SU534236A1 - The method of obtaining deoxyribomonucleotides - Google Patents

Description

(54) METHOD FOR OBTAINING DISOXYRIBOMONONUCLEOTIDE

one

The invention relates to the production of biologically active substances which can be used in medicine as physiologically active agents.

A known method for producing mononucleotides enzymatically, for example, by treating RNA with a cultured strain of the StoiplrvLococcus aurcus strains under conditions of reduced activity, contained in the phosphatase medium.

There is also known a method for producing 5-mononucleotides, consisting in the current that polymeric nucleic acids are used as raw materials: they are split into 5-phosphodiesterase mononucleotides by active extracts of plant origin, followed by isolation of the target product by known methods,

However, the known methods do not provide a high yield of the target product,

In order to expand the resource base and increase the yield of the target product in the proposed method, as a source of raw materials used waste production

Protamine sulfate from fish milt, extraction is carried out with acid, and enzymatic hydrolysis is carried out with nucleases from commercial fish liver.

For the production of deoxyribomonucleotides by the proposed method, enzyme preparations are used, which are obtained from the liver of marine commercial fish, such as flounder, pink and. Get them as follows.

Kambachi liver is homogenized in distilled water, acidified to pH 3.0 in a Waring homogenizer, then the homogenate is extracted for 3 days, freed from the upper layer of fat, and then centrifuged at 4 ° C and 250 O rpm for 40 minutes, then the supernatant is drained and adjusted to 25% saturation with dry ammonium sulphate. The precipitate is separated by centrifugation, and the supernatant is adjusted to 9 O% saturation. The precipitate is collected by filtration and dissolved in a minimum volume of distilled water. The resulting solution is again made up to 40% with ammonium sulphate, then the precipitate is separated by centrifugation and discarded, the supernatant is brought to 90% saturation with ammonium sulphate, then the precipitated precipitate is collected by suction filtration. The resulting precipitate is dissolved in a minimum amount of 0.01 M phosphate buffer, pH 8.0, and in this form the resulting preparation is stored for no loss of activity. Further purification of the enzyme preparation was carried out by gel filtration on a Sephadex 6-100 column. After purification of the enzyme preparation, fractionation was carried out on a DEA cellulose column and the combined phosphodiesterase fractions, deoxyribonuclease 1 and Ts were collected. Deoxyribomononucleotides from the production of protamine sulfate in the following way. Example Waste production of protamine sulfate from fish milk is ground to a powder in a porcelain mortar. Then, the resulting powder mass (250 g) is stirred into 5 liters of 7% trichloroacetic acid (TX the resulting solution is poured into 1 liter of the flask and shaken on a universal shaker to 4 ° minutes, then centrifuged for 20 minutes at 500 ° rpm, the precipitate is removed, and the sedimentary liquid is drained into a glass container. Thus, the acid-soluble products are removed — aligonucleotides, mononucleotides, and nitrogen bases.The resulting acid extract is neutralized with 4N, ammonia to pH 7.0 by adding 5g of 4g MgF / OD, then make n eparat nucleases {4OO mg) was isolated as described above. The reaction solution is incubated at 37 ° C for 24 hours, the pH of the incubation mixture is periodically monitored. After the enzymatic hydrolysis of the oligonucleotides is completed and the 10O 96 ° hydrolysis alcohol is added, the resulting solution is filtered, then evaporated to a syrupy liquid, which is diluted with distilled water and filtered through activated carbon to remove pigments. The separation of the mixture of deoxyribomonucleotides is carried out as follows. A 1 ml solution of a mixture of deoxy ribomononucleotides is taken and applied to a 50x12 cm ion exchange column containing a Dowex resin. the pH of the applied solution was preliminarily adjusted to 10 with ammonia. First, the column was washed with bidistilled water to remove ammonia, 1215 L of OjOlM ammonium chloride, to those. until the pH of the sample solution was lowered to 7.0, as a result of this washing, the nitrogenous bases and nucleosides were removed, then the column was washed with 0.01 M hydrochloric acid (15-20 liters), and all nucleotides were removed. Polynucleotides and residues of non-hydrolyzed DNA remain on the column. Thus, the initial separation of the products of DNA hydrolysis is carried out. Then the deoxyribomononucleotide fraction obtained by passing 0 through the O1M hydrochloric acid solution and concentrated by repeated reabsorption on Dowex-1 is basified with a solution of ammonia. a smaller column (10x2 cm) containing Dowex-1 to isolate O-specific deoxyribonucleotides by fractionating them as follows. About 0. 01 M ammonium chloride solution is passed until the pH of the solution flowing from the column drops to 6.0, then is passed through a 0 column, O01 M hydrochloric acid solution, and a fraction containing deoxycytidyl acid (including 5-methyldithylic acid), then a fraction containing deoxyadenylic acid was extracted with 0.002 M hydrochloric acid, a fraction containing thymidylic acid was separated with solution 0, and a fraction containing deoxyguanilic acid was separated with a solution of 0.005 M hydrochloric acid Lot identification of nucleotides collected from the column is carried out using the spectrum of photometry. The resulting solutions of deoxyribomonucleotides are individually concentrated by re-adsorption on ion-exchange columns, washed with water and upyoured with dilute hydrochloric acid solutions. In this case, the purine deoxyribomonucleotides are quickly neutralized with an aqueous solution of ammonia in order to avoid acid hydrolysis. A further concentration of each deoxyribomononucleotide obtained in solution is carried out by evaporation in vacuum of the solution, followed by precipitation with a mixture of ethanol and ether or a solution of barium with ammonia. The proposed method makes it possible to use as a raw material the production waste of protamine sulfate from the milk of fish, which until now has not been used in the whole world and is thrown away as unnecessary. The preparation of deoxyribomononucleotides from the proposed feedstock is twice as divided as compared to their production from polymeric nucleic acids. 36 F 3 o bb of the formula of ioretration The method of obtaining deoxyribomonucleotide by extracting the raw material, enzymatic hydrolysis of the obtained extract, followed by separation of the target product by known methods, characterized in that, in order to broaden the raw material base and increase the yield of the target product, waste is used as the starting material production of protamine sulphate from fish milt, extraction is carried out with acid, and enzymatic hydrolysis is carried out with nucleases from commercial fish liver.