The method of obtaining vysokolegirovannyh of nucleosides and nucleotides

 

(57) Abstract:

The use of biotechnology. The inventive method of obtaining vysokolegirovannyh of nucleosides and nucleotides, namely, that cultivated strains-producers of nucleosides and nucleotides of the species Bacillus subtilis and species of Bacillus amyloliquefaciens in fully deuterated medium (D2O). As a fully deuterated environment using hydrolysates produced with deuterium oxide and watermethanol biomass producer strain nuke, after separation of the target product. For the production of D-labeled inosine use strain Vila Bacillus subtilis, and for the production of D-labeled thymidine use a strain of the species Bacillus amyloliquefaciens. The method allows to obtain vysokodetalizarovannye nucleosides, with 62.5 percent of the hydrogen atoms in the molecule are replaced by deuterium. B. subtilis and B. amyloliquefaciens are able to produce and secrete in a fermentation medium inosine and thymidine. The method allows to simplify the procedure for obtaining deuterium-labeled nucleosides, reducing it to a single fermentation, and to make the process of obtaining the final products more environmentally friendly by eliminating the number of reactions with the use of harmful substances and radioactive from the get vysokolegirovannyh of nucleosides and nucleotides.

Nucleosides and nucleotides are widely used in medicine for treatment of diseases associated with abnormal activity of the heart, liver, and cellular metabolism. So, the drug Riboxin, created on the basis of the drug inosine, has a stimulating effect on the redox processes in the cell, improves the energy balance (EUR. Pat. N 0 617 423, Europe. Pat. N 0 423 618, GDR patent N 296 687).

Datetimezone analogues of these compounds required for various biochemical, pharmacokinetic and other studies, in particular for structural and functional studies of nucleic acids by NMR. For these purposes using drugs of nucleosides and nucleotides, in which at least half of the hydrogen atoms in the molecule must be replaced by deuterium. Because vysokodetalizarovannye nucleosides and nucleotides used in obtaining labeled diagnostic pharmaceuticals (AMP, ATP and others), the development of the most economical ways of obtaining these compounds in preparative quantities is a very important task.

To date, labeled with stable isotopes nucleosides and nucleotides receive the receipt of such compounds include method based on the hydrolysis and chemical transformation of the components of DNA biomass of microalgae, for example, Chlorela vulgaris grown in liquid media with heavy water level deuteranomaly to 9.9% (Application N 62-6793, Japan, Europe. Pat. N 0 220 951). Because the presence of high concentrations of deuterium oxide in the medium inhibits the growth and development of most types of microorganisms, the concentration of target products in a biological material is very low.

With the further development of the species of biotechnological approaches have the opportunity to improve the standard methods to obtain the labeled nucleosides and nucleotides, but these methods were used to obtain labeled with radioactive tritium compounds. So, in the patent literature describes a chemical-enzymatic methods of obtaining trailrunner nucleosides using extracts of cells and the cells of the microorganisms that possess enzymatic activity (Ed. mon. Czechoslovakia N 267 846).

In a number of patents have reported methods to obtain site-specific labeled with tritium of polyethoxylated (proc. application N 89/09780). Features of existing methods consist in the fact that obtaining these compounds are almost always produced in discordianism tritium in water in the presence of immobilized polyactides gel enzyme preparations, secreted from bacteria, for example, Esherichia alcalescens, etc.

Worth mentioning are also methods of obtaining nucleotides labeled different from non-radioactive tritium deuterium labels (proc. application N 92/00989, proc. application N 91/13902, GDR patent N 295 855). According to these patented methods enable labels in ribonucleoside does not exceed 40% and thus the desired products do not meet the requirements on the content label.

The main strategy of the biosynthesis of deuterium-labeled products is used for biotechnological purposes not conventional producers, and pre-adapted to high concentrations of the desired isotope forms of microorganisms.

The present invention allows to simplify the procedure for obtaining vysokolegirovannyh nucleosides, in particular inosine and thymidine.

The process of obtaining vysokolegirovannyh of nucleosides and nucleotides is that producing strains nucleotide of the genus Bacillus cultivated in fully deuterated environment.

As a fully deuterated components of the environment can be used hydrolysates produced with deuterium oxide and watermethanol biomass methylotrophic bacteria Brevibacterium met the ssy producer strain, grown on deuterated environment.

Strains producing nucleoside or nucleotide are special genetically-engineered strains that are able to produce and secrete nucleosides and nucleotides, in a fermentation medium.

The cultivation of these strains in fully deuterated environment leads to the formation of vysokolegirovannyh of nucleosides and nucleotides.

So, for example, to obtain vysokomehanizirovannogo inosine can be used a strain of the species Bacillus subtilis, and to obtain labeled thymidine - strain of the species Bacillus amyloliguefaciens,

The advantage of the proposed method of obtaining vysokolegirovannyh nucleosides is that genetically engineered strains of B. subtilis and B. amyloliquefaciens are able to grow on media containing the maximum concentration of deuterium oxide.

The method allows to replace glucose and essential amino acids in the hydrolysates deuterium-labeled biomass methylotrophic bacteria Brevibacterium methylicum or hydrolysates deuterium-labeled biomass of Bacillus subtilis, contain compounds that can serve as carbon sources and growth factors.

The method is characterized OTS is the super hydrolyzed in DCl, is returned to the cycle as growth factors.

The method is also characterized by a high degree of isotopic enrichment of deuterium-labeled nucleosides (62,5% of the hydrogen atoms in the molecule are replaced by deuterium) and high yields of the target products.

Example 1.

Preparing liquid sowing medium of the following composition, wt.%:

Glucose technical - 2,0;

The source of growth factors - protein-vitamin concentrate (PVC) - 1,0;

Peptone - 1,0,

NaCl - 0,25,

Heavy water is 100.

Seed grown in flasks with a capacity of 250 ml containing 20 ml of medium in a rocking chair, giving 240 - 260 rpm at 34oC within 24 hours of Seeding material in the amount of 4% is transferred into a fermentation medium of the following composition, wt.%:

Glucose technical - 12,0;

The source of growth factors avtorizovannaja at 0.8 ATM (D2O) deuterium-labeled biomass methylotrophic bacteria - 2,5,

NH4NO3to 3.0,

MgSO4- 0,5,

Chalk - 2,0,

Heavy water is 100.

Bring the pH of the medium to 7.0 to 7.2 with 2N KOH solution (in D2O) before sterilization and sterile 0.8 ATM for 30 minutes

Cells of B. subtilis N B3157 seeded in title 2107cells. Jr. the Process f is the same as the rate of dissolution of oxygen 4,0-O2l/h at 34oC. After 7 days fermentation culture liquid accumulates 8,2 g/l of inosine, not less than 60% hydrogen (C-H-bond), which are replaced by deuterium (data of NMR and MS).

Upon completion of the fermentation the cells are precipitated by centrifugation (5000 rpm, 20 min), the culture fluid containing the deuterium-labeled inosine evaporated to a volume of two times smaller than the original. Protein impurities are removed, precipitating their 2-fold excess of acetone to remove the acetone by evaporation in vacuum at 10 mm RT.article Clarification of the culture fluid conducting adsorption on 5 g of activated charcoal. Then the clarified culture fluid evaporated in vacuum at 10 mm RT.article to the dry residue. The inosine in the form of crystals emit, precipitating it from a saturated methanol.

Example 2.

Culture, the composition of the agar medium, the amount of inoculum and fermentation condition and the selection of inosine from the culture fluid are the same as in example 1. The difference is in the composition of the fermentation medium, namely deuterium-labeled biomass methylotrophic bacteria replaced by biomass of the producer strain B. subtilis obtained after fermentation on the environments, the maximum saturated with deuterium oxide. Output eno environment, the amount of inoculum and fermentation conditions are the same as in example 1. The difference is used as a producer of thymidine strain of Bacillus amyloliquefaciens N B6843. When the cultivation of the strain B. amyloliquefaciens in this environment is formed 3.0 g/l of thymidine labeled with deuterium, with the content of D is not less than 60%.

Thus, the claimed method allows you to:

to simplify the procedure for obtaining deuterium-labeled nucleosides and nucleotides, including inosine and thymidine, reducing it to fermentation and to direct the allocation of the nucleosides of the environment;

to make the method of obtaining the target product cleaner and radiation safe by eliminating radioactive isotopes, including tritium.

1. The method of obtaining vysokolegirovannyh of nucleosides and nucleotides, wherein the cultivated strains-producers of nucleosides and nucleotides of the species Bacillus subtilis and species of Bacillus amyloliquefaciens in fully deuterated medium (D2O).

2. The method according to p. 1, characterized in that as a fully deuterated environment using hydrolysates produced with deuterium oxide and watermethanol biomass methylotrophic bacteria Brevibacterium methylicum.

3. The method according to p. 1, distinguished by the nucleoside after separation of the target product.

4. The method according to p. 1, characterized in that for the production of D-labeled inosine use a strain of the species Bacillus subtilis NB3157.

5. The method according to p. 1, characterized in that for the production of D-labeled thymidine use a strain of the species Bacillus amyloliquefaciens N V.

 

Same patents:
The invention relates to the field of biotechnology and medicine, and in particular to methods of obtaining 2'-deoxyribonucleosides

The invention relates to veterinary Microbiology and sanitation and is a strain of the soil spore-forming bacteria Bacillus subtilis used for disinfection of poultry litter and manure from pathogenic microorganisms

The invention relates to a method of cleaning oil and oil products, including the application of contaminated surface peat

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus buchneri 600 is isolated from barley grains at milkwax stage of ripeness. The strain Lactobacillus buchneri 600 is registered in 05. 08. 2002 year in the All-Russian State collection of microorganism strains used in veterinary science and animal husbandry at number Lactobacillus buchneri VGNKI 02.08.04-DEP and deposited in the collection 000 "Biotrof". Invention provides the more effective multiplication of the strain in maize ensilaging green mass and preserving flattened grains with enhanced formation of lactic acid, inhibition of putrid microflora that allows preparing fodder from vegetable raw with enhanced quality. Invention can be used in fodder production for ensilage maize green mass and preserving flattened grains.

EFFECT: valuable properties of strain.

3 tbl, 2 ex

FIELD: biotechnology, agriculture, microbiology.

SUBSTANCE: invention relates to a new isolated strain of Lactobacillus acidophilus 1660/08 as a producer of the protein fodder. The strain Lactobacillus acidophilus 1660/08 is obtained by the selection method and selected by its ability to form significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.10.-DEP. Invention provides eliminating the environment pollution in producing the protein fodder, to enhance the specific protein yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process in its preparing, to simplify equipment fitting out and to utilize waste in manufactures using natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus plantarum 578/25 is obtained by method of step-by-step selection and selected by its ability to produce significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.09.-DEP. Invention provides eliminating the pollution of environment in producing the protein fodder, to elevate the protein specific yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process of its preparing, to simplify apparatus equipment, to utilize waste in manufacturing using the natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, microbiology, dairy industry.

SUBSTANCE: the strain of microorganism Lactobacillus lactis VKPM B-8354 is prepared without using mutagens and genetic methods and shows resistance against broad spectrum of lactophages. The strain ferments effectively milk from different trading sorts, with broad range of fatness and different methods of thermal treatment. Individual specific properties of the strain allows its applying as a monostrain ferment. Curd obtained with applying the strain L. lactis VKPM B-8354 shows good organoleptic qualities, nonacid taste and homogenous consistence. The strain is suitable especially for plants with small volume of manufacture but with varied assortment.

EFFECT: valuable properties of strain.

3 tbl, 2 dwg, 5 ex

FIELD: biotechnology, biochemistry, microbiology.

SUBSTANCE: the strain Bacillus circulans B-65 a producer of cyclodextrin glucanotransferase is isolated and selected form the soil sample by culturing in nutrient medium with amylolytic activity 12.17 U/ml and cyclodextrinogenic activity up to 0.530 U/ml. Cyclodextrin glucanotransferase isolated from B. circulans B-65 shows the high degree for conversion of starch to cyclodextrins and this enzyme is specific for formation of β-cyclodextrin. Invention can be used in food industry for preparing cyclodextrins and cyclodextrin glucanotransferase used in different branches of industry.

EFFECT: improved isolating method, valuable properties of strain and enzymes.

9 cl, 8 tbl, 2 ex

FIELD: biotechnology, microbiology, veterinary science.

SUBSTANCE: for preparing a preparation cells of microorganism Escherichia coli VKM CR-322D is cultured in nutrient medium containing Hottinger's broth, glucose, yeast extract, manganese sulfate, potassium hydrophosphate, sodium chloride and tap water in the content of amine nitrogen 125-155 mg%. Glucose is added by batch portions in the process of culturing cells that is carried out at temperature 30-31oC at stirring and aeration for 10-12 h. Prepared cultural fluid containing 3 x 109 bacterial cells/ml is mixed with protective sucrose-gelatin medium and subjected for lyophilic drying. Dried mass is stored under nitrogen that enhances safety of viable cells in the preparation. Applying the preparation for prophylaxis and treatment of agricultural and domestic animals and poultries with gastroenteric diseases provides its effectiveness.

EFFECT: improved preparing method, valuable veterinary properties of preparation.

17 cl, 8 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: method for preparing liquid lactobacterin involves regeneration, culturing passages of lyophilized culture and culturing ferment of lactobacilli in liquid lyophilized nutrient medium containing dry defatted milk enzymatic hydrolyzate with the content of amine nitrogen 1 485 mg%, 30.0 ± 3.0 g/l; yeast concentrated autolyzate, 110.0 ± 10 g/l; food agar, 0.8 g/l, and distilled water, up to 1 l. Culturing ferment is carried out up to accumulation of biomass of lactobacilli 109-1010 CFU/ml. Then 10-30% of supernatant liquid is removed from the ready product and replaced it with equal volume of fresh nutrient medium. Invention provides simplifying technology in preparing liquid lactobacterin and to elevate the storage period of viable lactobacilli. Invention can be used in producing probiotic preparations.

EFFECT: improved preparing method.

1 tbl, 3 ex

FIELD: biotechnology, food and medicinal industry, microbiology.

SUBSTANCE: the strain Bifidobacterium longum 379-IN is obtained by selection without using methods of genetic modification of the strain Bifidobacterium longum B379M and distinct by ability to utilize insulin. The strain is deposited in GKNM GU "MNIIEM named for G. N. Gabrichevskiy Russia Ministry of Public Health" at № 172. The strain shows high technological effectiveness, accumulates biomass with substrates of vegetable origin and artificial nutrient media for short periods with concentration of bifidobacteria, it elicits acid-forming and antagonistic properties with respect to pathogenic and putrid microflora. This allows its using in manufacturing bacterial preparations, biologically active supplements for food, fermented-dairy and nonfermented-dairy foodstuffs, ferments, hygienic and cosmetic agents providing probiotic effect and normalization of microbiocenosis in human body, among them in gastroenteric and urogenital tracts, cutaneous and mucosa integuments. Invention can be used in manufacturing bacterial preparations, biologically active supplements for food, fermented-dairy and nonfermented-dairy foodstuffs, hygienic and cosmetic agents.

EFFECT: valuable properties of strain, expanded assortment of similar agents.

6 ex

Up!