JP2002095494A - Method for producing ribose-1-phosphates and nucleoside compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently and industrially producing ribose-1- phosphates and a nucleoside compound by an enzyme. SOLUTION: This method for producing the ribose-1-phosphates comprises allowing microbial cells of microorganisms containing heat-stable phosphopentomutase and containing substantially no phosphatase, or the enzyme derived from the microorganisms to act on the ribose-5-phosphates. The method for producing the nucleoside compound comprises allowing the microbial cells of the microorganisms containing the heat-stable phosphopentomutase and a heat-resistant nucleoside phosphorylase, and containing substantially no phosphatase and nucleosidase, or the enzyme derived from the microorganisms to act on the ribose-5-phosphates and a nucleic acid base.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a nucleoside compound used as a raw material for an antiviral agent, an antisense drug and the like, and to ribose 1-phosphate, which is an important starting material for biochemical synthesis of the compound. The present invention relates to a method for producing acids.

[0002]

2. Description of the Related Art In industrial production of nucleoside compounds, since the yield of chemical synthesis other than thymidine is extremely low, DN
Extraction and isolation of A (deoxyribonucleic acid) from the hydrolyzate is mainly performed. However, salmon and herring milt currently used as a raw material for DNA are limited in resources, and are difficult to collect, and require low-temperature storage equipment. In addition, milt contains proteins and saccharides in addition to DNA, and the DNA must first be separated therefrom. Furthermore, in the hydrolyzate of DNA, four types of 2 '
-The presence of deoxynucleosides (deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine) complicates the separation step and increases the cost.

[0003] On the other hand, there has been reported a method of obtaining a desired nucleoside or 2'-deoxynucleoside by using a nucleoside or 2'-deoxynucleoside and a nucleic acid base as raw materials and performing a base exchange reaction by the action of nucleoside phosphorylase (Hori). N., Watana
be M. be. , Yamazaki Y .; , & Mikam
i Y. , Agric. Biol. Chem. , 53,
197-202 (1989), JP-A-11-46790
No.). The essence of this method is the ribosylation of nucleobases with ribose 1-phosphate or 2-deoxyribose 1-phosphate catalyzed by nucleoside phosphorylase. That is, starting from ribose 1-phosphate or 2-deoxyribose 1-phosphate and any nucleobase,
By the action of nucleoside phosphorylase, it is possible to prepare the corresponding nucleoside compound. The method for enzymatically synthesizing a nucleoside compound from ribose 1-phosphate or 2-deoxyribose 1-phosphate and a nucleobase is the simplest, and purification after synthesis is also easy.

[0004] However, ribose 1-phosphate and 2-deoxyribose 1-phosphate are compounds that are extremely difficult to chemically synthesize, and are not only chemically unstable but also easily decomposed by phosphatases. . Therefore, it was not possible to industrially synthesize a nucleoside compound using ribose 1-phosphate or 2-deoxyribose 1-phosphate and a nucleobase as starting materials. Under such circumstances, a method for producing ribose 1-phosphate or 2-deoxyribose 1-phosphate as a starting material was established, and ribose 1-phosphate or 2-deoxyribose 1-phosphate was used as a starting material. It has been strongly desired to establish a method for producing a nucleoside compound.

[0005]

Accordingly, an object of the present invention is, first, to provide a biochemical method for efficiently producing ribose 1-phosphate or 2-deoxyribose 1-phosphate. The second object is to provide a biochemical method for efficiently and industrially producing a nucleoside compound using ribose 1-phosphate or 2-deoxyribose 1-phosphate and a nucleobase as raw materials.

In making the present invention, the present inventors have paid attention to the following points and have repeatedly studied.

[0007] As an enzyme which efficiently produces ribose 1-phosphate or 2-deoxyribose 1-phosphate, phosphopentmutase (EC5.4.2.7 (hereinafter referred to as P
This enzyme catalyzes an equilibrium reaction represented by the formula (1) or (2).

[0008]

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Conventional reports (Smith, CG, an)
d Bernstein, I.D. A. , Biochimi
ca et Biophysica Acta, 52,
184-193 (1961)), the equilibrium of the formula (2) is remarkably deviated toward the 2-deoxyribose 5-phosphate-producing side, and 2-deoxyribose 5-phosphate and 2-deoxyribose 1- Phosphoric acid presence ratio is 9
5: 5.

However, in formulas (1) and (2), the ratio of the two compounds at equilibrium is theoretically equivalent to the ratio of the released energy (potential energy, △ 'G) when the phosphate group is cleaved. It is thought that it is estimated 70: 3
Since it is estimated to be about 0, the proportion of ribose 1-phosphate or 2-deoxyribose 1-phosphate in the reactions represented by the formulas (1) and (2) is expected to be 30%.

In addition, ribose 1-phosphate or 2-deoxyribose 1-phosphate is an unstable compound, and is a phosphatase (EC 3.1.3.1 alkaline phosphatase, and EC 3.1.3.2 acid phosphatase). Therefore, it is desirable that the microorganism used as the enzyme source contains a significant amount of phosphopentmutase and nucleoside phosphorylase and does not contain phosphatase. In this case, the nucleoside phosphorylase is a purine nucleoside phosphorylase (E
C2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.2.2).

Furthermore, nucleosidases (for example, EC 3.2.2.2.) Which degrade nucleoside compounds of the final product.
7 Microorganisms that do not contain adenosine nucleosidase) are more desirable.

2-deoxyribose 5-phosphate is deoxyribose phosphate aldolase (EC 4.1.2.4)
As a result, it is easily decomposed into glyceraldehyde 3-phosphoric acid and acetaldehyde. Therefore, when using 2-deoxyribose 5-phosphate as a substrate, it is desirable that the microorganism does not contain the enzyme.

Although the starting nucleic acid base is hardly soluble in water, the solubility generally increases with increasing temperature.
Preferably, the enzyme is a thermostable enzyme that can be used at high temperatures. For example, to dissolve 1 g of adenine, 2
L of water is required, but 40 mL of boiling water is sufficient (The Merck Index, 9th edition, 1983)
Annual Report, 138 pages).

[0015]

The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems. As a result, (1) a microorganism strain isolated from the natural world has strong phosphopentomtoses, purine nucleoside phosphorylases and pyrimidines. Exhibiting nucleoside phosphorylase activity and substantially exhibiting no phosphatase or nucleosidase activity; (2) the microorganism has a suitable growth temperature of 50 ° C. or higher, and the contained phosphopentomtose and nucleoside phosphorylase are used at 50 ° C. or higher. (3) When 2-deoxyribose 5-phosphate is used as a raw material, an aldehyde such as acetaldehyde or propionaldehyde is used when the cells of the microorganism strain or the enzyme derived from the cells are allowed to act. Coexist with deoxyribose phosphate aldolase That 2-degradation of deoxyribose 5-phosphate is suppressed, that the reaction yield is significantly improved,
(4) the microorganism strain belongs to the genus Bacillus, and one strain showing particularly high activity is identified as Bacillus coagulans, and the other strain is identified as Bacillus species;
(5) By the cells of the Bacillus strain or an enzyme derived from the cells,
The present inventors have found that a corresponding nucleoside compound is synthesized from ribose 5-phosphate or 2-deoxyribose 5-phosphate and a nucleobase at a value substantially close to the theoretical yield, and have completed the present invention.

That is, the gist of the present invention is as follows.

(1) A ribose 5-phosphate selected from the group consisting of ribose 5-phosphate and 2-deoxyribose 5-phosphate contains a thermostable phosphopentomthase and substantially contains a phosphatase Reacting with the cells of a microorganism that does not
Production of ribose 1-phosphates, characterized by obtaining ribose 1-phosphates corresponding to the respective ribose 5-phosphates selected from the group consisting of -phosphate and 2-deoxyribose 1-phosphate Method. (2) Ribose 5-phosphate and 2-deoxyribose 5
-A ribose 5-phosphate selected from the group consisting of phosphoric acid and a nucleobase, containing a thermostable phosphopentomtase and a thermostable nucleoside phosphorylase, and containing substantially no phosphatase and nucleosidase; A method for producing a nucleoside compound, characterized by obtaining an nucleoside compound corresponding to each of the aforementioned ribose 5-phosphates selected from the group consisting of nucleosides and 2'-deoxynucleosides by reacting the microorganism-derived enzyme. . (3) The method for producing ribose 1-phosphates according to the above (1), wherein an aldehyde is allowed to coexist when the cells of the microorganism or an enzyme derived from the microorganism are allowed to act. (4) The method for producing a nucleoside compound according to the above (2), wherein an aldehyde is allowed to coexist when the cells of the microorganism or an enzyme derived from the microorganism are allowed to act. (5) The method for producing ribose 1-phosphates according to the above (1) or (3), wherein the microorganism is a heat-resistant microorganism belonging to the genus Bacillus. (6) The method for producing a nucleoside compound according to (2) or (4), wherein the microorganism is a thermostable microorganism belonging to the genus Bacillus. (7) The microorganism is Bacillus coagulans YGK
-6054 (FERMP-17852) or Bacillus
Species YGK-6008 (FERMP-178
51) The method for producing ribose 1-phosphate according to any one of (1), (3) and (5) above. (8) The microorganism is Bacillus coagulans YGK
-6054 (FERMP-17852) or Bacillus
Species YGK-6008 (FERMP-178
51. The method for producing a nucleoside compound according to any one of (2), (4) and (6) above, which is 51). (9) Bacillus coagulans YGK-6054 (FERM P-1785) that produces thermostable phosphopentomtoses and thermostable nucleoside phosphorylases
2) and Bacillus species YGK-6008
(FERM P-17851).

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

<Raw Materials> Ribose 5-phosphate or 2-deoxyribose 5-phosphate (hereinafter referred to as ribose 5-phosphate) used as a raw material in the present invention is commercially available (for example, manufactured by Sigma). Ribose 5-phosphates can be prepared by treating ribose or 2-deoxyribose (hereinafter referred to as riboses) with a phosphorylating agent such as phosphorus oxychloride.

The nucleobase as another raw material includes natural thymine, uracil, adenine, guanine, hypoxanthine and the like, as well as artificial nucleobase analogs recognized by nucleoside phosphorylase (eg, 5-bromouracil). , 5-fluorouracil, 5-trifluoromethyluracil, 2-aminopurine, 2,6-diaminopurine, 2-chloropurine, 6-chloropurine, 2,6
-Dichloropurine, 2-amino-6-chloropurine, 6
-Mercaptopurine, 6-methylthiopurine, 1-deazaadenine, 3-deazaguanine, benzimidazole, glyoxal-guanine, etc.) can be used.

<Synthesis of ribose 1-phosphate or 2-deoxyribose 1-phosphate and a nucleoside compound by enzymatic reaction> A microorganism containing a heat-resistant phosphopentomthase is added to a buffer solution containing ribose 5-phosphate. When the cells or the enzyme derived from the cells are added and the mixture is stirred within a suitable temperature range of the enzyme, the reaction represented by the formula (1) or (2) occurs, and ribose 1-phosphate or 2-deoxyribose 1-phosphate (Hereinafter referred to as ribose 1-phosphates).

[0022]

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Next, when a nucleobase and a nucleoside phosphorylase are present in the reaction solution, ribosylation is performed to produce a nucleoside compound. For example, in the presence of adenine, which is a purine base, the reaction represented by the formula (3) or (4) occurs due to the action of purine nucleoside phosphorylase (hereinafter abbreviated as PUNP in the formula), thereby producing adenosine or deoxyadenosine. In addition, when thymine which is a pyrimidine base is present, a reaction represented by the formula (5) or (6) is caused by the action of pyrimidine nucleoside phosphorylase (hereinafter abbreviated as PYNP in the formula), and 5-methyluridine or thymidine is generated. .

[0024]

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[0025]

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Therefore, for example, 2--
When deoxyribose 5-phosphate and adenine or thymine, and as an enzyme phosphopentomase and purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase coexist in the reaction solution, formulas (2) and (4) are used.
Alternatively, the reaction of the formula (2) and the reaction of the formula (6) occur continuously, and deoxyadenosine or thymidine is generated.

Since Bacillus coagulans YGK-6054 and Bacillus sp. YGK-6008 used as preferred microorganism strains in the present invention each contain a phosphopentmutase, a purine nucleoside phosphorylase and a pyrimidine nucleoside phosphorylase, the starting materials are continuously reacted. Nucleoside compounds corresponding to the above nucleobases can be synthesized.

Since microorganisms are living organisms, they usually contain enzymes such as phosphatase, nucleosidase, and deoxyribose phosphate aldolase for survival, which are ribose 1-phosphate and ribose 5-phosphate, respectively. Catalyzes the elimination of acids from phosphoric acid (decomposition into ribose or 2-deoxyribose), decomposition of nucleoside compounds, and decomposition of 2-deoxyribose 5-phosphate (decomposition into glyceraldehyde 3-phosphate and acetaldehyde). Are undesirable enzymes for the present invention. Therefore, microorganisms having no such enzyme activity are preferred.

Although it is well known that sodium ethylenediaminetetraacetate (EDTA) is effective as a phosphatase inhibitor, phosphopentamutase generally uses manganese ion (divalent) as an essential element of its activity, so that EDTA is effective. It acts in an inhibitory manner and cannot be added (En
Zyme Handbook, Page 5.4.2.
7 Phosphopentamutase, Spri
nger-Verlag Berlin Heider
berg, 1990). However, Bacillus coagulans YGK used as a preferred strain in the present invention
-6054 and Bacillus species YGK-60
No. 08 has extremely weak phosphatase activity, and does not actually require the addition of EDTA. In addition, nucleosidase activity is practically not observed.

On the other hand, a small amount of deoxyribose phosphate aldolase (hereinafter abbreviated as DERA) exists,
When 2-deoxyribose 5-phosphate is used as a raw material,
The addition of the aldehyde can reduce the equilibrium in the following formula (7) by 2
-Deoxyribose 5-phosphate shifted to the right side (right side) and found to act as an inhibitor of 2-deoxyribose 5-phosphate degradation. Aldehydes include acetaldehyde, propionaldehyde, formaldehyde, glycolaldehyde, glyoxal, glyceraldehyde and the like. Among them, acetaldehyde and propionaldehyde are preferred, and acetaldehyde is most preferred.

[0031]

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When ribose 5-phosphate is used as a raw material, no decomposition reaction is caused by the enzyme even when deoxyribose phosphate aldolase is present. If the amount is sufficient, it will not hinder the whole enzymatic reaction.

The present invention has been accomplished by separating such a microorganism strain from the natural world and discovering reaction conditions.

<Reaction conditions and separation / purification and quantification of product> Next, the reaction conditions will be described. In the present invention, the initial concentration of ribose 5-phosphate as a raw material is 5 to 200 mM, preferably 5 to 50 mM. The initial concentration of the starting nucleobase is 5 to 200 mM, preferably 5 to 50 mM.
mM. The initial concentration of the nucleobase is preferably not less than the initial concentration of ribose 5-phosphate. Generally, the higher the molar concentration of the nucleobase relative to the ribose 5-phosphate, the higher the yield of the nucleoside compound relative to the ribose 5-phosphate. However, when a nucleic acid base having a concentration higher than the saturation concentration is added, the nucleic acid base may be added in portions while observing the progress of the reaction. Initial concentration of aldehydes is 5-600 mM
And preferably 200 to 350 mM.

The pH of the reaction solution (buffer) is 6.0 to 11.
0, and preferably 9.0 to 11.0. The buffer is preferably a Tris-HCl buffer. The reaction may be carried out at an appropriate temperature range of the thermostable enzyme of 40 to 65 ° C, preferably 50 to 60 ° C. The reaction time depends on the reaction conditions, but the reaction is usually completed in 2 to 24 hours.

The nucleoside compound can be collected from the reaction solution by ultrafiltration, ion exchange separation, adsorption chromatography, or the like.

Qualitative / quantitative analysis of the reaction product can be performed by TLC, and more precise quantification can be performed by HPLC equipped with a UV detector and / or a refractometer.

<Method of Measuring Phosphopentomtase Activity in Screening> In screening of microorganisms from natural soil, phosphopentomthase activity was determined as follows.

In ribose 5-phosphate, which is a reaction substrate of phosphopentmutase, ribose 5-phosphate is easily decomposed by phosphatase, and 2-deoxyribose 5-phosphate is deoxyribose phosphate aldolase. It is easily decomposed by phosphatase. Further, the quantification of the reaction product, ribose 1-phosphate, was complicated. For this reason, it has been difficult to measure the phosphopentombase activity by quantifying the reaction substrate or the product.

Therefore, in screening, ribose 5
-Phosphoric acid, hypoxanthine and nucleoside phosphorylase were added to the reaction solution, and ribose 1-phosphate generated by phosphopentmutase was further converted to inosine or deoxyinosine, and the amount was quantified to evaluate the phosphopentomidase activity.

<Enzyme-producing microorganism and its identification> The microorganism used in the present invention may be of any origin. In the case of producing ribose 1-phosphate,
It is not particularly limited as long as it is a microorganism that contains a thermostable phosphopentomtose and does not substantially contain a phosphatase, and when a nucleoside compound is produced, it contains a thermostable phosphopentomtase and a thermostable nucleoside phosphorylase. The microorganism is not particularly limited as long as the microorganism is substantially free of phosphatase and nucleosidase. In the present invention, "substantially does not contain an enzyme" means that the enzyme does not contain the enzyme, or even if it does, the enzyme has only a weak activity that does not affect the production method of the present invention. Means no. As a microorganism satisfying this condition, a heat-resistant microorganism belonging to the genus Bacillus is preferably exemplified. More specific strains are preferably Bacillus coagulans (Bacil).
rus coagulans) YGK-6054 (F
ERM P-17852) and Bacillus sp. YGK-6008 (F
ERM P-17851). These strains have been deposited in Japan at the Patent Microorganisms Depositary Center of the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry under the above deposit number.

[0042] These microorganisms grow on a culture medium for bacteria, and addition of a sugar such as ribose or a nucleoside such as inosine to the culture medium is effective in increasing the production of phosphopentomtoses and nucleoside phosphorylases. As the nitrogen source, inorganic nitrogens such as ammonium chloride and ammonium sulfate or organic nitrogens such as peptone can be used. It is desirable to add manganese and magnesium to the medium. The cultured cells can be used as is in this enzyme reaction, but it is necessary to obtain and use enzymes derived from the microorganisms by the usual methods (ultrasonic or milling, centrifugation, ammonium sulfate separation, membrane separation, etc.). Can also.

The bacteriological properties of the deposited strain were determined by
The results of a study based on the Manual of Systematic Bacteriology Volume 1 (1984) and the Bergey's Manual of Deterministic Bacteriology Ninth Edition (1994) are as follows. The experiment was mainly performed by Takeharu Hasegawa, revised edition, "Classification and Identification of Microorganisms" (Academic Publishing Center, 1985)
It carried out by the method of description.

Bacillus coagulans (Bacill)
us coagulans) YGK-6054 (FE
RM P-17852) [hereinafter abbreviated as YGK-6054].

1. Morphological properties (1) Cell shape and size: Bacillus, 0.7 × 2 μm (2) Gram stainability: positive (3) Presence or absence of cell polymorphism: No (4) Motility: Yes (5) 1. Flagellated state: perihair (6) Spores: oval endogenous sporulation, located at end (7) Acid resistance: none Cultural properties (1) Gravy agar plate culture: irregular, whole edge wavy, low and flat, slightly glossy, cream color (2) Gravy agar slant culture: creamy, translucent, spreads over the entire medium and has good growth (3) Broth liquid culture: Medium turbidity and uniform (4) Broth gelatin puncture culture: Liquefaction of whole (when cooled) (5) Litmus milk: coagulation, pH 8 Physiological properties (1) Nitrate reduction: positive (2) Denitrification reaction: positive (3) MR test: negative (4) VP test: positive (5) Indole formation: negative (6) Hydrogen sulfide formation: negative (7) Starch hydrolysis: Negative (8) Use of citric acid: Positive (9) Use of inorganic nitrogen source:-Nitrate: Positive-Ammonium salt: Positive (10) Pigment production: Negative (11) Urease: Negative (12) Oxidase: Positive (13) Catalase: Positive (14) Range of growth ・ Growth at pH 5 to 6.8: grew ・ NaCl concentration: Grew at 1-2%, did not grow at 5% ・ Temperature range : Grown at 42-59 ° C (optimal 52-55)
℃), does not grow at 30 ℃ (15) Attitude to oxygen: facultative anaerobic (16) OF test ・ Glucose: F (no gas production) Necessary to show the characteristics of other species (1) Use of various carbon sources ・ Lactose:-・ Maltose: + ・ D-xylose: + ・ Mannitol: + ・ D-raffinose:-・ Sorbitol:-・ Sucrose: --Inositol:--Adonitol:--Rhamnose:--L-arabinose:--D-mannose:--Ribose: +-Galactose: +-D-glucose: +-D-fructose: +-N-acetylglucosamine : + Trehalose: + (2) β-galactosidase: positive (3) Arginine decarboxylation: negative (4) Lysine decarboxylation: negative (5) Ornithine decarboxylation: negative (6) Esculin hydrolysis: positive (7) Indole Production: negative (8) Arginine dihydrolase: negative (9) Gelatin hydrolysis: positive Chemical taxonomic properties (1) GC content: 50-52 mol% (HPLC method) Based on the above mycological properties, this strain was identified as Bacillus coagulans.

Bacillus species
s sp. ) YGK-6008 (FERM P-17)
851) [hereinafter abbreviated as YGK-6008].

1. Morphological properties (1) Cell shape and size: Bacillus, 0.8 × 2 to 3 μm (2) Gram staining: positive (3) Cell polymorphism: none (4) Motility: yes ( 5) Epiphytic state of flagella: perihair (6) Spore: oval endogenous spore formation, position at terminal (7) Acid resistance: none Cultural properties (1) Gravy agar plate culture: elliptical, smooth, low convex, slightly glossy, creamy (2) Gravy agar slant culture: creamy, opaque, grows and grows along the streaks (3) Gravy liquid culture: Medium turbidity and uniform (4) Gravy gelatin stab culture: Liquefaction of whole surface (when cooled) (5) Litmus milk: coagulation, pH 8 Physiological properties (1) Nitrate reduction: positive (2) Denitrification reaction: positive (3) MR test: negative (4) VP test: positive (5) Indole formation: negative (6) Hydrogen sulfide formation: negative (7) Starch hydrolysis: Negative (8) Use of citric acid: Positive (9) Use of inorganic nitrogen source:-Nitrate: Positive-Ammonium salt: Positive (10) Pigment production: Negative (11) Urease: Negative (12) Oxidase: Positive (13) Catalase: Positive (14) Range of growth • Growth at pH 5 to 6.8: Growing at pH 6.8 but not at pH 5 to 5.7 • NaCl concentration: 1% Grew, did not grow at 2-5% ・ Temperature range: Grew at 42-59 ° C (optimal 52-55
℃), does not grow at 30 ℃ (15) Attitude to oxygen: facultative anaerobic (16) OF test ・ Glucose: F (no gas production) Necessary to show characteristics of other species (1) Use of various carbon sources ・ Lactose:-・ Maltose: + ・ D-xylose: + ・ Mannitol: + ・ D-Raffinose:-・ Sorbitol:-・ Sucrose: --Inositol:--Adonitol:--Rhamnose:--L-arabinose:--D-mannose:--Ribose: +-Galactose: +-D-glucose: +-D-fructose: +-N-acetylglucosamine : + Trehalose: + (2) β-galactosidase: positive (3) Arginine decarboxylation: negative (4) Lysine decarboxylation: negative (5) Ornithine decarboxylation: negative (6) Esculin hydrolysis: positive (7) Indole Production: negative (8) Arginine dihydrolase: negative (9) Gelatin hydrolysis: positive Chemical taxonomic properties (1) GC content: 48 to 50 mol% (HPLC method) Based on the above mycological properties, this strain was identified as Bacillus sp.

[0048]

EXAMPLES The present invention will be described more specifically with reference to experimental examples and examples.

The identification of ribose 5-phosphate, ribose 1-phosphate and ribose was performed by TLC analysis. The analysis conditions are as follows.

[Method] TLC plate: Kieselgel 60F ₂₅₄ (manufactured by Merck) Developing solution: n-butanol / 2-propanol / water = 3/12 /
4 (v / v / v) Detection: ethanol / p-anisaldehyde / acetic acid / sulfuric acid = 9
0/5/1/5 (v / v / v / v) Color development: 90 to 100 ° C In addition, quantification of nucleobases and nucleoside compounds is performed by HPLC.
Performed by analysis. The analysis conditions are as follows.

[Method] Column: Inertsil ODS-2 (GL Science) φ4.6 mm × 250 mm Eluent: 8% methanol / 0.1M-NH ₄ H ₂ PO
₄ (pH 7.0) Flow rate: 1 mL / min Column temperature: 40 ° C. Detection: UV 260 nm The yield of the nucleoside compound was expressed in terms of percent molar concentration based on ribose-5-phosphate.

EXPERIMENTAL EXAMPLE 1: Isolation of strain from soil sample
Primary screening using Bose 5-phosphate as substrate Soil suspension (Soil is Kyoto City, Hirakata City, Osaka Prefecture, Hisahime Ehime Prefecture)
Inosine, thymidine or ribose
Medium containing a single carbon source (inosine,
2 g of midin or ribose, K_TwoHPO_Four 1 g, (NH
_Four)_TwoSO_Four 5 g, MnCl_Two・ 4H_TwoO 0.1 g, M
gSO_Four・ 7H_TwoO 0.8g, containing 25g agar, pH
7.0). Culture at 30 ° C, 60 ° C or 70 ° C
The grown and grown colonies were isolated. These isolated strains
Liquid medium containing the same carbon source as the separation medium (1 L medium)
Or 2 g of inosine, thymidine or ribose,_TwoH
PO_Four 1 g, (NH_Four)_TwoSO_Four 5 g, MnCl_Two・ 4
H_TwoO 0.1g, MgSO _Four・ 7H_TwoO 0.8g, yeast
Inoculate pH 7.0) containing 2 g of mother extract and culture
Was.

The obtained cells were assayed for phosphopentomidase activity as follows. 2 mL of the culture solution of the test microorganism was centrifuged to obtain stationary cells. 0.1 M Tris-HCl buffer (pH 7.4), 10 mM ribose 5-phosphate, 5 mM hypoxanthine, 0.1 mM-M
nCl ₂ .4H ₂ O, 0.02 mM glucose 1,6-
Diphosphate, nucleoside phosphorylase (Sigma)
0.06 units [pH 7.4, 1 μm per minute at 25 ° C.]
The amount of the enzyme that converts mol inosine to hypoxanthine and ribose 1-phosphate is defined as one unit. Was added to the above-mentioned static cells, and the mixture was shaken in a 2 mL Eppendorf tube for 6 hours (MnCl 2).
₂ · 4H ₂ O, and glucose 1,6-diphosphate has been reported as an activator of the reaction by phosphodiester pen mutase (Hammer-Jesperson, K., Mu
nch-Petersen, A .; , Eur. J. Bio
chem. , 17, 397-407 (1970)). The generated inosine was quantified by HPLC analysis to evaluate the phosphopentomthase activity. The culture temperature and reaction temperature were the same as the separation temperature. As a result, 30
Phosphopentomtase activity was observed in the strains isolated at 60 ° C and 60 ° C. Inosine was obtained in a yield of 2% or more,
16 strains isolated at 30 ° C. and 33 strains isolated at 60 ° C. were selected (Table 1).

[0054]

[Table 1]

Experimental Example 2 Investigation of Culture and Reaction Conditions of Primary Screening Selected Strains The optimal culture and reaction temperature of the primary screening selected strains were examined. Phosphopentomtase-active strains isolated at 60 ° C., when cultured and reacted at 50 ° C., had increased phosphopentmutase activity in many strains. On the other hand, the phosphopentmutase-active strain isolated at 30 ° C. was suitable for culture and reaction at 30 ° C. When the pH of the reaction solution was examined, the use of Tris-HCl buffer (pH 8.5) increased the activity.

Experimental Example 3: Secondary screening using ribose 5-phosphate as a substrate The strains selected in the primary screening were cultured in the same manner as in the primary screening. 2 mL of the culture solution was centrifuged to obtain stationary cells. 0.166 M Tris-HCl buffer (pH 8.5), 50 mM ribose 5-phosphate, 1
0mM hypoxanthine, 0.1mM-MnCl ₂ · 4H
0.5 mL of a reaction solution containing 0.06 units of ₂ O, 0.02 mM glucose 1,6-diphosphate, and nucleoside phosphorylase (manufactured by Sigma) was added to the quiescent cells described above.
The mixture was shaken for 6 hours in a mL Eppendorf tube. The generated inosine was quantified by HPLC analysis to evaluate the phosphopentomthase activity. The culture temperature and reaction temperature were set to 50 ° C. for the strain isolated at 60 ° C.
The temperature of the strain isolated at 0 ° C was 30 ° C. As a result, a high strain of phosphopentomthase was observed in the strain isolated at 60 ° C. Thirteen strains isolated at 60 ° C. from which inosine was obtained at a yield of 5% or more were selected (Table 2).

[0057]

[Table 2]

Experimental Example 4: Tertiary screening using 2-deoxyribose 5-phosphate as a substrate Strains selected in the secondary screening were cultured in the same manner as in the primary screening. 0.4 mL of the culture solution was centrifuged to obtain stationary cells. 0.166 M Tris-HCl buffer (pH 8.5), 5 mM 2-deoxyribose 5-phosphate, 5 mM hypoxanthine, 0.1 mM-M
nCl ₂ .4H ₂ O, 0.02 mM glucose 1,6-
Diphosphate, nucleoside phosphorylase (Sigma)
0.1 mL of the reaction solution containing 0.012 units is added to the above-mentioned quiescent cells, and 50 mL is added to the cells in a 2 mL Eppendorf tube.
The mixture was shaken at 6 ° C. for 6 hours. Deoxyinosine produced was quantified by HPLC analysis. The yield of the generated deoxyinosine was 0 to 6%. After the reaction, 2-
Deoxyribose 5-phosphate hardly remained, and it was considered that deoxyribose phosphate aldolase was decomposed into glyceraldehyde 3-phosphate and acetaldehyde.

When 333 mM acetaldehyde was added to the above reaction solution and the reaction was carried out in the same manner, the conversion efficiency to deoxyinosine was greatly increased in most of the strains. Among them, YGK-6054 and YGK-600
8 efficiently produced deoxyinosine in yields of 26.5% and 16.4%, respectively. After the reaction, the substrate 2-deoxyribose 5-phosphate remains almost unreacted,
By adding acetaldehyde, 2-deoxyribose 5-deoxyribose phosphate aldolase was used.
It was considered that the decomposition of phosphoric acid was suppressed. YGK-60
54 and YGK-6008 were selected as highly active phosphopentmutase bacteria.

Example 1: YGK-6054 and YGK-6
008-Degrading activity of 2-deoxyribose 5-phosphate at a final concentration of 166 mM Tris-HCl buffer (pH 9.
0), 50 mM 2-deoxyribose 5-phosphate,
0.1 mM glucose 1,6-diphosphate, 1 mM-M
Static cells of nCl ₂ , YGK-6054 or YGK-6008 (obtained by the method described in Example 9 described later)
A reaction solution containing 8% (w / v) was prepared. Further, a reaction solution was prepared by adding 0.1 M acetaldehyde at a final concentration to the above reaction solution. Apply 50 μL of each reaction solution to 2 m
The mixture was shaken at 55 ° C. for 2 hours in an L eppendorf tube. After the reaction, the amount of 2-deoxyribose produced and the amount of 2-deoxyribose 5-phosphate remaining were measured by TLC analysis (Table 3). In both strains, the production of 2-deoxyribose was minute and the phosphatase activity was weak.

In both strains, the amount of reduction of 2-deoxyribose 5-phosphate was large in the reaction solution without addition of acetaldehyde, but decreased in the reaction solution with addition of acetaldehyde. Both strains have deoxyribose phosphate aldolase activity, but by adding acetaldehyde, 2-deoxyribose phosphate aldolase can be used.
It was considered that the decomposition of deoxyribose 5-phosphate was suppressed.

[0062]

[Table 3]

Example 2 YGK-6054 and YGK-6
008 Nucleic acid substrates available for enzyme Various final nucleobases shown in Table 4 at 50 mM, 166
mM Tris-HCl buffer (pH 9.0), 50 mM 2
-Deoxyribose 5-phosphate, 0.1 mM glucose 1,6-diphosphate, 0.3 M acetaldehyde, YG
Static cells of K-6054 or YGK-6008 (obtained by the method described in Example 9 described later) 8% (w / v)
Was prepared and reacted by shaking at 55 ° C. for 2 hours in a 2 mL Eppendorf tube. The generated various nucleoside compounds were quantified by HPLC analysis. As a result, YGK-6054 and YGK-6008
Enzyme systems other than hypoxanthine, adenine, guanine, uracil, thymine, glyoxal-guanine, 5
-Fluorouracil and 5-bromouracil are used as substrates, and 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyuridine, thymidine, glyoxal-guanine-2'-deoxyriboside and 2'-deoxy-5, respectively. It was found that -fluorouridine and 2'-deoxy-5-bromouridine could be produced (Table 4).

[0064]

[Table 4]

Unlike the screening in the experimental examples, no commercially available nucleoside phosphorylase was used. Nevertheless, the results in Table 4 were obtained with YGK
This shows that -6054 and YGK-6008 produce thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase together with thermostable phosphopentomtoses, respectively. It was also shown that the enzyme systems of both strains utilize not only natural nucleobases but also artificial nucleobases as substrates.

Example 3 YGK-6054 and YGK-6
Effect of Culture Conditions and Medium Composition on Enzyme Production of 008 2 g of sugar or nucleoside shown in Table 5 per liter of K ₂
HPO ₄ 1 g, NH ₄ Cl 5 g, MnCl ₂ .4H ₂ O
0.1 g, was placed MgSO ₄ · 7H ₂ O 0.8g, medium (pH 7.0) 5 mL containing yeast extract 2g in a test tube, and sterilized. YGK-6054 or YGK-
6008 was inoculated and cultured at 50 ° C. overnight. Culture solution
4 mL was centrifuged to obtain quiescent cells. 0.166M
Tris-HCl buffer (pH 8.5), 5 mM 2-deoxyribose 5-phosphate, 5 mM hypoxanthine, 3
33 mM acetaldehyde, 0.1 mM MnCl _2.
0.1 mL of a reaction solution containing 4H ₂ O and 0.02 mM glucose 1,6-diphosphate was added to the above quiescent cells, and shaken at 50 ° C. for 6 hours in a 2 mL Eppendorf tube. Deoxyinosine produced was quantified by HPLC analysis.

As a result, when any of the strains was cultured in a medium to which no sugar or nucleoside was added, production of deoxyinosine was not observed. In the case of YGK-6054, a medium supplemented with inosine or thymidine, YGK-6
In the case of 008, the yield of deoxyinosine was particularly high when cultured in a medium to which ribose or 2-deoxyribose was added (Table 5).

[0068]

[Table 5]

Example 4: YGK-6054 and YGK-6
Effect of culture temperature on enzyme production of 008 2 g of inosine, 1 g of K ₂ HPO ₄ , NH ₄ per liter
Cl 5 g, MnCl ₂ .4H ₂ O 0.1 g, MgSO
₄ · 7H ₂ O 0.8g, medium containing yeast extract 2 g (p
H7.0) 10 mL was put into an L-shaped test tube and sterilized.
GK-6054 was inoculated. Similarly, YGK-6008 was inoculated into the above medium in which inosine was replaced with ribose. These were cultured with shaking at 42 to 63 ° C., and the change over time in the degree of growth (OD660) was observed. As a result, the two strains grew at 42 to 59 ° C., and the optimal culture temperature was 52 to 55 ° C. in view of the growth rate, and the maximum growth (OD660) was:
YGK-6054 was obtained at 52 ° C., and YGK-6008 was obtained at 55 ° C. (FIGS. 1 and 2).

Example 5: YGK-6054 and YGK-6
Effect of culture time on synthesis of nucleoside compounds by 008 2 g of inosine, 1 g of K ₂ HPO ₄ , NH ₄ per liter
Cl 5 g, MnCl ₂ .4H ₂ O 0.1 g, MgSO
₄ · 7H ₂ O 0.8g, medium containing yeast extract 2 g (p
H7.0) 10 mL was put into an L-shaped test tube and sterilized.
GK-6054 was inoculated. Similarly, YGK-6008 was inoculated into the above medium in which inosine was replaced with ribose. These are cultured with shaking at 52 ° C. and grown (OD6
60) The change with time was observed.

Further, a part of the culture solution was collected during the culture, and 0.4 mL of the culture solution was centrifuged to obtain stationary cells. 0.1
66 M Tris-HCl buffer (pH 8.5), 5 mM 2
-Deoxyribose 5-phosphate, 5 mM hypoxanthine, 333 mM acetaldehyde, 0.1 mM-Mn
Cl ₂ · 4H ₂ O, the reaction solution 0.1mL containing 0.02mM glucose 1,6-diphosphate in addition to the stationary cells,
The mixture was shaken at 50 ° C. for 6 hours in a 2 mL Eppendorf tube. Deoxyinosine produced was quantified by HPLC analysis.

As a result, in both strains, almost no production of deoxyinosine was observed in the cells (vegetative cells) at 0 to 5 hours of culture, which is the early stage of the growth phase. The use of bacterial cells (spore spore cells having endogenous spores formed therein) at 7 to 9 hours of culture, which corresponds to the late growth phase to the stationary phase, maximized the yield of deoxyinosine. The yield of deoxyinosine was low in the cells (spores) at 23 hours of culture, which corresponds to the death phase (FIGS. 3 and 4).

Example 6: YGK-6054 and YGK-6
Effect of Aldehydes on the Synthesis of Nucleoside Compounds by 008 0 to 1 M acetaldehyde or propionaldehyde at final concentration, 166 mM Tris-HCl buffer (pH
9.0), 50 mM 2-deoxyribose 5-phosphate, 50 mM hypoxanthine, 0.05 mM glucose 1,6-diphosphate, YGK-6054 or YGK-6
008 quiescent microbial cells (obtained by the method described in Example 9 below) each containing 50% of a reaction solution containing 8% (w / v).
L in a 2mL Eppendorf tube at 50 ° C
After shaking reaction for 2 hours, the generated deoxyinosine was quantified by HPLC analysis. As a result, the yield of deoxyinosine was significantly increased by adding 0.2 to 0.3 M acetaldehyde or propionaldehyde in both strains (FIGS. 5 and 6).

The reaction was carried out by replacing 2-deoxyribose 5-phosphate with ribose 5-phosphate in the above reaction solution, and the amount of inosine produced was quantified.
Addition of 0.3 M acetaldehyde had no effect on inosine yield (FIG. 7).

Example 7: YGK-6054 and YGK-6
Effect of Buffer Type and pH on Synthesis of Nucleoside Compounds by 008 166 mM Tris-HCl Buffer (pH 8.0-11.
0) 166 mM boric acid-sodium hydroxide buffer (pH 8 to 10), 166 mM glycine-sodium hydroxide buffer (pH 7.0 to 10.0), 5 mM potassium phosphate buffer (pH 6.0 to 7.5) ) Was considered. The buffer at the final concentration and the above concentration, 50 mM 2
-Deoxyribose 5-phosphate, 50 mM hypoxanthine, 0.05 mM glucose 1,6-diphosphate,
0.2M acetaldehyde, YGK-6054 or Y
A reaction liquid containing 8% (w / v) of 50% of a quiescent cell of GK-6008 (obtained by the method described in Example 9 described later)
L in a 2mL Eppendorf tube at 50 ° C
After shaking reaction for 2 hours, the generated deoxyinosine was quantified by HPLC analysis. As a result, in both strains, the yield of deoxyinosine was high when Tris-HCl buffer (pH 9.0 to 11.0) was used (FIGS. 8 and 9).

Example 8: YGK-6054 and YGK-6
Effect of Reaction Temperature on the Synthesis of Nucleoside Compounds by 008 at a Final Concentration of 166 mM Tris-HCl Buffer (pH 9.
0), 50 mM 2-deoxyribose 5-phosphate, 5
0 mM hypoxanthine, 0.05 mM glucose 1,
6-diphosphate, 0.2 M acetaldehyde, YGK-
50 μL of a reaction solution containing 8% (w / v) of quiescent bacterial cells of 6054 or YGK-6008 (obtained by the method described in Example 9 described later) was prepared, and the mixture was placed in a 2 mL Eppendorf tube at 30 to 70 ° C. For 2 hours with shaking. Deoxyinosine produced was quantified by HPLC analysis.
As a result, in both strains, the yield of deoxyinosine was high when the reaction was performed at 50 to 60 ° C (FIG. 10).

Example 9: Synthesis of nucleoside compound under optimum culture conditions and optimum reaction conditions ・ Synthesis of nucleoside compound by YGK-6054 2 g of inosine, 1 g of K ₂ HPO ₄ , NH ₄ per liter
Cl 5 g, MnCl ₂ .4H ₂ O 0.1 g, MgSO
₄ · 7H ₂ O 0.8g, medium containing yeast extract 2 g (p
H7.0) 10 mL was placed in a 50 mL Erlenmeyer flask and sterilized. One platinum loop of YGK-6054 was inoculated with this, and 5
The mixture was shaken back and forth at 2 ° C. and 130 rpm for 9 hours to obtain a pre-culture solution. 100 mL of the above medium was placed in a 500 mL Erlenmeyer flask and sterilized.
Reciprocal shaking was performed for 18.5 hours at rpm. The culture was centrifuged to obtain quiescent cells.

100 mL of 0.85% sodium chloride solution
The cells were suspended and centrifuged again to obtain washed cells. The wet bacteria weighed 0.66 g and was in the state of spore cells.
A final concentration of 166 mM Tris-HCl buffer (pH 9.
0), 50 mM 2-deoxyribose 5-phosphate, 5
0 mM hypoxanthine, 0.1 mM glucose 1,6
- _{diphosphate, 1mM-MnCl 2 · 4H 2} O, 0.3M
Reaction solution 1 containing 8% (w / v) of acetaldehyde, YGK-6054 quiescent cells (obtained by the above culture)
80 μL was prepared. This was shaken at 55 ° C. in a 2 mL Eppendorf tube to perform a reaction. When the generated deoxyinosine was quantified over time by HPLC,
Four hours after the reaction, 17.8 mM of deoxyinosine was produced, and the yield was 35.6% (FIG. 11).

[0079] In addition, final concentration 166mM Tris - HCl buffer (pH 9.0), 50 mM ribose 5-phosphate, 50 mM hypoxanthine, 0.1 mM glucose 1,6-diphosphate, 1mM-MnCl ₂ · 4H ₂ O, YG
Resting cells of K-6054 (obtained by the above culture)
When 180 μL of a reaction solution containing 8% (w / v) was prepared, and the reaction was carried out in the same manner as above, and the amount of inosine produced was quantified, 11.7 mM of inosine was produced in the fourth hour of the reaction.
The yield was 23.3% (FIG. 12).

Synthesis of nucleoside compound by YGK-6008 2 g of ribose, 1 g of K ₂ HPO ₄ , NH ₄
Cl 5 g, MnCl ₂ .4H ₂ O 0.1 g, MgSO
₄ · 7H ₂ O 0.8g, medium containing yeast extract 2 g (p
H7.0) 10 mL was placed in a 50 mL Erlenmeyer flask and sterilized. One platinum loop of YGK-6008 was inoculated into this, and 5
Shake back and forth at 5 ° C and 130 rpm for 9 hours to obtain a preculture solution. 100 mL of the above medium is placed in a 500 mL Erlenmeyer flask and sterilized.
Shake back and forth for 14.5 hours at rpm. The culture was centrifuged to obtain quiescent cells. The cells were suspended in 100 mL of a 0.85% sodium chloride solution and centrifuged again to obtain washed cells. The wet bacterium weighed 0.72 g and was in the state of spore sac cells.

The reaction was carried out using YGK-6008 in the same manner as in the reaction conditions for YGK-6054. When 2-deoxyribose 5-phosphate was used as a substrate, the reaction 4
At the time, 16.7 mM of deoxyinosine was produced, and the yield was 33.3% (FIG. 11). Also, ribose 5
-When phosphoric acid is used as a substrate, 8.95 at 4 hours after the reaction
mM inosine was produced and the yield was 17.9% (FIG. 12).

[0082]

According to the present invention, 2-deoxyribose 5-phosphate or ribose 5 which is a ribose 5-phosphate is used.
-Ribose 1-phosphate, 2-deoxyribose 1-phosphate or ribose 1-phosphate corresponding to each of the above-mentioned ribose 5-phosphates efficiently and efficiently by causing an enzyme of a heat-resistant microorganism to act on phosphate. Acids can be produced. In addition, a nucleoside compound can be efficiently produced with high yield from 2-deoxyribose 5-phosphate or ribose 5-phosphate, which is a ribose 5-phosphate, and a nucleic acid base by the enzyme of the microorganism.

[Brief description of the drawings]

Fig. 1 Bacillus coagulans YGK-605
4 is a graph showing the influence of the culture temperature on the growth degree of No. 4.

Fig. 2 Bacillus species YGK-6008
5 is a graph showing the influence of the culture temperature on the growth degree of A.

FIG. 3 Bacillus coagulans YGK-605
4 is a graph showing the influence of the culture time on the synthesis of a nucleoside compound by No. 4.

Fig. 4 Bacillus species YGK-6008
4 is a graph showing the influence of the culture time on the synthesis of a nucleoside compound by the method.

FIG. 5: Bacillus coagulans YGK-605
4 is a graph showing the effect of aldehydes on the synthesis of nucleoside compounds from 2-deoxyribose 5-phosphate and nucleobases by No. 4.

FIG. 6: Bacillus species YGK-6008
3 is a graph showing the effect of aldehydes on the synthesis of nucleoside compounds from 2-deoxyribose 5-phosphate and nucleobases by HPLC.

FIG. 7: Bacillus coagulans YGK-605
4 is a graph showing the effect of aldehydes on the synthesis of nucleoside compounds from ribose 5-phosphate and nucleobases by Bacillus species YGK-6008.

FIG. 8: Bacillus coagulans YGK-605
4 is a graph showing the effect of the type of buffer and pH on the synthesis of nucleoside compounds according to Example 4.

FIG. 9 is a graph showing the effect of buffer type and pH on the synthesis of nucleoside compounds by Bacillus species YGK-6008.

FIG. 10: Bacillus coagulans YGK-60
The graph which shows the influence of the reaction temperature on the synthesis | combination of the nucleoside compound by 54 or Bacillus species YGK-6008.

FIG. 11: Time course of the synthesis of nucleoside compounds from 2-deoxyribose 5-phosphate and nucleobases by Bacillus coagulans YGK-6054 or Bacillus species YGK-6008 under optimal culture conditions and optimal reaction conditions A graph showing.

FIG. 12: Ribose 5-mediated by Bacillus coagulans YGK-6054 or Bacillus species YGK-6008 under optimal culture conditions and optimal reaction conditions.
The graph which shows the time-dependent change of the synthesis | combination of the nucleoside compound from a phosphoric acid and a nucleobase.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12R 1:07) C12R 1:07) (C12N 1/20 (C12N 1/20 E C12R 1:07) C12R 1:07 ) (C12N 1/20 (C12N 1/20 A C12R 1:07) C12R 1:07) (C12P 19/02 (C12P 19/02 C12R 1:07) C12R 1:07) (72) Inventor Yonosuke Sunaga Tokyo 3-37-1, Sakashita, Itabashi-ku, Tokyo Organic Synthetic Pharmaceutical Co., Ltd., Tokyo Research Laboratory (72) Inventor Toyoki Sato 3-37-1, Sakashita, Itabashi-ku, Tokyo Organic Synthetic Pharmaceutical Co., Ltd. 4B064 AF33 CA03 CA21 CD05 DA01 4B065 AA15X AC14 BB05 BB11 BB15 CA23 CA44

Claims (9)

[Claims] 1. Ribose 5 selected from the group consisting of ribose 5-phosphate and 2-deoxyribose 5-phosphate.
-Phosphates containing thermostable phosphopentomtoses,
The respective ribose 5 selected from the group consisting of ribose 1-phosphate and 2-deoxyribose 1-phosphate by the action of a cell of a microorganism substantially free of phosphatase or an enzyme derived from the microorganism. -A method for producing ribose 1-phosphate, which comprises obtaining ribose 1-phosphate corresponding to phosphates. 2. Ribose 5 selected from the group consisting of ribose 5-phosphate and 2-deoxyribose 5-phosphate.
-Phosphates and nucleobases, containing a thermostable phosphopentomtose and a thermostable nucleoside phosphorylase, and an enzyme derived from the microorganism or a microorganism substantially free of phosphatase and nucleosidase,
A method for producing a nucleoside compound, comprising obtaining a nucleoside compound corresponding to each of the aforementioned ribose 5-phosphates selected from the group consisting of nucleosides and 2'-deoxynucleosides. 3. The method for producing ribose 1-phosphates according to claim 1, wherein an aldehyde is allowed to coexist when the cells of the microorganism or an enzyme derived from the microorganism are allowed to act. 4. The method for producing a nucleoside compound according to claim 2, wherein an aldehyde is allowed to coexist when the cells of the microorganism or an enzyme derived from the microorganism are allowed to act. 5. The method for producing ribose 1-phosphates according to claim 1, wherein the microorganism is a thermostable microorganism belonging to the genus Bacillus. 6. The method for producing a nucleoside compound according to claim 2, wherein the microorganism is a thermostable microorganism belonging to the genus Bacillus. 7. The method according to claim 7, wherein the microorganism is Bacillus coagulans YGK-6054 (FERM P-17852) or Bacillus species YGK-6008 (FERM).
The method for producing ribose 1-phosphates according to any one of claims 1, 3 or 5, which is P-17851). 8. The microorganism according to claim 1, wherein the microorganism is Bacillus coagulans YGK-6054 (FERM P-17852) or Bacillus species YGK-6008 (FERM).
The method for producing a nucleoside compound according to any one of claims 2, 4 or 6, which is P-17851). 9. A Bacillus bacterium which produces a thermostable phosphopentomtase and a thermostable nucleoside phosphorylase.
Coagulans YGK-6054 (FERM P-1
7852) and Bacillus species YGK-6
008 (FERM P-17851).