DE2462314A1 - PROCESS FOR ENZYMATIC HYDROLYSIS OF RIBONUCLEIC ACID - Google Patents

Description

¹¹ Process for the enzymatic hydrolysis of ribonucleic acid "

Priority: February 14, 1973, Japan, No. 17 449/73 March 24 ^: 1973, Japan, No. 33 088/73

The invention relates to a process for enzymatic hydrolysis of ribonucleic acid (RNA) to form nucleoside-3 ', 5'-cyclophosphates.

Adenosine 3 ', 5'-cyclophosphate is known to have a mediator role in hormone-related metabolic processes. 3 ^f , 5'-c-AMP and the other hucleoside-3 ¹ , 5 ^l -cyclophosphates are important as drugs in addition to their importance as reagents for biochemical and medical investigations. - ·

So far, the enzymatic hydrolysis of RNA has been to nucleoside S'.S'-cyclophosphates not described in the literature. It is only known that 3 ', 5'-C-AMP by conversion of adenosine triphosphate with adenyl cyclase, which has been obtained from microorganisms or by fermentative processes.

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The object of the invention is to provide a process for hydrolysis of enzymatisehen IINS to form nucleoside 3 ^1, 5'-cyclic phosphates of jointing, which is easy to carry out industrially.

It was found according to the invention that when intact microorganism cells which have a ribonuclease activity have, obtained in the presence of a chelating agent and phosphate ions on RNA nucleoside-3 ', 5'-cyclophosphate can be.

The invention relates to a process for the enzymatic hydrolysis of ribonucleic acid to nucleoside-3 ¹ , 5'-eyclophosphates, characterized in that ribonucleic acid with intact cells of ribonuclease activity having microorganisms of the species Escherichia coli or Aerobacter aerogenes ATCG 8329, Salmonella typhosa ATCC 9992, Pseudomonas aeruginosa ATCC 15246, Hansenula anomala ATCC 20144, Kloeckera apiculata ATCC 18212 or Torulopsis sphaerica ATCC 8549 in an aqueous medium in the presence of ethylenediaminetetraacetic acid, cyclohexanediamine tetraacetic acid in the presence of at least 0.17 methylenediamine tetraacetic acid in the presence of chhosphate-formed and hydroxyethylenediaminate.

. Examples of nucleoside-3 ', S'-cyclophosphates which can be obtained by the process according to the invention are 3 ·, S'-cyclophosphate (3', 5'-C-MP), guanosine-3 ', S'-cydophosphate (3 ^!, 5'-C-GMP), uridine-S '^' -cyclophosphate (3 ¹ , 5'-C-UMP) and cytidine-3 ', 5'-cyclophosphate.

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When carrying out the reaction at higher concentrations of phosphate ions selectively nucleoside-3 ', 5'-cyclophosphate educated. When maintaining a medium phosphate ion concentration also arise in addition to the 3 ', 5'-cyclophosphates 2 ', 3' -cyclophosphate.

In the method according to the invention, all microorganisms of the species Escherichia coli can be used which have a Have ribonuclease activity. To see if a For a certain microorganism exhibiting ribonuclease activity, conventional methods can be used.

The microorganisms used in the method according to the invention are grown according to the usual breeding methods for bacteria or yeast. For example, the microorganisms in a a carbon source, a nitrogen source, inorganic salts and other nutrient-containing nutrient media. Examples of carbon sources are carbohydrates, such as Glucose and sucrose. Organic acids, alcohols and hydrocarbons can also be used if the one used Microorganism for the assimilation of these carbon sources is capable. Examples of nitrogen sources are organic

Nitrogen.

and inorganic / compounds, as well as natural organic substances, such as peptone * corn steep liquor and yeast extract. Further

can the nutrient medium with phosphates, such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate and metal salts, such as Magnesium sulfate.

The cultivation conditions are different depending on the microorganism used selected. After cultivation is complete, the cells are separated from the fermentation mash, for example by centrifugation .

For carrying out the RNA hydrolysis process according to the invention v / ground the cells in an aqueous medium at a concentration of 10 to 40 mg / ml, preferably about 20 mg / ml on the dry weight of the cells. The suspension is filled with RNA, a chelating agent and a certain amount of '· Phosphate ion added.

The RNA used in the process according to the invention is processed according to conventional Process obtained and generally derived from microorganisms. For hydrolysis it is used in an amount of 1 to 40 mg / ml, preferably 10 to 20 mg / ml is used.

■ The chelating agents are in the process according to the invention in a concentration of 5 to 30 millimolar, preferably 10 to 30 millimolar used.

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As already mentioned, “in the process according to the invention, the amount of nucleoside 3 ·, 5 ^f -cyclophosphates in addition to nucleoside 2 '^' - cyclophosphates is influenced by the concentration of the phosphate ions in the reaction medium.

For example, Escherichia coli ATCC 10798 is in 50 ml of a 30 mg / ml glucose, 6 mg / ml yeast extract, 8.5 mg / ml potassium dihydrogen phosphate, 11 mg / ml dipotassium hydrogen phosphate and Oi25 mg / ml magnesium sulfate buffer containing pH 6.8 Cultivated for 16 hours at 38 ^° C. in a 3 liter Erlenmeyer flask. When the cultivation is complete, the cells are separated from the fermentation mash by centrifugation. Then 2.5 g (dry weight) of these cells are suspended in 100 ml of water, 0.10 m, 0.15 m, 0.16 m, 0.17 m and 0.3 m phosphate buffer with a pH of 7.5 . The suspensions are mixed with 0.5 g of RNA and 292 mg of ethylenediaminetetraacetic acid and reacted at 40 ° C. for 48 hours. The amount of nucleoside-2 ¹ , 3'-cyclophosphates and nucleoside-3 ¹ , 5'-cyclophosphates formed is then determined. The results are shown in Table I.

Table I.

Phosphate ions Formation of nucleoside Formation of nucleoside _r concentration, m 2 ^f , 3'-cyclophosphates,
mg / ml 3 ', 5'-cyclophosphates,
mg / ml 0 traces 0.10 2 traces 0.15 1.7 0.25 Q, 16 0.96 0.77 0.17 traces 1.2 0.3 traces 1.2

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The above results show that the fluid is selectively hydrolyzed to nucleoside-2 ', 3'-cyclophosphates in the absence of phosphate ions. With increasing phosphate ion concentration, the formation of nucleoside-2 ¹ , 3'-cyclophosphates decreases, while the amount of nucleoside-3 ¹ , 5'-cyclophosphates formed increases. At a phosphate ion concentration of 0.17 m or more, the RNA is hydrolyzed practically selectively to nucleoside-3 ', 5'-cyclophosphates.

The process according to the invention thus gives reaction products at phosphate ion concentrations of 0.17 m and higher, which mainly consist of ITucleoside-3 ', 5'-cyclophosphates exist.

Various phosphates can be used as a source of phosphate ions v / earth, which release phosphate ions to the aqueous medium. Particularly suitable sources of phosphate are in the following examples listed.

In general, the reaction between RI ¹ IS, the microorganism cells, the chelating agent and the phosphate ions is from 1 to 96 hours, preferably 24 to 42 ⁰ C, carried out to 48 hours, at temperatures from 35 to 45 ⁰ C, Advantages-.reis ο 33 . The pH value is kept in the range from 4 to 9 during the reaction.

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Pur the selective formation of nucleosides *, i'-cyclophosphates the pH is preferably kept in the range of 8.

Suitable aqueous reaction media are various buffer solutions, such as acetate buffer and phosphate buffer. With regard to the adjustment of the pH value, the use of phosphate buffers is recommended "preferred. . ·

After the reaction has ended, the reaction products are isolated and purified by customary methods. For example, the cells are removed from the reaction mixture by filtration. The cell-free filtrate is adsorbed on activated carbon or a synthetic adsorbent and then eluted with alkaline, aqueous methanol or a mixture of acetone and water. The eluate is concentrated and adsorbed on an ion exchange resin, for example Dowex 1x2 (Cl ~ form) (strongly basic anion exchange resin from The Dow Chemical Co.). It is then eluted with a neutral salt solution, for example an aqueous ammonium hydrogen carbonate solution. ^{A mixture of nucleoside-2 1} , 3 ^! Is obtained from the eluate by freeze-drying or crystallization. -cyclophosphates containing 2 ¹ , 3'-C-AMP, 2 ·, 3'-C-GMP, 2 ', 3'-C-TOlP and 2', 3'-C-CMP or e; Ln mixture of Mucleoside-3 ', 5'-cyolophosphates, the 3', 5'-c-AMP, 3 '', 5'-c-GMP, 3 ', 5'-C-UI-IP and 3', 5'- C-CMP contains.

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If the reaction product is a mixture of nucleoside-2 ', 3'-cyclophosphates and nucleoside-3 ', 5'-cyclophosphates is obtained, this can easily be converted into the individual nucleoside-cyclophosphates be separated. For example, the cells

* filtered off from the reaction mixture. The filtrate is then with an acid such as hydrochloric acid or phosphoric acid to pH set from 3.5 to 4.0. After the filtrate has been concentrated, the nucleoside 3 ', 5'-cyclophosphates precipitate. .:;

". - -_- - - ': ^ The as festival

Nucleoside 3 ', 5 ^f -cyclophosphates obtained from the substance can then be dissolved in a weakly alkaline solvent with a pH of at least 7.5. Which are obtained in this way. tenen. "" j ', 5 ·' -Cyclophosphates can refer to the above • '. be cleaned in the manner described.

The 3 ¹ , 5'-cyclophosphates obtained can be

easily separate into the individual components according to known methods. For example, a mixture of

•,. 3 '^' - cyclophosphates on one with an anion exchange resin,

such as Dowex 1x2 (Cl "" form) loaded column. The elution is carried out in stages using 0.1 to 1.0 M solutions of neutral salts such as ammonium hydrogen carbonate. By repeated column chromatography, the

3 ', 5'-cyclophosphates into the individual components cut open.

The examples illustrate the invention.

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- 9 Example 1

EschericMa coli ATGG 10798 is available in 500 ml of a 30 mg / ml Glucose, 50 mg / ml corn steep liquor, 8.5 mg / ml potassium dihydrogen phosphate, 11 mg / ml dipotassium hydrogen phosphate and 0.25 mg / ml Nutrient medium containing magnesium sulphate with a pH value of 6.5 16 Hours at 38 G in a 3 liter Erlenmeyer flask bred. The cells are then separated off by centrifugation.

4.4 g of the cells obtained are suspended in 200 ml of 0.33% phosphate buffer with a pH of 8.0. The suspension is mixed with 2 g of RNA and 876 mg of ethylenediaminetetraacetic acid. Then, the mixture was reacted for 48 hours at 4O ⁰ G. In the reaction mixtures of 7.2 mg / ml to obtain a mixture of nucleoside 3 ^1, 5'cyclophosphates.

The cells are then filtered off. The filtrate is adsorbed on a synthetic resin. Elution is carried out with 50% acetone and the eluate obtained is concentrated. The concentrated solution is adsorbed on Dowex 1x2 (Cl "form. The elution is carried out with a calcium chloride-hydrochloric acid buffer solution. The eluate is then concentrated and freeze-dried. 0.6 g of a powdery mixture of nucleoside-3 ¹ , 5 is obtained '-cyclophosphates of 86 percent purity.

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Example 2 ^

Aerobacter aerogenes ATCC 8329 is dissolved in 500 ml of a nutrient medium containing 10 mg / ml glucose, 6 mg / ml yeast extract, 8.5 mg / ml potassium dihydrogen phosphate, 11 mg / ml dipotassium hydrogen phosphate and 0.25 mg / ml magnesium sulfate at pH-Y. ^r ert 6.8 for 8 hours at 38 ⁰ C in a 3 liter Erlenmeyer flask grown. The cells are then centrifuged off.

2.5 g of the cells thus obtained are suspended in 100 ml of 0.40 M phosphate buffer with a pH of 8.0. 800 mg of RNA and 584 mg of ethylenediaminetetraacetic acid are added to the suspension. The mixture is ^{reacted at 40 °} C. for 36 hours. After the reaction is obtained 4.3 mg / ml of a mixture aps nucleoside 3 ^1, 5'-cyclic phosphates.

The reaction mixture is then worked up as in Example 1. This gives 133 mg of a powdery mixture of 3-cyclohexene ¹ i5' phosphates of 83prozentiger purity.

Example 3

Salmonella typhosa ATCC 9992, in 500 ml of a nutrient medium of the same composition as bred 116 hours at 28 ⁰ C in a 3 liter Erlenmeyer flask in an example. The cells are then centrifuged off.

2 g of the cells obtained are suspended in 100 ml of 0.36 M phosphate buffer with a pH of 8.0. 1.5 g of RNA and 730 mg of ethylenediaminetetraacetic acid are added to the suspension. The mixture is ^{reacted at 43 °} C. for 44 hours. 9> 1 mg / ml of one are obtained

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Mixture of nucleoside 3 '> 5'-cyclophosphates.

Example 4

The procedure is as in Example 1 but using Pseudomonas aeruginosa ATCC 15246 Escherichia coli ATCC instead of 10,798th obtained 371 / ig / ml of a mixture of nucleoside 3 ^1, 5'-cyclic phosphates.

Example5

Hansenula anomala ATCC 20144 is dissolved in 500 ml of a 40 mg / ml glucose, 5 mg / ml peptone, 2 mg / ml yeast extract, 2 mg / ml potassium dihydrogen phosphate, 2 mg / ml dipotassium hydrogen phosphate, 1 mg / ml magnesium sulfate and 20 mg / ml malt extract culture medium containing pH 6.5 for 28 hours at 28 ° C in a 3 liter Erlenmeyer flask. When the cultivation is complete, the cells are centrifuged off. 2.5 g of the cells are then suspended in 100 ml of 0.33 M phosphate buffer, pH 8.0. The suspension is mixed with 1 g of RNA and 500 mg of hydroxyethylenediamine triacetic acid and reacted at 40 ° C. for 72 hours. After the reaction has ended, 3> 6 mg / ml of a mixture of nucleoside-3 ^f , 5'-cyclophosphates are obtained. The reaction mixture is then worked up as in Example 4. 47 mg of a powdery mixture of nucleoside-3 ¹ , 5 ^f -cyclophosphates of. 76 percent purity.

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Examples

Kloeckera apiculata ATCC 18212 is grown according to Example 5, with the exception that the cultivation time is 32 hours. The cells are then centrifuged off. Then 3 g of the cells obtained are suspended in 100 ml of 0.33 M phosphate buffer of pH 8.0. 1 g of RNA and 692 mg of cyclohexanediaminetetraacetic acid are added to the suspension. The mixture is ^{reacted at 40 °} C. for 72 hours. After the reaction has ended, 3> 5 mg / ml of a mixture of nucleoside-3 ', 5'-cyclophosphates are obtained. The reaction mixture is worked up as in Example 1. This gives 42 mg of a powdery mixture of 3 ^1, 5'-cyclic phosphates of 74prozentiger purity.

B e i s ρ i e 1 7 "

2.5 g of Torulopsis sphaerica ATCC 8549 cells obtained according to Example 5 were suspended in 100 ml of 0.33 M phosphate buffer of pH 8.0. 1 g of RNA and 438 mg of ethylenediaminetetraacetic acid are added to the suspension. The mixture is then reacted at 40 ° C. for 48 hours. After completion of the reaction, 523 of a mixture of nucleoside 3 ¹ receives ug / ml, 5'-cyclic phosphates.

Γ). fl fi U R / Π R. U 1

Claims (5)

Claims 1i Process for the enzymatic hydrolysis of ribonucleic acid to nucleoside 3 ', 5' -cyclophosphates, characterized in that ribonucleic acid with intact cells of ribonuclease activity exhibiting microorganisms of the species Escherichia coil or of Aerobacter. _; aerogenic ATCG 8329, Salmonella typhosa ATCG 9992, Pseudomonas aeruginosa ATCC 15246, Hansenula anomala ATCC.20144, Kloeckera apiculata ATCC 18212 or Torulopsis sphaerica ATCC 8549 in aqueous Kediura in the presence of ethylenetylenediainetetraacetic acid in the presence of at least 0.1 ethylenediainetetriacetic acid or in the presence of ethylenetylenediainetetraacetic acid or 0.1 m converts phosphate ions. 2. The method according to claim 1, characterized in that the microorganism is Escherichia coli • ATCC 10? 98 · used. · ··· ..-. . . .3. Method according to claim 1, characterized in that that 1 to 40 mg / ml ribonucleic acid is reacted with 10 to 40 mg / ml intact microorganism cells. 4. The method according to claim 1, characterized in that that one adjusts the concentration of the chelating agent in the medium to 5 to 30 millimolar. 5. The method of claim 1f, characterized in that ^the pH value of the medium to about 8, a * 60984 5/0841