has the ability to utilization of glycerol and

- resistant to growth inhibition by glyoxylate, being.

6. The method of obtaining Riboflavin, including the stage of growth of bacteria in accordance with 1-5 in a nutrient medium and extraction of Riboflavin from the culture fluid.

In more detail the present invention will be described below.

(1) the Bacterium according to the present invention.

The bacterium according to the present invention is the bacterium Bacillus sublilis, with capacity for production of Riboflavin, in which the production of Riboflavin is increased by introducing into the chromosome of the bacterium heterologous rib operon from Bacillus amyloliquefaciens.

Used here, the term "bacteria with the ability to produce Riboflavin" means a bacterium capable of production and accumulation in nutrient Riboflavin in the amount of bol is Shem, than the natural or parent strain .subtilis, such as strain .subtilis 168, and preferably means that the microorganism is able to produce and accumulate in the medium is 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, which belongs to the species Bacillus subtilis according to the classification known to a person skilled in the field of Microbiology. And the term "bacterium Bacillus amyloliquefaciens" means a bacterium, which belongs to the species Bacillus amyloliquefaciens in accordance with the classification known to a person skilled in the field of Microbiology.

The term "rib operon" means a fragment of a chromosome that contains genes encoding proteins essential for the production of Riboflavin, rib operon in bacteria belonging to the genus Bacillus contains the following genes: gene ribO, encoding a regulatory element, ribG gene encoding deaminase/reductase, ribB gene encoding ariboflavinosis (α-subunit), ribA gene encoding GTP-cyclohydrolase/3,4-dihydroxy-2-butanone-4-fosfolinazou, ribH gene encoding limosininae, and gene ribT, encoding a protein with unknown function (Morozov and others, they Say. Genet. Mick. Virusol., 7:42, 1948). Nucleotide sequence of the genes ribG, ribB, ribA, ribH and ribH from Bacillus subtilis submitted to GenBank under the numbers gi:16079385, gi:16079384, gi:16079383, gi:16079382 and gi:16079381, with the NGOs (nucleotides with 2429492 on 2430577, with 2428834 on 2429481, with 2427623 on 2428819, with 2427126 on 2427590 and 2426639 on 2427013 in the sequence NC_000964.1).

The term "heterologous operon", used here, this means that the operon was isolated from a chromosome of an organism other than Bacillus sublilis. Bacteria containing heterologous operon in the chromosome can be obtained by standard methods recombinantly DNA, transformation, transfection.

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

Deregulation of the regulatory system for the synthesis of Riboflavin can be done by changing the regulatory section of the operon, replace the specified regulatory area other strong plot with a constitutive level of expression, inactivation of the gene encoding the repressor, obtaining mutations in the protein - repressor, 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 what mutagenesis, the destruction of the gene using homologous recombination and/or insertion-deletion mutagenesis.

The term "capacity for utilization of glycerol" means the ability to metabolize glycerol is more effective than the parent strain, for example, the ability of the strain .subtilis used in the present invention, to grow faster than the parent strain, when these strains are grown on medium containing glycerol as the sole carbon source. More specifically this means that the strain .sublilis has the ability to utilization of glycerol, if this strain grows faster than the parent strain, when these strains are grown in suitable conditions on a medium containing glycerol as the sole carbon source, for example, in a liquid medium containing 0.1% glycerol. More specifically, we can say that the strain of B. subtilis has the ability to utilization of glycerol, if this strain forms a colony for 2 days at 37°when growing this strain on agar medium containing glycerol as the sole carbon source, for example, on a medium containing 0.1% glycerol and agar, in suitable conditions. The term "appropriate conditions" refers to temperature, pH, oxygen Il the optional presence of essential nutrients and other conditions, necessary for the cultivation of strain .subtilis.

It is known that enzymes glyconeogenesis have the level of physical activity, insufficient for recycling products of glycolysis, such as glycerophosphate. It is also known that strains .subtilis grow very poorly on media containing glycerol as the sole carbon source. On this basis it has been suggested that the selection of mutants able to grow on medium containing glycerol as the sole carbon source, will allow to obtain mutants with increased level of activity of enzymes involved in glyconeogenesis, and, consequently, to increase the production of Riboflavin in the course of the fermentation process on the sucrose.

The term "bacterium is resistant to growth inhibition by glyoxylate, being" means the bacterium obtained from the parent strain by genetic modification of the properties in such a way that the strain is able to grow on a nutrient medium containing glyoxylate, being. Specifically, resistance to glyoxylate, being refers to the ability of bacteria to grow on minimal medium containing glyoxylate, being at a concentration at which the parent strain specified bacteria can't grow or the ability of bacteria to grow faster on a nutrient medium containing glyoxylate, being than the natural or parent strain of the decree is Noah bacteria. For example, a bacterium which is able to form colonies when grown for 3-5 days at 34°on cups with agar medium containing 0.5 mg/ml or more, preferably 1.0 mg/ml or more glyoxylate is resistant to glyoxylate, being.

Glyoxylate, being is a very important intermediate connection glyoxylate shunt required for growth on these carbon sources, as acetate or fatty acids as specified path allows directly convert acetyl-COA into the intermediate products of metabolism. The specified path contains three enzyme. Two of them, isocitrate and palatinase And make some intermediates of the tricarboxylic acid cycle from isocitrate in malate. It was shown that, in contrast to E. coli, the enzyme activity glyoxylate shunt in .subtilis very low. In addition, glyoxylate, being inhibited the growth of natural cells .subtilis at a concentration of 1 mg/ml To increase the activity of enzymes glyoxylate shunt was removed to obtain mutants that are resistant to glyoxylate, being.

The bacterium according to the present invention can be obtained from bacteria, have the ability to the production of Riboflavin by giving it stability and/or ability to dispose of glycerol. In addition, the bacterium according to the present invention can be obtained by giving FPIC is the competitiveness of the products of Riboflavin bacteria, already having a specific resistance and/or capacity for utilization of glycerol.

Thus, any known strain of bacteria belonging to the genus Bacillus having the ability to produce Riboflavin, can be subjected to one or more of the above mutagenic treatments, and then checks the resulting mutant strains for compliance with the above requirements of the present invention, which includes the utilization of glycerol and resistance to glyoxylate, being, and, therefore, suitable for use in the present invention. The resulting strains were tested by growing in a nutrient medium, and the strains with the capacity for the production of Riboflavin with output greater than the parent strain, selected and used in the present invention.

As the parent strains which can be improved with the aim of obtaining bacteria according to the present invention, can be used strains of bacteria belonging to the genus Bacillus, the producers of Riboflavin, such as strains of Bacillus subtilis Vniigenetika-304 and a (patent of the USSR 908092), the Bacillus subtilis strain 304/RMH (CMPM IN-2694) (French patent 2546907), a strain of Bacillus subtilis 62/pMX30riO186 (VKPM B-6797) (RF patent 2081906), the Bacillus subtilis strain RB50::[pRF69]60(Ade+) (U.S. patent 5,925,538), a strain of Bacillus subtilis FERM BP-3855 and FERM BP-3855 (European patent EP W) and the like.

The method according to the present invention is a method of obtaining Riboflavin, including the stage of growth of bacteria according to the present invention in a nutrient medium and extraction of Riboflavin from the culture fluid.

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

The nutrient medium for production of Riboflavin can be both synthetic and natural, provided that it contains carbon sources, nitrogen, inorganic ions and other organic components necessary. As a carbon source can be used sugars such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose and starch hydrolysis; alcohols, such as glycerol, mannitol and sorbitol; and organic acids such as gluconic, fumaric, citric and succinic acid, and similar compounds. As the nitrogen source can be used inorganic salts of nitrogen, such as ammonium sulfate, ammonium chloride and ammonium phosphate; organic compounds of nitrogen such as soybean hydrolysate; ammonia gas; wagnerstr ammonia and similar compounds. It is desirable that vitamins, such as vitamin B1other compounds, for example, nucleic acids such as adenine and RNA, or yeast extract, and these compounds were present in suitable amounts as organic nutritional supplements. In addition, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese, and such compounds may be added, if necessary.

Cultivation carried out preferably under aerobic conditions from 16 to 72 hours, and the temperature during the growing support within 30 to 45°With pH ranging from 5 to 8. pH support using inorganic and organic acidic and basic compounds, and ammonia gas.

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

The best way of carrying out the invention

Example 1. Construction of Bacillus subtilis strain containing ariboflavinosis operon from Bacillus amyloliquefaciens in the chromosome.

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

In order to clone the genes of the biosynthesis of Riboflavin, chromosomal DNA of strain .amyloliquefaciens A50 was processed restricts the th HindIII. The resulting mixture of DNA fragments was Legerova in the vector pACYC184 (NBL Gene Sciences Ltd., UK, GenBank/EMBL accession number X06403), pre-treated with the same restriction enzyme. The resulting product was used to transform E. coli strain BSV821. Were selected transformants capable of growth on a nutrient medium containing chloramphenicol. From these transformants was isolated plasmid and the fragment containing the rib operon was periglomerular in vector RSV. The obtained plasmid was named rvh. Plasmid rvh has complementable mutations in all genes of the rib operon, except for genes ribTD.

To derive mutants with constitutive expression of genes of the biosynthesis of Riboflavin plasmid rvh was treated with hydroxylamine in standard mutagenesis. Laboratory auxotrophic strain .subtilis ribG850 was transformed mutant DNA plasmid and assessed the ability of the obtained transformants to produce Riboflavin. Using PCR, it was found that the production of Riboflavin was caused by mutations in the gene ribO. Mutation ribO1 (replacing T With position +87) was identical to known mutations in the operon .subtilis. Mutation ribO2 was a new mutation With a T at position +149. A plasmid containing the mutation ribO2, was named rvh. Strain .subtilis ribG850 transformed by the plasmid rvh produced to 65 mg/l of Riboflavin, despite the fact that the same strain, rasformirovaniya the plasmid rvh, did not produce Riboflavin.

Besplatniy strain of Bacillus subtilis Vniigenetika-304 (a patent of the USSR 908092) was used as the parent strain to construct a strain of Bacillus subtilis GM49 containing chromosome deregulated ariboflavinosis operon from Bacillus amyloliquefaciens. For integration deregulated Riboflavinum operon from Bacillus amyloliquefaciens into the chromosome of Bacillus subtilis strain 304 was designed vector for integration REC (KmRApRRib+). Vector RC consists of the E. coli plasmid pUC19, gene kanamycin nucleotidyltransferase (kan) from plasmid pUB110 and deregulated (mutation ribO2) Riboflavinum operon of Bacillus amyloliquefaciens of plasmids rvh. Plasmid REC not capable of replication in .subtilis.

For integration REC in the chromosome was used the method of "random integration.

First, a BamHI-fragment of plasmid REC was Legerova with BamHI-fragments of chromosomal DNA from strain .subtilis 168. A plasmid containing areas homologous to the genome .subtilis, was able to integrate into the chromosome by homologous recombination. Then, obtained after ligation, the mixture of fragments was used for transformation laboratory auxotrophic strain .subtilis rib G850. Transformants were selected by phenotype KmRRib+. Transformation frequency was about 100 to 1 γ DNA. Analyzed 60 clones with what enation Km RRib+.The majority of integrants was unstable or auxotrophic. They lost tokens KmRand Rib+in the process of growth in a nutrient medium without antibiotics. We have found one stable integrative clone, named VK. Strain .subtilis BK53 ribG850 aab::REC (RibBam+KmR) contained ariboflavinosis operon from .amyloliquefaciens (RibBam+) and the gene for resistance to kanamycin KmR(kan), integrated into the chromosome .subtilis in locus, called "aab".

Integrated operon .amyloliquefaciens was transferred into the recipient chromosome besplodnogo strain .subtilis 304 - producer of Riboflavin from a strain of B. subtilis BK53 ribG850 of AAB::REC (PibBam+KmRusing the method of transduction using phage R9. The gene for resistance to kanamycin, integrated into the chromosome .subtilis together with Riboflavinum the operon from .amyloliquefaciens, was used as a selection marker. The resulting strain was named GM49. The specified strain GM49 aab::REC (RibBam+KmRcontains two Riboflavin operon in the chromosome: own operon and operon from .amyloliquefaciens.

The ability of strain .subtilis GM49 to the production of Riboflavin was tested in flasks with culture medium described below in the section "Conditions of fermentation". The process of fermentation (batch fermentation) were carried out in flasks with a capacity of 850 ml Volume was 150 ml Strain GM49 produced 3 g/l RIBO the avalanche 72 hours.

Example 2. Construction of Bacillus subtilis strain containing both ariboflavinosis operon from .amyloliquefaciens in the chromosome, and ariboflavinosis operon of B. subtilis on the plasmid.

Strain .subtilis GM51/pMX45 was obtained from besplodnogo strain .subtilis GM49 - producer of Riboflavin.

First was the resulting strain, deficient in the recombination system.

To obtain strain GM51 with insertional mutation recE::cat in the recE gene was used plasmid rut. Plasmid RVT, a derivative of plasmid RVT containing recE gene from Bacillus subtilis (Gassel, M. and Alonso J.C., "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 gene for resistance to chloramphenicol (cat), encoding chloramphenicol acetyltransferase. Plasmid RWT was integrated into the recE gene in the areas of recognition of restrictase ClaI (recE::cat), present in the gene. Mutant gene recE::cat was integrated into the chromosome of strain .subtilis GM49 recombination by double crossing-over. Thus was obtained a strain of B. subtilis GM51 (aab::REC recE::cat (RibBam+KmRCmR)).

Then deficient in the recombination system (recE::cat) CmRstrain .subtilis GM51 was transformed with plasmid RMH containing ariboflavinosis operon from strain .subtilis ribO335. The indicated plasmid RMH contains a large (10 kb) DNA fragment from chromosome .subtilis. Plasmid RMH is miscompiles the plasmid (1-2 copies of naklejka). Plasmid RMH described in detail in the French patent 2546907.

Strain GM51/pMX45 produced 5.8 g/l of Riboflavin within 72 hours of fermentation in flasks and 15 g/l of Riboflavin in over 70 hours of fermentation in the fermenter (jar fermentors) volume 1 liter

Example 3. Excretion of Bacillus subtilis strain capable of utilizing glycerol as the sole carbon source.

Strain .subtilis GM41/pMX45 was obtained from ariboflavinosis producer .subtilis GM51/pMX45 as a spontaneous mutant, capable of utilizing glycerol (0.1%) as the sole carbon source. Strain .subtilis GM41/pMX45 was obtained by selection on minimal nutrient Spizizen containing glycerol (0.1%) instead of glucose. Strain .subtilis GM41/pMX45 was deposited in the Russian national Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1st Road travel, 1) in accordance with the Budapest Convention on September 17, 2002 under inventory number VKPM B-8386.

Strain .subtilis GM41/pMX45 produced 8.8 g/l of Riboflavin within 72 hours of fermentation in flasks and 18 g/l of Riboflavin in over 70 hours of fermentation in the fermenter (jar fermentors) volume 1 liter

Example 4. Excretion of Bacillus subtilis strain resistant to glyoxylate, being.

Strain .subtilis GM44/pMX45 was obtained from strain .subtilis GM41/pMX45 as a spontaneous mutant resistant to glyoxylate, being in a concentration of 1 mg/ml Strain .subtilis GM44/pMX45 was received Selecci the th minimum nutrient Spizizen, containing 1 mg/ml glyoxylate and glycerol (0.1%) instead of glucose.

Strain .subtilis GM44/pMX45 was deposited in the Russian national Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1st Road travel, 1) in accordance with the Budapest Convention on September 17, 2002 under inventory number VKPM B-8387.

Strain .subtilis GM44/pMX45 produced 9.6 g/l of Riboflavin within 72 hours of fermentation in flasks and 21 g/l of Riboflavin in over 70 hours of fermentation in the fermenter (jar fermentors) volume 1 liter

Section: the conditions of fermentation

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

Sowing environment:

Sucrose20.0 g/l
Yeast autolysate10.0 g/l
MgSO4×6H2O1.0 g/l
Erythromycin10 γ/ml
pH before sterilization7.1-7.2

Process of production of Riboflavin was conducted on a laboratory fermenter Marubishi capacity of 1 l with the bioprocessor. Cultivation conditions were as follows:

mixing 1100 rpm, temperature 39°, aeration 1:1 (v/v), the initial volume of 300 ml, inoculation seeds - 10% of the initial volume of medium for fermentation, pH supports rival at 7.0± 0.2 using a 6% aqueous solution of ammonia and 5% sulfuric acid. A mixture of sugars added during fermentation together with dressing.

In the course of the fermentation was controlled by the residual concentration of sugars (fructose, glucose, sucrose and maltose). Typically, the mode of feeding was chosen so that the total concentration of sugars did not exceed 10 g/L.

The composition of the initial environment:

Sucrose30.0 g/l
Yeast powder32.0 g/l
Grain extract10.8 g/l
KN2PO43.0 g/l
To2HPO49.0 g/l
MgSO4×7H2O0.6 g/l
Urea6.0 g/l

The composition of the feed:

Molasses (type C)227 g/l
Syrup VHM227 g/l
Sucrose419 g/l

Dressing contains about 720 g/l of sugars (glucose, sucrose, maltose, and others)

Preparation of the initial environment

Yeast powder for nutrient medium were 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 fool was performed 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 the autoclave.

The initial volume (AS)300 ml
Seed30 ml
Dressing (0-120 h)about 170 ml
Water (28-48 h)90 ml
Aqueous ammonia 1:1about 20-30 ml
Antifoam (20% solution in water)if you want
The final volume (theoretical)620 ml
(real)550-570 ml
Dressing:1.2 ml/h (7-24 h)
1.5 ml/h (25-72 h)

The duration of fermentation (up to 120 hours.

The number of produced Riboflavin was determined spectrophotometrically at 464 nm and confirmed by reversed-phase HPLC (eluent - 34% methanol).

1. The method of obtaining Riboflavin, including the stage of growth of the bacteria Bacillus subtilis producer Riboflavin and extraction of Riboflavin from the culture fluid, characterized in that as the bacteria - producers of Riboflavin using the bacterium Bacillus subtilis, with at least one of the following characteristics:

content is t rib operon from Bacillus amyloliquefaciens in the chromosome of the bacterium;

has the ability to utilization of glycerol or

resistant to growth inhibition by glyoxylate, being.

2. The strain of Bacillus subtilis GM51/pMX45 containing chromosome rib operon from Bacillus amyloliquefaciens, producing Riboflavin.

3. The strain of Bacillus subtilis GM41/pMX45 (VKPM B-8386)with the capacity for utilization of glycerol, producing Riboflavin.

4. The strain of Bacillus subtilis GM44/pMX45 (VKPM B-8387), resistant to growth inhibition by glyoxylate, being, producing Riboflavin.



 

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6 cl, 60 dwg, 5 ex

FIELD: microbiology, biotechnology, chemical industry.

SUBSTANCE: invention relates to strain Klebsiella pneumoniae containing conjugative pBK1 plasmid, which determines resistance of this microorganism community to arsenic compounds.

EFFECT: strain for production of bacterium strains useful as arsenic-containing compound biological destructors.

FIELD: biotechnology, animal breeding, in particular probiotic production.

SUBSTANCE: claimed method includes production of nutrient medium, its fermentation with strain consortium of Clostridium cellobioparum, Ruminococcus flavefaciens, Lactobacillus acidophilus, Propionibacterium acnes and collection of biomass. Said biomass is mixed with protective medium and dried.

EFFECT: cow milk productivity.

2 cl, 1 tbl, 3 ex

FIELD: biotechnology, food processing and medicine industry , in particular production of bacterium preparations.

SUBSTANCE: Strain Bifidobacterium bifidum is obtained by selection without using of genetic modification methods and is capable to utilize of inulin and cellobiose. Strain accumulates biomass on plant-origin substrates and artificial broth for short period with high bifidobacterium concentration. Strain has acid-forming properties and represents antagonist of pathogenic and saprogenic microflora. Bacterium preparations, bioactive food supplements, ferments, fermented milk products, fermented and non-fermented foodstuffs, hygienic and cosmetic products containing the same have probiotic effect and provide normalization of microbiocenose in human organism, including gastrointestinal and urogenital tracts, skin integument and mucilaginous.

EFFECT: new strain Bifidobacterium bifidum with probiotic and acid-forming properties.

2 tbl, 5 ex

FIELD: biotechnology, in particular food processing, dairy and medicine industry.

SUBSTANCE: claimed method includes broth preparation, sterilization and cooling. In prepared broth probiotic strain together with two non-lysogenic strains of thermophylic streptococcus in ratio 4:1 and 3:2, respectively, are cultivated. Mass of each probiotic strain Streptococcus salivarius subsp. Thermophilus is built up separately from one another. Then strains are mixed in equal amounts, pre-packed, dried and packed.

EFFECT: increased assortment of probiotic agents with improved properties.

4 cl, 2 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: claimed method includes bacteria cultivation of genus Bacillus in broth followed by isolation of collected inosine from cultural liquid. As producer bacteria with resistance to growth inhibition with polymyxin B and/or colistin is used. Also disclosed are method for production of inosine-5'-monophosphate and two new strains of inosine producers: Bacillus subtilis and Bacillus amyloliquefaciens.

EFFECT: inosine production with increased yield.

8 cl, 4 tbl, 3 ex

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention proposes the strain Enterobacter agglomerans/FERM BP-7207 that is able to metabolize the carbon source at pH value when L-glutamic acid precipitates in liquid cultural medium containing L-glutamic acid in the saturation concentration and the carbon source. Also, the strain is able to accumulate L-glutamic acid in the amount exceeding the amount that corresponds to its saturation concentration in liquid cultural medium at this pH value. Also, invention relates to a method for preparing L-glutamic acid by fermentation that involves culturing this microorganism in liquid cultural medium wherein pH value is brought about to the value when L-glutamic acid precipitates to prepare and accumulate L-glutamic acid and to precipitate L-glutamic acid in this cultural medium. The proposed microorganism is prepared by addition of a sample comprising microorganisms to acid cultural medium containing L-glutamic acid in the saturation concentration and the carbon source followed by removal of strain that is able to metabolize this carbon source. Invention provides preparing L-glutamic acid with the high degree of effectiveness.

EFFECT: improved preparing method, valuable properties of strain.

20 cl, 9 dwg, 3 tbl, 8 ex

FIELD: biotechnology, in particular method for construction and production of mutant transglutaminases (MTG).

SUBSTANCE: invention relates to method for construction and production of mutant transglutaminases based on space structure of transglutaminase obtained from Streptoverticillium mobaraense, as well as to mutant MTG obtained by said method. Also disclosed are method for MTG modification based on space structure and modified transglutaminase with enhanced reactivity relative to substrate. Method of present invention makes it possible to predict MTG binding site to substrate on the base of space structure that is determined by MNG crystal X-ray analysis, and to design mutant transglutaminases by replacement, insertion or deletion of amino acid residues disposed on transglutaminase substrate-binding site.

EFFECT: new method for production and modification of mutant transglutaminases.

6 cl, 60 dwg, 5 ex

FIELD: genetic engineering, in particular production of human granulocyte colony-stimulating factor.

SUBSTANCE: Recombinant plasmid DNA pES3-7 with molecular weight of 3.63 MDa (5907 b.p.) is constructed. Said DNA consists DNA Ndel/Notl-fragment containing sequence of recombinant G-CSF artificial gene, β-lactamase gene; and plasmid pET22b(+) DNA Ndel/Notl-fragment containing promoter and terminator of T-RNA-polymerase transcription, amplifier of 17 phage 10 gene translation. Plasmid pES3-7 contains as genetic marker β-lactamase gene which determines resistance of E.coli cells transformed with plasmid pES3-7 to ampicillin, and unique restriction endonuclease recognition sites existing on the next distance to the right from Ndel-site: Xbal - 38 b.p.; Hpal - 1332 b.p.; Pstl - 4065 b.p.; Pvul - 4190 b.p.; Xhol - 5363 b.p. Obtained plasmid is used in transformation of Escherichia coli cells to produce strain E.coli BL21(DE3)/pES3-7 as subproducer of recombinant G-CSF. Method of present invention makes in possible to produce recombinant G-CSF with high yield (20-30 % based on total cell protein content).

EFFECT: simplified method for production of recombinant G-CSF with high yield.

2 cl, 2 dwg, 2 ex

FIELD: genetic engineering, molecular biology, biochemistry.

SUBSTANCE: method involves construction of recombinant plasmid DNA pES4-4 with molecular mass 3.64 Mda (5917 base pairs) and consisting of Nde I/Bam HI-fragment of DNA comprising sequence of artificial gene of recombinant human interferon α-2b, β-lactamase gene and Nde I/Bam HI-fragment of plasmid pET22b(+)DNA comprising promoter and transcription terminator of T7-RNA polymerase, translation enhancer of phage T7 gene 10. Plasmid pES4-4 comprises β-lactamase as a genetic marker determining resistance of E. coli cells transformed with this plasmid to ampicillin, and unique recognition sites for endonuclease restriction enzymes located in the following distance to the right from site Nde I: Xba I - 38 base pairs; Hpa I - 1332 base pairs; Pst I - 4065 base pairs; Pvu I - 4190 base pairs, and Xho I - 5363 base pairs. Escherichia coli cells are transformed with prepared plasmid and the strain E. coli BDEES4 is obtained that is a super producer of recombinant human interferon α-2b. Invention provides preparing recombinant human interferon α-2b with high yield and by simplified technology. Invention can be used for preparing recombinant human interferon α-2b.

EFFECT: improved preparing method.

3 cl, 2 dwg, 2 ex

FIELD: genetic engineering, molecular biology, biochemistry.

SUBSTANCE: recombinant plasmid DNA pTES-His-OPH is constructed for expression of polypeptide eliciting properties of organophosphate hydrolase comprising Cla I/Hind III fragment of plasmid pTrcTEGF, fragment of plasmid pTES-OPH with nucleotide sequence that encodes amino acid sequence of the matured form of organophosphate hydrolase, and nucleotide sequence encoding 6 histidine residues that is located by 5'-end of nucleotide sequence encoding organophosphate hydrolase. Based on indicated plasmid the strain Escherichia coli TSKMIBKH-29 - a producer of polypeptide eliciting properties of organophosphate hydrolase is obtained. Applying the invention provides preparing polypeptide with properties of organophosphate hydrolase by simplified technology and this polypeptide elicits the improved catalytic effectiveness of action with respect to thio-containing phosphoric acid triesters. Invention can be used for carrying out hydrolysis of organophosphate compounds.

EFFECT: valuable biochemical properties of producer.

2 cl, 4 dwg, 2 tbl, 4 ex

FIELD: biochemistry, gene engineering, in particular amidation of hormones and other peptides used in medicine and agriculture.

SUBSTANCE: peptide sequence obtained in process of triptic digesting of peptidylglycine alpha-amidating monooxygenase (PAM) from fat thyroid encephaloid carcinoma is determined. On the base of obtained data oligonucleotide sequences are synthesized and used for isolation of full-sized PAM encoding DNA. Expression of obtained DNA fragments in heterogeneous systems makes it possible to obtain recombinant PAM useful in amidation reaction of peptide substrate with C-terminated glycine providing the same effect that for native enzyme.

EFFECT: effective method for production of amidated peptides.

12 dwg, 2 tbl, 9 ex

FIELD: biotechnology, in particular virulent genes and proteins.

SUBSTANCE: peptide with Neisseria meningitidis antigen activity and polynucleotide encoding the same are used in preparation of drug for prevention or treatment of diseases and conditions associated with Neisseria or gram-positive infections. Antibodies is obtained by using disclosed peptide. Vaccines for prevention or treatment of diseases and conditions associated with Neisseria meningitides contain attenuated mutant strain of Neisseria meningitides having gene mutation, insertion or deletion which disturbs expression of certain nucleotide sequence.

EFFECT: method for prevention or treatment of Neisseria infection with increased effectiveness.

13 cl, 1 tbl

FIELD: gene and protein engineering for various luminescent assays.

SUBSTANCE: new luciferase mutant forms have been obtained. Said mutant forms have increased thermostability and optionally different emission wave length in contrast with respective wild type enzymes. In all disclosed muteins natural amino acid residue in position equivalent to 357-position in Photinus pyralis luciferase sequence is replaced with other residue, preferably with uncharged polar amino acid (in particular tyrosine) residue. Mutant luciferases of present invention are useful in various analytical systems as reporter agent.

EFFECT: Mutant luciferases with new properties.

22 cl, 15 dwg, 6 tbl, 12 ex

FIELD: biotechnology, in particular provision storage.

SUBSTANCE: bacteriocin represents polypeptide isolated from lactobacillus sakei 2512 and is capable to suppress lysteria growth and reproducing. Bactericin has specific amino acid sequence represented in claims. Nuclear acid sequence encoding said polypeptide is disclosed. Also disclosed is a vector including nuclear acid sequence for cloning and/or expression of polypeptide, for example in transformed cells, selected from Lactococcus, Lactobacillus, etc. Method for production of recombinant polypeptide is developed. Claimed bactericin or strain 2512 are used as component of bactericide composition, being capable to suppress growth of gram positive pathogenic bacteria, in particular Listeria monocytogenes.

EFFECT: large scale application of bacteriocin against pathogenic or undesired flora in food industry.

13 cl, 2 dwg, 1 tbl, 3 ex

FIELD: biotechnology, in particular method for production of L-amino acid except L-glutamic acid.

SUBSTANCE: claimed method includes cultivation of bacteria Methylophilus, which is capable to grow utilizing methanol as a main carbon source and to produce L-amino acid; and collection L-amino acid from culture. For example, bacteria Methylophilus with increased activity of dihydrodipicolinate synthase and aspartokinase is used. Said activity is increased by introducing DNA encoding dihydrodipicolinate synthase which is not inhibited with L-lysine by negative back coupling, and DNA encoding aspartokinase which is not inhibited with L-lysine by negative back coupling, into cells.

EFFECT: increased yield of L-amino acids.

10 cl, 7 dwg, 6 tbl, 7 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing inosine and 5'-inosinic acid that are prepared by using microorganism Escherichia coli. Production of inosine by indicated microorganisms is elevated due to the enhanced activity of protein encoded by gene yijE. Invention provides increasing yield of inosine and 5'-inosinic acid.

EFFECT: improved preparing method, valuable properties of strain.

8 cl, 3 dwg, 2 tbl, 3 ex

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