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Publication numberUS3920519 A
Publication typeGrant
Publication dateNov 18, 1975
Filing dateFeb 11, 1974
Priority dateFeb 14, 1973
Also published asCA1023290A1, DE2406811A1, DE2406811B2, DE2462314A1, DE2462314B2
Publication numberUS 3920519 A, US 3920519A, US-A-3920519, US3920519 A, US3920519A
InventorsYuh Norimoto, Shigeru Ohmura, Yoshiaki Shimizu, Toshio Tatano
Original AssigneeYuh Norimoto, Shigeru Ohmura, Yoshiaki Shimizu, Toshio Tatano
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Enzymatic hydrolysis of ribonucleic acid
US 3920519 A
Abstract
Ribonucleic acid is hydrolyzed in an aqueous reaction medium into nucleoside 2', 3'-cyclic phosphates and/or nucleoside 3', 5'-cyclic phosphates with intact cells of microorganisms having a ribonuclease activity. The formation ratio of nucleoside 2', 3'-cyclic phosphates and nucleoside 3', 5'-cyclic phosphates is selectively altered by varying the concentration of phosphate ion in the culture medium.
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United States Patent 1191 Norimoto et al.

[45] Nov. 18, 1975 ENZYMATIC HYDROLYSIS OF RIBONUCLEIC ACID Inventors: Yuh Norimoto, 1-2, Kyowa-cho,

Hofu, Yamaguchi; Shigeru Ohmura, 543, Negoya, Numazu, Shizuoka; Yoshiaki Shimizu; Toshio Tatano, both of 1188, Shimotogari, Nagaizumi-cho, Sunto, Shizuoka, all of Japan Filed: Feb. 11, 1974 Appl. No.2 441,309

Foreign Application Priority Data 3,630,842 12/1971 lshiyama et al 195/28 N OTHER PUBLICATIONS Mantani et al., Agr. Biol. Chem, Vol. 36, No. 2, pp. 242-248, (1972).

Primary Examiner- -Alvin E. Tanenholtz Attorney, Agent, or Firm-Fitzpatrick,Cel1a, Harper & Scinto [57] ABSTRACT Ribonucleic acid is hydrolyzed in an aqueous reaction medium into nucleoside 2, 3-cyclic phosphates and- /or nucleoside 3, 5-cyclic phosphates with intact .cells of microorganisms having a ribonuclease activity.

The formation ratio of nucleoside 2', 3'-cyc1ic phosphates and nucleoside 3, 5'-cyclic phosphates is selectively altered by varying the concentration of phosphate ion in the culture medium.

19 Claims, 1 Drawing Figure US. Patent Nov. 18, 1975 3,920,519

O aw :912

O 0/0 i il r i i O 0 4 17 75 ,2 H

O O OOOOOOOO QZZZEZ O OOOO ENZYMATIC HYDROLYSIS OF RIBONUCLEIC ACID I BACKGROUND OF THE INVENTION The present invention relates to enzymatic hydrolysis 'of ribonucleic acid (hereinafter referred to as RNA),

and more specifically, to the enzymatic hydrolysis of RNA into nucleoside 2', 3'-cyclic phosphates and/or nucleoside 3', 5-cyclic phosphates with intact cells of microorganisms having a ribonuclease activity.

Adenosin 2', 3-cyclic phosphate (hereinafter referred to as 2', 3-c AMP), which is a nucleoside 2, 3-

cyclic phosphate has recently been subject to considerable investigation. This is because 2, 3'-c AMP is considered to be important since the substance is present in the central nervous system of higher animals. Al-

though the role of 2, 3'-c AMP in the central nervous system has not yet been clarified, the substance is considered to play an important role in regard to nerve conduction. Therefore, 2', 3'-c AMP and other nucleonucleotido-2'-transferase such as ribonuclease T acid ribonuclease, leaf ribonuclease, etc., RNA is first hydrolyzed into intermediates such as nucleoside 2', 3'- cyclic phosphates including adenosin 2, 3'-cyclic phosphate (2, 3'-c AMP), guanosin 2, 3-cyclic phosphate (hereinafter referred to as 2', 3'-c GMP), uridin 2', 3-cyclic phosphate (hereinafter referred to as 2', 3-c UMP), and cytidin 2, 3-cyclic phosphate (hereinafter referred to as 2, 3-c CMP). The intermediates are finally decomposed into nucleoside 3'-phosphates, i.e., adenosin 3'-phosphate, guanosin 3'-phosphate, uridin 3'-phosphate and cytidin 3-phosphate.

It is also known that, in enzymatic hydrolysis of RNA, the presence of a chelating agent inhibits decomposition of nucleoside 2', 3-cyclic phosphates into nucleoside 3-phosphates. For instance, in a known method,

as reported by S. Mantani et al., RNA is hydrolyzed into nucleoside 2, 3-cyclic phosphates with crystalline ribonuclease derived from Rhizopus nivens in the presence of a chelating agent [Agr. Biol. Chem, Vol. 36, No. 2, pages 242-248 (l972)]. However, the use of a purified or partially purified ribonuclease for the hydrolysis of RNA is not practical in that the preparation thereof involves complicated steps and that various byproducts in addition to the desired products are formed in the reaction mixture.

Accordingly, a process for the hydrolysis of RNA on an industrially feasible scale is in high demand.

DESCRIPTION OF THE INVENTION In accordance with the present invention, it has been found that when RNA is subjected to the action of intact cells of microorganisms having a ribonuclease activity in the presence of a chelating agent, the RNA is hydrolyzed into nucleoside 2',3'-cyclic phosphates in a high yield without significant formation of by-products.

in comparison with the paper chromatograms of 2",

The paper chromatograms of FIG. 1 are obtained as follows:

Escherichia coli, ATCC 10798 is cultured in a medium comprising 30 mg/ml glucose, 6 mg/ml yeast extract, 8.5 mg/ml potassium dihydrogenphosphate, 11 mg/ml dipotassium hydrogenphosphate and 0.25 mg/ml of magnesium sulfate (pH 6.8) at 38C for 16 hours. Thereafter, the cells are collected by centrifugation.

25 mg/ml (as dry cells) of the cells are suspended in water containing 15 mg/ml of RNA and 15 mM of EDTA and the mixture is allowed to react at 40C for 48 hours.

A similar reaction is carried out using 25 mg/ml (as dry cells) of raptured cells obtained by treating the cells prepared above with ultrasonic waves of 28 kilocycle/sec at 8C for 10 hours. 7

The reaction mixtures are subjected to paper chromatography. Development is carried out with the upper layer of isobutyric acid.n-BuOI-I.O.5N NH OH (12121) by ascending method at 25C for 16 hours.

Asis shown in FIG. 1, when intact cells of a microorganism are used to hydrolyze RNA, the resulting reaction mixture contains only the desired nucleoside 2', 3'-cyclic phosphates, except for the unreacted RNA.

On the other hand, when a partially purified ribonuclease is used for the hydrolysis of RNA, it is evident that various byproducts are formed in the reaction mixture in addition to the desired nucleoside 2, 3-cyclic phosphates.

It is considered that in the case of a purified ribonuclease, the ribonuclease specimen is contaminated by other enzymes, and, therefore, various by-products are formed due to the occurence of side reactions. In this respect, in the case of the intact cells of microorganisms of the present invention, it is considered that the RNA is hydrolyzed with a ribonuclease specimen present on the surface-of the microbial cells and the desired nucleoside 2', 3'-cyclic phosphates are produced practically selectively.

In accordance with the present invention, it has also been found that, in the hydrolysis of RNA with intact cells of microorganisms, nucleoside 2, 3'-cyclic phosphates and nucleoside 3, 5-cyclic phosphates including adenosin 3', 5-cyclic phosphate (hereinafter referred to as 3', 5'-c AMP), guanosin 3, 5-cyclic phosphate (hereinafter referred to as 3', 5-c GMP), uridin 3, 5-cyclic phosphate (hereinafter referred to as 3', 5'-c UMP) and cytidin 3, 5-cyclic phosphate (hereinafter referred to as 3, 5 '-c CMP), can be obtained in a preselected ratio by controlling the amount of phosphate ion in' the reaction medium. More specifically, when the hydrolysis is carried out in the absence or in the presence of a very low concentration of phosphate ion,RNA is selectively decomposed into nucleoside 2, 3'-cyclic phosphates; and when the reaction is carried out in the presence of a relatively higher concentration of phosphate ion, RNA is selectively decomposed into nucleoside 3', 5 -cyclic phosphates. Obviously, when the phosphate ion concentration is adjusted to a median level, both nucleoside 2, 3'- and 3', 5'-cyclic phosphates are formed and accumulated in the reaction medium.

Adenosin 3', 5'-cyclic phosphate is one of the components of nucleoside 3', 5'-cyclic phosphates and is 3 known to function as a mediator of hormon-induced changes in the metabolism. 3, 5'-c AMP and other nucleoside 3, 5'-cyclic phosphates are not only useful as reagents for the medical studies but are expected to be useful as medicaments.

Heretofore, there have been no reports on enzymatic hydrolysis of RNA to prepare nucleoside 3', 5-cyclic phosphates. It has been only known that 3, 5'-c AMP is produced by reaction of adenosin triphosphate with adenyl cyclase obtained from a microbial source or is produced by fermentation reaction.

In accordance with the present invention, any microorganism that exhibits a ribonuclease activity may be used. Any of the methods well known in the art may be utilized for the determination of whether a specific microorganism exhibits a ribonuclease activity. Once a microorganism has been determined to exhibit the required activity it may be employed in the present invention; and it is to be understood that any microorganism possessing such activity is contemplated by the invention.

Preferred microorganisms are Gram-negative bacteria particularly, those belonging to the genera Escherichia, Salmonella, Aerobacter or Pseudomonas. The genus Aerobacter is generally characterized by short rods; motile or nonmotile, the motile species possessing peritrichous flagella; gramnegative; grow readily on ordinary media; ferment glucose and lactose with the production of acid and gas; produce two or more times as much carbon dioxide as hydrogen from glucose; methyl-red test negative; Voges-Proskauer test positive; trimethyl-eneglycol not produced from glycerol by anaerobic fermentation; citric acid and salts of citric acid are utilized as sole sources of carbon; aerobic, facultatively anaerobic.

The genus Escherichia is generally characterized by short rods; motile or nonmotile; gram-negative; glucose and lactose are fermented with the production of acid and gas; acetylmethylcarbinol is not produced; methylred test positive; carbon dioxide and hydrogen are produced in approximately equal volumes from glucose; generally not able to utilize uric acid as a sole source of nitrogen.

The genus Salmonella is generally characterized by rods that are usually motile by means of peritrichous flagella, although nonmotile forms may occur. Gramnegative. gelatin not liquefied; indole not produced; bydrogensulfide production is variable; acid is produced from glucose, mannitol, maltose, and sorbitol; gas production is usually observed (exceptions are Salmonella typhosa and Salmonella gallinarum, but gas production may also be absent in other species or serotypes; lactose, sucrose, salicin, and adonitol are not attacked; the fermentation of other carbohydrates is variable; acetylmethylcarbinol is not produced; methyl-red test is positive; nitrites are produced from nitrates; ammonium citrate is usually utilized; urea not hydrolyzed; KCN-sensitivity is negative.

The genus Pseudomonas is generally characterized by cells monotrichous, lophotrichous, or nonmotile; Gramnegative; frequently develop fluorescent, diffusible pigments of a greenish, bluish, violet, lilac, rose, yellow, or other color; sometimes the pigments are bright red or yellow and nondiffusible; there are many species that fail to develop any pigmentation; the majority of species oxidize glucose to gluconic acid, 2- ketogluconic acid, or other intermediates; usually inac- 4 tive in the oxidation of lactose; nitrates are frequently reduced either to nitrites, ammonia, or free nitrogen.

In addition, Gram-positive yeasts belonging to the genera Hansenula, Kloechera and Torulopsis are also preferred. The genus Hansenula belongs within the subfamily Saccharomycetaideae and is generally characterized by budding cells and pseudomycelium, spores hat-shaped, Saturn-shaped or round, 1 to 4 per ascu, dissimilative oxidative (pellicle formation) also fermentative, nitrate assimilated.

The genus Torulopsis is found within the Family Cryptococcaceae and is generally characterized by multilateral budding, no pseudomycelium or mycelium, no formation of starchlike compounds, mostly fermentative.

The genus Kloeckera is also found within the Family Cryptococcaceae and is generally characterized by bipolar budding, lemon-shaped, cells and fermentative.

The microorganisms are propagated in manners well known in the art for culturing bacteria or yeasts, as the case may be. For example, the microorganism is propagated into a medium usually used for culturing the specific microorganism, containing a carbon source, a nitrogen source, inorganic materials and other nutrients. Suitable carbon sources include carbohydrates, such as glucose, sucrose, etc. Organic acids, alcohols and hydrocarbons may also be used depending upon the assimilability possessed by the microorganism to be employed. As the nitrogen source, organic and inorganic nitrogen compounds usually used for cultun'ng and natural organic substances, such as peptone, corn steep liquor, yeast extract, etc. may be used. Further, phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate and a metal source such as magnesium sulfate may be added to the medium.

Culturing may be carried out under the conditions suitable for the microorganisms to be employed. After the completion of culturing, the cells are collected, for example, by centrifugation.

In carrying out the hydrolysis of RNA, the cells are suspended in an aqueous medium at a concentration of 10 to 40 mg/ml, preferably, about 20 mg/ml, determined as dry weight. To the suspension is added RNA, a chelating agent and a predetermined amount of phosphate ion.

The RNA to be employed in the present invention may be obtained in any well known manner, and is usually obtained from microorganisms. For hydrolysis, a concentration of 1 to 40 mg/ml, preferably, 10 to 20 mg/ml of RNA is used.

As the chelating agent, any of those usually used for chelating metal ion may be used. (see for example the aforementioned Mantani publication). Preferred are, ethylenediaminetetraacetic acid (hereinafter referred to as EDTA), cyclohexanediaminetetraacetic acid (hereinafter referred to as Cy-DTA) and hydroxyethylenediaminetriacetic acid (hereinafter referred to as EDTA-OH). The chelating agent is employed at a concentration of 5 to 30 mM, preferably 10 to 30 mM.

As described above, in the present invention, the formation ratio of nucleoside 2', 3'-cyclic phosphates and nucleoside 3', 5'-cyclic phosphates is controlled by the concentration of phosphate ion in the reaction medium.

To illustrate, a strain of Escherichia coli, ATCC 10798, is cultured in 500 ml of a medium comprising 30 mg/ml glucose, 6 mg/ml yeast extract, 8.5 mg/ml po- 5. tassium dihydrogenphosphate, 11 mg/ml dipotassium hydrogenphosphate and 0.25 mg/ml magnesium sulfate (pH 6.8) in a 3 L-Erlenmeyer flask at 38C for 16 hours. After the completion of culturing, the cells are allowed to react at 40C for 48 hours. Following the reaction, the formation of nucleoside 2, 3'-cyclic phosphates and that of nucleoside 3, 5-cyclic phosphates are determined. The results are shown in the following table.

Table Concentration Formation of nucleo- Formation of nucleoof phosphate side 2',3-cyclic side 3',5-cyclic ion (M) phosphate (mg/ml) phosphate (mg/ml) 2 trace 0.10 2 trace 0.15 1.7 0.25 0.16 0.96 0.77 0.17 trace 1.2

0.3 trace 1.2

As is evident from the results of the above experiment, when phosphate ion is absent from the reaction system, RNA is decomposed selectively into nucleoside 2', 3-cyclic phosphates. As the concentration of phosphate ion is increased, the formation of nucleoside 2, 3'-cyclic phosphates decreases while that of nucleoside 3, '-cyclic phosphates increases. When the concentration of phosphate ion is as high as 0.17M or more, RNA is decomposed almost selectively into nucleoside 3', 5'-cyclic phosphates.

Thus, in accordance with the present invention, when the concentration of phosphate ion is in the medium is from 0 to 0.15M, the reaction products are rich in nucleoside 2', 3'-cyclic phosphates; and when the con- 'centration is 0.17M and higher, the reaction products are rich in nucleoside 3, 5'-cyclic phosphates. Where the concentration is between 0.15M and 0.17M, nucleoside 2', 3'- and 3, 5-cyclic phosphates are produced almost equally.

As a source of phosphate ion, various phosphates that liberate phosphate ion in an aqueous medium such as phosphoric acid, sodium phosphate, potassium phosphate, ammonium phosphate, etc., may be employed.

Particularly suitable sources are evident from the following examples.

Generally speaking, the aqueous medium containing RNA, microbial cells, a chelating agent and various concentrations of phosphate ion is allowed to react at 35C to 45C, preferably, 38C to 42C, for 1 to 96 hours, preferably, 24 to 48 hours. During the reaction, the pH is maintained at 4 t0 9. Where the selective production of nucleoside 2, 3-cyclic phosphates is desired, the pH is preferably kept at around 5 and where the selective production of nucleoside 3', 5'cyclic phosphate is desired, the pH is preferably kept at around 8.

Suitable aqueous reaction media may be various buffer solutions such as an acetate buffer and a phosphate buffer. In view of the pH adjustment, the use of a phosphate buffer solution is most convenient.

After completion of the reaction, the products are isolated and purified by well known means. For example, the cells are removed from the reaction mixture by filtration. The cell-free filtrate is subjected to adsorption on activated carbon or a synthetic adsorbent and thereafter elution is carried out with aqueous methanol of an alkaline pH or a mixture of acetone and water. The eluate is concentrated and adsorbed on an anion exchange resin, for example, Dowex 1X2 (Cl) (trade name for a strongly basic anion exchange resin, produced by The Dow Chemical Co., U.S.A.). Then elution is carried out with a neutral salt solution such as an aqueous ammonium bicarbonate solution. After freezedrying or crystallization of the eluate, a mixture of nucleoside 2', 3'-cyclic phosphates containing 2', 3'-c AMP, 2, 3-c GMP, 2, 3'-c UMP and 2, 3-c CMP or a mixture of nucleoside 3', 5'-cyclic phosphates containing 3, 5-c AMP, 3', 5-c GMP, 3, 5-c UMP and 3, 5' CMP is obtained.

Where the reaction products are obtained as a mixture of nucleoside 2, 3-cyclic phosphates and nucleoside 3', 5'-cyclic phosphates, the mixture can be readily separated into each nucleoside cyclic phosphate. For example, the cells are removed from the reaction mixture by filtration. The filtrate is adjusted to a pH of 3.5

I to 4.0 with a suitable acid, such as hydrochloric acid or phosphoric acid; By concentrating the filtrate, nucleoside 3', 5-cyclicphosphates separate out. After recovering nucleoside 3', 5'-cyclic phosphates by filtration, nucleoside 2', 3'-cyclic phosphates are obtained as the filtrate. The nucleoside 3', 5-cyclic phosphates obtained as a solid may then be dissolved in a solvent having a weakly alkaline pH of 7.5 or more. Nucleoside 2', 3- or 3, 5'-cyclic phosphates separated in this manner can be purified, respectively, in the same manner as described above.

The thus obtained nucleoside 2', 3'- or 3', 5'-cyclic phosphates are readily separated into each component by any means well known in the art. For example, a mixture of nucleoside 2, 3 or 3', 5-cyclic phosphates is passed through a column of an anion exchange resin such as Dowex 1X2 (Cl). Stepwise elution is carried out using 0.1 to 1.0M solutions of a neutral salt such as ammonium bicarbonate. By repeatedly carrying out the similar steps of column chromatography, nucleoside 2', 3- or 3, 5'-cyclic phosphates can completely be separated into each component.

Practice of certain specific embodiments of the present invention is illustrated by the following representative examples.

EXAMPLE 1 In this example, Escherichia coli, ATCC 10798, is cultured in 500 ml of a medium comprising 30 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 (pH 6.8) in a 3 L-Erlenmeyer flask at 38C for 16 hours. Thereafter, the cells are collected from the culture liquor by centrifugation.

Then, 4 g (in all of the examples, the weight of the cells is dry weight) of the cells are suspended in 200 ml of water. To the suspension are added 3 g of RNA and 876 mg of EDTA. The mixture is allowed to react at 40C for 48 hours. After the reaction, 14 mg/m1 of a mixture of nucleoside 2, 3'-cyclic phosphates is formed in the reaction mixture.

The cells are removed from the resulting reaction mixture by filtration and the filtrate is passed through a column of Dowex 1X2 (Cl). Elution is carried out with 7 an aqueous solution of ammonium bicarbonate and the eluate is concentrated and freeze-dried. As a result, 1.8 g of a powder of a mixture of nucleoside 2', 3-cyclic phosphates of 90% purity comprising 2', 3-c AMP, 2, 3'-c GMP, 2', 3'-c UMP and 2', 3'-c CMP is obtained.

EXAMPLE 2 In this example, 2.5 g of the cells of Escherichia coli, ATCC 10798, obtained in the same manner as described in Example 1 are suspended in 100 ml of water. To the suspension are added 1 g of RNA and 519 mg of Cy-DTA. The mixture is allowed to react at 40C for 48 hours. After the reaction, 6.2 mg/ml of a mixture of nucleoside 2, 3'-cyclic phosphates is formed in the medium.

EXAMPLE 3 In this example, Escherichia coli, ATCC 10798, is cultured in 500 ml of a medium comprising 30 mg/ml glucose, 50 mg/ml corn steep liquor, 8.5 mg/ml potassium dihydrogenphosphate, 11 mg/ml dipotassium hydrogenphosphate and 0.25 mg/ml magnesium sulfate (pH 6.5) in a 3 L-Erlenmeyer flask at 38C for 16 hours. Thereafter, the cells are collected by centrifugation.

Then 2 g of the cells are suspended in 100 ml of water. To the suspension are added 1.5 g of RNA and 500 mg of EDTA-OH. The mixture is allowed to react at 40C for 48 hours. After the completion of the reaction, 9.7 mg/ml of a mixture of nucleoside 2', 3-cyclic phosphates is formed in the reaction medium.

EXAMPLE 4 In this example, 4.4 g of the cells of Escherichia coli, ATCC 10798, obtained in the same manner as in Example 3 are suspended in 200 ml of 0.33M phosphate buffer solution having a pH of 8.0. To the suspension are added 2 g of RNA and 876 mg of EDTA and the mixture is allowed to react at 40C for 48 hours. As a result, 7.2 mg/ml of a mixture of nucleoside 3', 5- cyclic phosphates is formed in the reaction mixture.

The cells are then removed by filtration and the filtrate is subjected to adsorption on a resin. Elution is carried out with 50% acetone and the eluate is concentrated. The concentrate is subjected to adsorption on Dowex 1X2 (Cl) and elution is carried out with a calcium chloride hydrochloric acid buffer solution. The eluate is concentrated and freezedried. As a result, 0.6 g of a powder of a mixture of nucleoside 3, 5'-cyclic phosphates of 86% purity is obtained.

EXAMPLE 5 In this example, Aerobacter aerogenes, IAM 1133, ATCC 8329, is cultured in 500 ml of a culture medium comprising mg/ml glucose, 10 mg/ml corn steep liquor, 8.5 mg/ml potassium dihydrogenphosphate, 11 mg/ml dipotassium hydrogenphosphate and 0.25 mg/ml magnesium sulfate (pH 6.8) in a 3 L-Erlenmeyer flask at 28C for 8 hours. After culturing, the cells are collected by centrifugation.

The, 7.5 g of the cells are suspended in 300 ml of 0.05M acetate buffer solution having a pH of 5.5. To the suspension are added 3 g of RNA and 1752 mg of EDTA. The mixture is then allowed to react at 40C for 24 hours. After completion of the reaction, 5.6 mg/ml of a mixture of nucleoside 2', 3'-cyclic phosphates is formed in the reaction mixture.

EXAMPLE 6 In this example, Aerobacter aerogenes ATCC 8329, is cultured in 500 ml of a medium comprising 10 mg/ml glucose, 6 mg/ml yeast extract, 8.5 mg/ml potassium dihydrogenphosphate, 11 mg/ml dipotassium hydrogenphosphate and 0.25 rug/ml magnesium sulfate (pH 6.8) in a 3 L-Erlenmeyer flask at 38C for 8 hours. Thereafter, the cells are collected by centrifugation.

Then, 2.5 g of the cells are suspended in ml of 0.40M phosphate buffer having a pH of 8.0. To the suspension are added 800 mg of RNA and 584 mg of EDTA and the mixture is allowed to react at 40C for 36 hours. After completion of the reaction, 4.3 mg/ml of a mixture of nucleoside 3', 5-cyclic phosphates if formed.

The reaction mixture is then treated in the same manner as in Example 4 resulting in 133 mg of a powder comprising a mixture of nucleoside 3, 5-cyclic phosphates of 83% purity being obtained.

EXAMPLE 7 In this example, Salmonella typhosa, ATCC 9992, is cultured in 500 ml of a medium having the same com- .position as that of Example 1 in a 3 L-Erlenmeyer flask at 28C for 16 hours. Thereafter, the cells are collected by centrifugation.

Then, 1.8 g of the cells are suspended in 100 ml of 0.05M phosphate buffer solution having a pH of 6.8. To the suspension are added 1 g of RNA and 438 mg of EDTA. The mixture is allowed to react at 40C for 15 hours resulting in the formation of 6.3 mg/ml of a mixture of nucleoside 2', 3 -cyclic phosphates in the reaction mixture.

The reaction mixture is then treated in the same manner as in Example 5. As a result, mg of a powder comprising a mixture of 2', 3'-cyclic phosphates of 82% purity is obtained.

EXAMPLE 8 In this example, 2 g of cells of Salmonella typhosa, ATCC 9992, obtained in the same as in Example 7 are suspended in 100 ml of 0.36M phosphate buffer solution having a pH of 8.0. To the suspension are added 1.5 g of RNA and 730 mg of EDTA and the mixture is allowed to react at 43C for 44 hours. As a result, 9.1 mg/ml of a mixture of nucleoside 3, 5'-cyclic phosphates is formed in the reaction mixture.

EXAMPLE 9 In this example, Pseudomonas aeruginosa, ATCC 15246, is cultured in the same manner as in Example 1. After culturing, the cells are collected by centrifugation. Then 2.3 g of the cells are suspended in 100 ml of 0.05M acetate buffer solution having a pH of 5.0. To the suspension are added 1.5 g of RNA and 584 mg of EDTA. The mixture is allowed to react at 38C for 43 hours, resulting in 9 mg/ml of a mixture of nucleoside i 2, 3'-cyclic phosphates being formed in the reaction mixture.

The reaction mixture is treated in the same manner as in Example 5. As a result, 144 mg of a powder comprising a mixture of nucleoside 2', 3-cyclic phosphates of 78% purity is obtained. I

EXAMPLE 10 EXAMPLE 1 1 In this example, Hansenula anomala, ATCC 20144, is cultured in 500 ml of a medium comprising 40 mg/ml glucose, mg/ml peptone, 2 mg/ml yeast extract, 2 mg/ml potassium dihydrogenphosphate, 2 mg/ml dipotassium hydrogenphosphate, 1 mg/ml magnesium sulfate and 20 mg/ml malt extract (pH 6.5) in a 3 L-Erlenmeyer flask at 28C for 28 hours. After culturing, the cells are collected by cetrifugation. Then, 2.5 g of the cells are suspended in 100 ml of 0.33M phosphate buffer solution having a pH of 8.0. To the suspension are added 1 g of RNA and 500 mg of EDTA-OH and the mixture is allowed to react at 40C for 72 hours. After the completion of the reaction, 3.6 mg/ml of a mixture of nucleoside 3', 5'-cyclic phosphates is formed in the reaction mixture. The reaction mixture is then treated in the same manner as in Example 4. As a result, 47 mg of a powder comprising a mixture of nucleoside 3', 5-cyclic phosphates of 76% purity is obtained. I

EXAMPLE 12 In this example, Kloechera apiculata, ATCC 18212, is cultured in the same manner as in Example 11 except that the culturing is carried out for 32 hours. Thereafter, the cells are collected by centrifugation. Then 3 g of the cells are suspended in 100 ml of 0.33M phosphate buffer solution having a pH of 8.0. To the suspension are added 1 g of RNA and 692 mg of Cy-DTA and the mixture is allowed to react at 40C for 72 hours. After completion of the reaction, 3.5 mg/ml of a mixture of nucleoside 3, 5'-cyclic phosphates is formed in the reaction mixture. The reaction mixture is then .treated in the same manner as in Example 4. As the result, 42 mg of a powder comprising a mixture of 3', 5'- -cyclic phosphates of 74% purity is obtained.

EXAMPLE 13 In this example, 2.5 g of the cells of Torulopsis sphaerica, ATCC 8549, obtained in the same manner as in Example 11 are suspended in 100 ml of 0.33M phosphate buffer solution having a pH of 8.0. To the suspension are added 1 g of RNA and 438 mg of EDTA and the mixture is allowed to react at 40C for 48 hours. After completion of the reaction, 523 ug/ml of a mixture of nucleoside 3, 5-cyclic phosphates if formed in the reaction mixture.

What is claimed is:

1. A process for producing nucleoside 2', 3'-cyclic phosphates and nucleoside 3', 5-cyclic phosphates which comprises reacting ribonucleic acid with intact cells of a microorganism having a ribonuclease activity in an aqueous reaction medium which includes a source of phosphate ion, in the presence of a chelating agent to enzymatically hydrolyze ribonucleic acid into nucleoside 2', 3'-cyclic phosphates and nucleoside 3,

5'-cyclic phosphates and isolating-the nucleoside 2',

3'-cyclic phosphates and nucleoside 3, 5'-cyclic phosphates from the resulting aqueous medium.

2. A process for producing nucleoside 2', 3-cyclic phosphates which comprises reacting ribonucleic acid with intact cells of a microorganism having a ribonuclease activity in an aqueous medium in the presence of a chelating agent and phosphate ion wherein the con'centration of phosphate ion in the aqueous medium is 0 to 0.15M to enzymatically hydrolyze ribonucleic acid into nucleoside 2', 3-cyclic phosphates and isolating the nucleoside 2', 3-cyclic phosphates from the resulting aqueous medium.

3. A process for producing nucleoside 3', 5'-cyc1ic phosphates which comprises reacting ribonucleic acid with intact cells of a microorganism having a ribonuclease activity in an aqueous medium which consists essentially of, in addition to said ribonucleic acid and said intact cells, an aqueous buffer solution, a chelating agent and phosphate ion wherein the concentration of phosphate ion in the aqueous medium is at least 0.17M to enzymatically hydrolyze ribonucleic acid into nucleoside 3, 5-cyclic phosphates and isolating the 3', 5'-

' cyclic phosphates from the resulting aqueous medium.

4. A process according to claim 1, wherein said microorganism belongs to a genus selected from the group consisting of Escherichia, Salmonella, Aerobacter, Pseudomonas, Hansenula, Kloeckera and Torulopsis.

5. A process according to claim 1, wherein said microorganism belongs to a species selected from the,

group consisting of Escherichia coli, Aerobacter aerogenes, Salmonella typhosa, Pseudomonas aeruginosa,

Hansenula anomala, Kloeckera apiculata and Torulopsis sphaerica.

6. A process according to claim 5, wherein said microorganism is selected from the group consisting of Escherichia coliATCC 10798, Aerobacter aeroge'nes ATCC 8329, Salmonella typhosa ATCC 9992, Pseudomonas aeruginosa ATCC 15246, Hansenula anomala ATCC 20144, Kloeckera apiculata ATCC 18212, and Torulopsis sphaerica ATCC 8549.

7. A process according to claim 1, wherein said chelating agent is selected from the group consisting of ethylenediaminetetraac etic acid, cyclohexanediaminetetraacetic acid and hydroxyethylenediaminetriacetic acid.

8. A process according to claim 1, wherein said medium contains from 0.15M to 0.17M phosphate ion whereby nucleoside 2, 3'-cyclic phosphates and nucleoside 3', 5-cyclic phosphates are about equally produced in said medium.

9. A process according to claim 1, wherein from 1 to 40 mg/ml of ribonucleic acid is reacted with from 10 to 40 mg/ml of intact microorganism cells.

10. A process according to claim 1, wherein said chelating agent is present in said medium at a concentration of from 5 to 30mM.

11. A process according to claim 2, wherein the pH of said medium is maintained at about 5.

12. A process according to claim 3 wherein the pH of said medium is maintained at about 8.

13. A process according to claim 2, wherein the source of phosphate ion is phosphoric acid, sodium 1 1 phosphate, potassium phosphate or ammonium phosphate.

14. A process according to claim 3, wherein the source of phosphate ion is phosphoric acid, sodium phosphate, potassium phosphate, or ammonium phosphate.

15. A process according to claim 1, wherein the temperature of reaction is from 35 to 45C. and wherein said reaction is conducted for a period of time of from 1 to 96 hours.

16. A process according to claim 1, wherein the temperature of reaction is from 38 to 42C. and wherein said reaction is conducted for a period of time of from 24 to 48 hours.

17. A process according to claim 1, wherein the pH of said aqueous reaction medium during reaction is from 4 to 9.

18. A process according to claim 1, wherein said aqueous reaction medium consists essentially of a buffered aqueous solution containing said chelating agent and said phosphate ion, and wherein said aqueous reaction medium has a pH of from 4 to 9.

19. A process according to claim 1, wherein the source of phosphate ion is phosphoric acid, sodium phosphate, potassium phosphate or ammonium phosphate.

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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US5064758 *Dec 20, 1988Nov 12, 1991Institut Moreologii ChelovekaMethod of preparing a mixture of ribonucleotides
US6773885 *Oct 1, 2001Aug 10, 2004Integrated Dna Technologies, Inc.Compositions and methods for visual ribonuclease detection assays
WO1989012689A1 *Dec 20, 1988Dec 28, 1989Inst Morfolog Cheloveka Ak MedMethod of obtaining a mixture of ribonucleotides
Classifications
U.S. Classification435/89, 435/92, 435/911, 435/199, 435/828, 435/879, 435/849, 435/944, 435/875, 435/930
International ClassificationC12P19/30, C07H1/00
Cooperative ClassificationY10S435/875, C07H1/00, Y10S435/849, Y10S435/879, Y10S435/944, Y10S435/828, Y10S435/93, C12P19/30, Y10S435/911
European ClassificationC07H1/00, C12P19/30