The method of obtaining deoxyribonucleotide

 

(57) Abstract:

Usage: Microbiology, engineering Enzymology.

The inventive method of producing deoxyribonucleotides, which is carried out by growing without additional aeration bacteria Propionibacterium freudenreichii ssp. shermanii and differs in that for 2 - 2.5 hours before the end of the cultivation contribute antibiotic, bleomycin, the cells are then separated from the environment, resuspended in potassium-phosphate buffer, permeabilized Triton X-100 with freezing in liquid nitrogen and rapid thawing, treated with glutaraldehyde and after dialysis placed in incubation medium containing a specific ribonucleotide. After 2-3 hours of incubation, the corresponding 2'-deoxyribonucleoside-5'-diphosphate separated from the environment. The method allows the one-step transformation is directed to receive individual 2'-deoxyribonucleoside-5'-diphosphate (with a given basis) using a previously not used for these purposes in biotechnology cheaper substrates - ribonucleotides (output relative to the substrate 25 - 37%).

The invention relates to the field of Microbiology and engineering Enzymology, namely to methods for de the production and the medical industry.

A method of obtaining of deoxyribonucleotides, based on the enzymatic hydrolysis of DNA from microbial nucleases [I]

The method has the following disadvantages: 1) by the action of the microbial DNA nucleases get a mixture of products of deoxyribonucleosides, deoxyribonucleotides and oligodeoxyribonucleotides, 2) the total yield of 4-2'-deoxyribonucleoside-5'-monophosphate low, and if the target individual 2'-deoxyribonucleotide output relative to the substrate (DNA) is not more than 15% 3) used substrate (DNA) extracted from salmon ROE fish, roads and inaccessible.

A method of obtaining of deoxyribonucleotides by aerobic cultivation of microorganisms that produce enzymes that cleave DNA in the presence of DNA secreted also by microorganisms. After culturing separately and then jointly, or immediately together these microorganisms on nutrient medium from the culture medium emit formed deoxyribonucleosides, deoxyribonucleotides and oligodeoxyribonucleotide. This way it is advisable to take for the prototype [2]

The prototype has the following disadvantages: 1) low yield of the target product in connection with n is) the complexity of the method, due to the need for aerobic cultivation of several microorganisms with subsequent significant clearance from those present in the culture broth compounds, 3) low efficiency of the way if necessary directional receive a certain deoxyribonucleotide.

The aim of the invention is to simplify, improve the efficiency of the method aimed to provide individual (base) 2'-deoxyribonucleotides and increasing the yield of the target product.

The principle of the proposed method of obtaining deoxyribonucleotides is to implement one-stage transformation process ribonucleotide with a given basis into the appropriate deoxyribonucleotide using permeabilizing cells propionic acid bacteria Propionibacterium freudenreichii ssp. shermanii. The enzymatic conversion is carried out by ribonucleotides, part of the membrane enzyme complex. Soft destruction of the cytoplasmic membrane removes barrier permeability (permeability) and carries out the availability of exogenous substrate (ribonucleotide) to the enzyme, resulting in its conversion into the desired d is.

The method is as follows. Culture Propioni-bacterium freudenreichii ssp. shermanii grown for 46 to 48 hours at 28-30owithout additional aeration in the medium of known composition. For 2 - 2.5 hours before the end of cultivation in culture add an antibiotic of the bleomycin concentration 0,00004-0,00005% of the cells are Then separated from the environment, resuspended in potassium-phosphate buffer 0,08 0,1 M, pH 7.0 to 10 12% concentration (dry weight) and permeabilized Triton X-100 at a concentration of 0,08-0,10% After which the cells are frozen in liquid nitrogen for 10 to 12 minutes, quickly thawed at 30oand treated with glutaraldehyde at a concentration of 0.08 to 0.10% After dialysis cells at a concentration of 2.5 3,05 (dry weight) are placed in the incubation medium of the composition, ribonucleoside-5'-diphosphate 0,07 0,09; adenosylcobalamin 0,003 -0,005% dithiothreitol 0,40 0,47% rest of potassium phosphate buffer 0.08 to 0.10 M, pH 7.0, and incubated at a temperature of 36 to 38owithin 2 to 3 hours, after which 2'-deoxyribonucleoside-5'-diphosphate (with a given basis) was isolated from the environment.

The proposed method is simplified in comparison with the prototype, since no joint operations aerobic cultivation of several microorganisms and purification of the secreted desired product is aqueous and inexpensive substrate of ribonucleotides, enabling the one-step transformation is directed to receive individual 2'-deoxyribonucleoside-5'-diphosphate (with a given basis). The accumulation of 2'-deoxyribonucleoside-5'-diphosphate is be 0,55 0,45 the mmol per liter of incubation medium (200 to 250 mg/l), which is the output relative to the substrate 27 of 35% as for the prototype, it is theoretically possible that the output of one individual deoxyribonucleotide regarding DNA may not exceed 25%

Example 1.

The culture of P. shermanii VKM-103 is grown for 48 hours at 30owithout additional aeration in the medium of the following composition, glucose -2; ammonium sulfate 0,3; potassium phosphate (one-deputizing) 0.1; magnesium sulfate 0,02; casein hydrolysate (or tripton)- 0,2; mg/l cobalt chloride - 3; manganese sulfate 5; sodium chloride 5; ferric chloride 0,002; zinc sulfate 0,002; Biotin 0,001; thiamine 0,2; pattent calcium and 1.0. Distilled water. Maintain a pH value of 6.8 and 7.1.

2.5 hours before the end of the cultivation grown in culture add an antibiotic of the bleomycin in 0,00005% concentration. Then the cells in the cold separated from the medium and washed with potassium phosphate buffer (0.03 M, pH 7.0) by centrifugation at 10,000 rpm and Then tile the Oia add Triton X-100 0.10% concentration, freeze in liquid nitrogen (soaking for 10 min) and rapidly thawed at a temperature of 30o. Later in the suspension permeabilizing cells add 0,09% glutaraldehyde for 10 15 minutes at 4 10othen cialiswhat against potassium phosphate buffer (0.10 M, pH 7.0) for 14 to 16 hours at 4 of 10o(dialysis ratio of 1:100). The thus treated cells at a concentration of 3% (by dry weight) contribute to the incubation medium of the following composition, adenosine-5'-diphosphate (ADP) -0,09; adenosylcobalamin (doCbl) 0,005; Dimitrios (DTT) 0,47; the rest potassium phosphate buffer 0.10 M, pH 7.0, and incubated at 38o3 hours. From a liter of incubation medium emit 250 mg of 2'-deoxyadenosine-5'-diphosphate, which corresponds to the output relative to the substrate 27%

Example 2.

The culture of P. shermanii VKM-103 is grown as described in example 1, 46 hour at a temperature of 28o.

2 hours before the end of the cultivation type antibiotic of the bleomycin in 0,00004% of the cells are Then separated from the medium, washed as in example 1, and resuspended in 0.08 M potassium phosphate buffer, pH 7.0 to 10% concentration (dry weight). In the resulting suspension add Triton X-100 in 0.08% concentration, then frozen in liquid nitrogen and quickly rosmarie subjected to dialysis, as indicated in example 1. The thus treated cells at a concentration of 2.5% (by dry weight) contribute to the incubation medium of the following composition, ADP 0,07; AdoCbl 0,003; DTT - 0,40, the rest potassium phosphate buffer, 0.08 M, pH 7.0, and incubated for 2 hours at 36o. From a liter of incubation medium emit 225 mg of 2'-deoxyadenosine 5'-diphosphate, which corresponds to the output relative to the substrate 35%

Example 3.

The culture of P. shermanii VKM-103 is grown as described in example 1, 47 hours at a temperature of 29o. 2.5 hours before the end of the cultivation injected antibiotic of the bleomycin concentration 0,00004% of the cells are Then separated from the medium, washed as in example 1, and resuspended in potassium-phosphate buffer 0,09 M, pH 7.0 to 11% concentration (dry weight). In the resulting suspension add Triton X-100 0,09% concentration, then frozen in liquid nitrogen and rapidly thawed as described in example 1. Later in the suspension is added glutaric aldehyde concentration of 0.085% and the suspension is subjected to dialysis as described in example 1. The thus treated cells at a concentration of 2.7% (by dry weight) contribute to the incubation medium of the following composition, guanosin-5'-diphosphate (GDF) -0,08; AdoCbl 0,004; DTT 0,044; the rest potassium phosphate buffer 0,092'-deoxyguanosine-5'-diphosphate, which corresponds to the output relative to the substrate 29%

1 Way to get deoxyribonucleotide by culturing bacteria in a nutrient medium, followed by separation of the target product, characterized in that as a producer take Propionibacterium freudenreichii ssp. chermanii, they are grown for 46 48 h at 28 to 30C, 2,0 2,5 hours before the end of cultivation contribute antibiotic of the bleomycin concentration 0,00005 0,00004% after which the cells are separated from the medium, washed and resuspended in potassium-phosphate buffer 0.08 to 0.10 M, pH 7.0 to 10 - 12% concentration (by dry weight), permeabilized Triton X-100 at a concentration of 0.08 to 0.10%, after which the cell suspension is frozen in liquid nitrogen for 10 to 12 min, thawed at 30 ° C, treated with glutaraldehyde at a concentration of 0.08 and 0.09% and dialist, then the resulting biocatalyst placed in the incubation medium containing ribonucleoside-5'-diphosphate, cobalamin, a reducing agent and buffer, and incubated for 2 to 3 h at 36 - 38C, and then get deoxyribonucleotide separated from the environment.

 

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