JP2003070494A - Method of producing 2'-deoxynucleoside compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of selectively producing 2'-deoxynucleoside compounds by using inexpensively and readily available dihydroxyacetone phosphate and acetaldehyde as starting raw materials or by using glyceraldehydes-3- phosphate and acetaldehyde as starting raw materials. SOLUTION: Glyceraldehyde-3-phosphate and acetaldehyde or dihydroxy- acetone phosphate and acetaldehyde are allowed to react with each other in the presence of the cell bodies of a microorganism including 2-deoxyribose-5- phosphate aldolase and triose phosphate isomerase or an enzyme originating from the microorganism to form 2-deoxyribose-5-phosphate. Then, the 2- deoxyribose-5-phosphate and the nucleic acid base are allowed to react with each other of the presence of the cell bodies of microorganism including phosphopentomutase and nucleoside phosphorylase to produce the 2'- deoxynucleoside compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for selectively producing a 2′-deoxynucleoside compound useful as a raw material for antiviral agents, antisense drugs and the like and a substrate for nucleic acid synthesis by a biochemical method. Regarding

[0002]

2. Description of the Related Art Conventionally, as a method for producing a 2'-deoxynucleoside compound, a method of extracting and producing from natural fish sperm is known as a conventional method. However, the starting DNA (deoxyribonucleic acid) is expensive and the separation and purification process is complicated, so that the 2'-deoxynucleoside compound cannot be obtained at low cost.

On the other hand, a method for obtaining a desired nucleoside by carrying out a base exchange reaction with a nucleoside phosphorylase using a 2'-deoxynucleoside compound and a nucleobase as a raw material has been reported (Hori. N., Water.
nabe, M .; , Yamazaki, Y .; , Mikam
i, Y. , Agric. Biol. Chem. , 53,
197-202 (1989)).

This method is the ribosylation of nucleobases with ribose 1-phosphate or 2-deoxyribose 1-phosphate, catalyzed by nucleoside phosphorylase. That is, according to this method, it is possible to prepare the corresponding 2'-deoxynucleoside compound by the action of nucleoside phosphorylase, using 2-deoxyribose 1-phosphate and an arbitrary nucleic acid base as raw materials.

However, a method of biochemically synthesizing a 2'-deoxynucleoside compound using an inexpensive and easily available substance as a starting material without using the existing 2'-deoxynucleoside compound as a raw material has not been established.

[0006]

Therefore, first, 2-deoxyribose 1-phosphate is replaced with 2-deoxyribose 5-phosphate.
We have completed an invention relating to a method for producing 2-deoxyribose 1-phosphate, which is biochemically produced from phosphoric acid by phosphopentomase, and an invention relating to a method for producing 2'-deoxynucleoside (Japanese Patent Application No. 2000-28918).
7).

Then, 2-deoxyribose 5-phosphate, which is a raw material of the invention described above, is converted from glyceraldehyde 3-phosphate (or dihydroxyacetone phosphate) and acetaldehyde into a catalyst for 2-deoxyribose 5-phosphate aldolase. Biochemically produced by the action, the invention relating to a method for producing 2-deoxyribose 5-phosphate was completed (Japanese Patent Application No. 2001-58902).

However, in either method,
Glyceraldehyde 3-phosphate (or dihydroxyacetone phosphate) and acetaldehyde, which are inexpensive and easily available, are used as starting materials, and the intermediate product (2-deoxyribose 5-phosphate) is not isolated but 2'- At present, the method for obtaining the deoxynucleoside compound, that is, the method for coupling the reaction by the enzyme derived from the microorganism has not been studied.

Therefore, an object of the present invention is to selectively produce a 2'-deoxynucleoside compound using glyceraldehyde 3-phosphate and acetaldehyde as starting materials or dihydroxyacetone phosphoric acid and acetaldehyde as starting materials. Is to provide.

[0010]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that glyceraldehyde 3-phosphate and acetaldehyde can be coupled by coupling a reaction by a microorganism-derived enzyme. As a starting material, or using dihydroxyacetone phosphoric acid and acetaldehyde as starting materials, 2-deoxyribose 5-phosphoric acid was produced, and then a method for selectively producing a 2'-deoxynucleoside compound was found to complete the present invention. Came to do.

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

(1) Glyceraldehyde 3-phosphate and acetaldehyde are reacted with cells of a microorganism containing 2-deoxyribose 5-phosphate aldolase or an enzyme derived from the microorganism to give 2-deoxyribose 5- A phosphoric acid is produced, and then the 2-deoxyribose 5-phosphate and a nucleobase are reacted with a microbial cell containing a phosphopentomtase and a nucleoside phosphorylase or an enzyme derived from the microorganism. Method for producing a'-deoxynucleoside compound.

(2) Dihydroxyacetone phosphate and acetaldehyde are reacted with a microbial cell containing a 2-deoxyribose 5-phosphate aldolase and a triose phosphate isomerase or an enzyme derived from the microorganism,
2-deoxyribose 5-phosphate is formed and then the 2
-A method for producing a 2'-deoxynucleoside compound, which comprises reacting deoxyribose 5-phosphate and a nucleobase with a microbial cell containing a phosphopentomase and a nucleoside phosphorylase or an enzyme derived from the microorganism.

(3) The microorganism containing 2-deoxyribose 5-phosphate aldolase belongs to the genus Klebsiella, the genus Enterobacter, or the genus Escherichia, and the microorganism containing phosphopentomase and nucleoside phosphorylase belongs to the genus Bacillus. The method for producing a 2'-deoxynucleoside compound according to (1) above, which is a microorganism to which the microorganism belongs.

(4) A microorganism containing 2-deoxyribose 5-phosphate aldolase and triosephosphate isomerase is a microorganism belonging to the genus Klebsiella, Enterobacter, or Escherichia, and contains phosphopentomase and nucleoside phosphorylase. Is a microorganism belonging to the genus Bacillus, 2'of the above (2)
-A method for producing a deoxynucleoside compound.

(5) The microorganism containing 2-deoxyribose 5-phosphate aldolase is Klebsiella pneumoniae B-44 (IFO 16579), and the microorganism containing phosphopentomase and nucleoside phosphorylase is Bacillus coagulans YGK-605.
4 (FERM P-17852), or Bacillus species YGK-6008 (FERM P-1785)
The method for producing a 2′-deoxynucleoside compound according to the above (1) or (3), which is 1).

(6) The microorganism containing 2-deoxyribose 5-phosphate aldolase and triosephosphate isomerase is Klebsiella pneumoniae B-44 (IF
O16579) and a microorganism containing phosphopentomase and nucleoside phosphorylase is Bacillus coagulans YGK-6054 (FERM P-17
852), or Bacillus species YGK-60
08 (FERM P-17851), the method for producing the 2′-deoxynucleoside compound according to the above (2) or (4).

(7) Coexistence of aldehydes when 2-deoxyribose 5-phosphate and a nucleobase are reacted with microbial cells containing a phosphopentomtase and a nucleoside phosphorylase or an enzyme derived from the microorganism The method for producing the 2'-deoxynucleoside compound according to any one of the above (1) to (6), characterized in that

(8) When 2-deoxyribose 5-phosphate and a nucleobase are reacted with a microbial cell containing a phosphopentomtase and a nucleoside phosphorylase or an enzyme derived from the microorganism, ammonium acetate or ammonium formate is added. The above (1) characterized by coexistence
~ The method for producing a 2'-deoxynucleoside compound according to any one of (7).

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

<Synthesis of 2'-deoxynucleoside compound by enzymatic reaction> In the present invention, first, as shown in the following formula (1), glyceraldehyde 3-phosphate and acetaldehyde are used as starting materials and 2-deoxyribose 5 is used. -2-deoxyribose 5-phosphate is produced by the catalytic action of a microbial cell containing a phosphate aldolase (hereinafter abbreviated as DERA in the formula) or an enzyme derived from the microorganism.
The equation (1) is an equilibrium reaction, but the equilibrium is biased toward the production side of 2-deoxyribose 5-phosphate.

[0022]

[Chemical 1]

Further, as shown in the following formula (2), glyceraldehyde 3-phosphate is obtained by converting dihydroxyacetone phosphate into triosephosphate isomerase (hereinafter, TP in the formula).
It can be produced by isomerizing by using the catalytic action of the bacterial cells of a microorganism containing I) or an enzyme derived from the microorganism.

[0024]

[Chemical 2]

In the present invention, when triose phosphate isomerase and 2-deoxyribose 5-phosphate aldolase are present at the same time, dihydroxyacetone phosphate and acetaldehyde are used as raw materials for the microorganism of the microorganism or the enzyme derived from the microorganism. By the catalytic action, the isomerization of the formula (2) and the subsequent aldolase reaction of the formula (1) continuously occur to produce 2-deoxyribose 5-phosphate.

Next, as shown in the following formulas (3) and (4), the above reaction solution (that is, a reaction solution containing 2-deoxyribose 5-phosphate), a nucleobase (adenine in the formula), and Phosphopentmutase (hereinafter abbreviated as PPM in the formula) and nucleoside phosphorylase (hereinafter N in the formula)
A 2′-deoxynucleoside compound can be produced via 2-deoxyribose 1-phosphate by the catalytic action of a microbial cell containing P) or an enzyme derived from the microorganism.

[0027]

[Chemical 3]

[0028]

[Chemical 4]

In the present invention, the transition of the reaction from the formula (1) to the formula (3) is carried out continuously without isolating the 2-deoxyribose 5-phosphate obtained by the reaction of the formula (1) from the reaction solution. You can do it. The continuous reaction from the formula (1) to the formula (3) is referred to as "reaction coupling" or "reaction coupling" in the present invention. First, the reaction coupling is (1).
After the reaction of the formula (or the reactions of the formulas (2) and (1)) is performed, and after a reaction time such that 2-deoxyribose 5-phosphate is produced, the formulas (3) and ( It can be carried out by adding a microorganism which catalyzes the reaction of the formula 4) and a nucleobase to the reaction solution. Alternatively,
When carrying out the reaction of the formula (1) (or when carrying out the reactions of the formulas (2) and (1)), the microorganisms which catalyze the reactions of the formulas (3) and (4) and the nucleobase have already been added. It may be added. However, the microorganisms which catalyze the reactions of the formulas (3) and (4), and the nucleobase are preferably added after the production of 2-deoxyribose 5-phosphate (1)
It is desirable in that the reaction of the formula (or the reactions of the formulas (2) and (1)) smoothly proceeds.

In the description of the present invention, the reaction leading to the formation of 2-deoxyribose 5-phosphate (ie, (1)
The reaction of the formulas and the formula (2)) and the subsequent reaction for producing the 2′-deoxynucleoside compound (that is, the reactions of the formulas (3) and (4)) are described separately for convenience. Is capable of continuously performing each reaction without isolating an intermediate product (2-deoxyribose 5-phosphate).

In order to continuously carry out the reaction from the formula (1) to the formula (3), it is preferable to carry out the reaction of the formula (3) in the presence of aldehydes. When the reaction of formula (3) is performed in the presence of aldehydes, the yield of the final product, 2′-deoxynucleoside, can be increased.
Due to the presence of aldehydes, 2-deoxyribose 5-
In the reaction system in which phosphate aldolase coexists, (1)
This is because the equilibrium reaction in the formula can be shifted to the production side (right side) of 2-deoxyribose 5-phosphate. Aldehydes include acetaldehyde, propionaldehyde, formaldehyde, glycolaldehyde,
Examples include glyoxal and glyceraldehyde. Of these, acetaldehyde and propionaldehyde are preferable, and acetaldehyde is most preferable.

Glucose 1,6-diphosphate has been reported as an activator of the reaction by phosphopentomase (Hammer-Jesperson, K.,
Munch-Petersen, A .; , Eur. J. B
iochem. , 17, 397-407 (1970)
Year)), it is desirable to add to the reaction system of formula (3).

<Raw material> The acetaldehyde, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, and nucleobase used as raw materials in the present invention are all commercially available (eg, Sigma-Aldrich Japan Co., Ltd., 20).
(2000 general catalog). It suffices that the compound used as the raw material of the present invention has a purity not lower than that which is usually used as an industrial raw material.

As the glyceraldehyde 3-phosphate, D-glyceraldehyde 3-phosphate and DL-glyceraldehyde 3-phosphate can be used. Dihydroxyacetone phosphoric acid is synthetically available. For example, dihydroxyacetone and acetyl phosphoric acid used as industrial raw materials are used as raw materials, and biochemically using dihydroxyacetone kinase (EC 2.7.1.19) as a catalyst. Can be synthesized (eg, Itoh, N., Tuj
ibata, Y. Liu, J .; Q. Appl. Mi
crobiol. Biotechnol. , 51 volumes, 1
93-200, 1999).

Nucleic acid bases which are another raw material include natural thymine, uracil, cytosine, adenine, guanine,
In addition to hypoxanthine and the like, artificial nucleobase analog compounds 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, benz Imidazole, glyoxal-guanine, etc.) can be used.

<Microorganisms and enzymes> 2 used in the present invention
-Deoxyribose phosphate aldolase (EC 4.1.
2.4), triosephosphate isomerase (EC5.
3.1.1), phosphopentmutase (EC 5.4.
2.7), and nucleoside phosphorylase (purine nucleoside phosphorylase (EC 2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.
2.2) which means both) can in principle be of any microbial origin. It should be noted that the microorganism used in the present invention does not use dihydroxyacetone phosphate as a substrate, and when glyceraldehyde 3-phosphate is used, the reaction does not need to contain triose phosphate isomerase. , May be contained.

All the microorganisms used in the present invention are preferably those substantially free of phosphatase and nucleosidase. In the present invention, the term "substantially free of phosphatase and nucleosidase" means that the enzyme is not contained, or even if the enzyme is contained, the enzyme shows only a weak activity that does not affect the production method of the present invention. Means no.

As the microorganisms satisfying the above-mentioned conditions and containing 2-deoxyribose 5-phosphate aldolase and triosephosphate isomerase, microorganisms preferably belonging to the family Enterobacteriaceae are mentioned, and specifically, Klebsiella spp.
Microorganisms belonging to the genus Enterobacter or the genus Escherichia are mentioned. As a more specific strain, Klebsiella pneumoniae B-44 (IFO 165 is preferable.
79). This strain is a fermentation research institute (In
stitute for Fermentation, Osaka; 2-17-85, juso-hon
machi, yodogawa-ku, Osaka, 532-8686, Japan) (March 1, 2001). According to the biosafety level of microorganisms created by the National Institute of Infectious Diseases, this strain belongs to Level 2, so the National Institute of Advanced Industrial Science and Technology, which is the international depositary authority under the Budapest Treaty (former AIST) ) The deposit was refused by the Institute of Biotechnology, and it was explained on February 27, 2001.

As the microorganisms which satisfy the above-mentioned conditions and contain phosphopentomtase and nucleoside phosphorylase, heat-resistant microorganisms preferably belonging to the genus Bacillus can be mentioned. Further, as a specific strain, preferably Bacillus coagulans
lans) YGK-6054 (FERM P-178
52), and Bacillus species
s sp. ) YGK-6008 (FERM P-17
851). This strain has been domestically deposited on May 12, 2000 at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, with the above deposit number.

In the present invention, the term “with a microbial cell of a microorganism or an enzyme derived from the microorganism” refers to a reaction using a suspension containing the microorganism (bacterial cell suspension) or an enzyme produced from the microorganism. Means to do. That is, the present invention is
The reaction may be carried out using the cell suspension itself, or the reaction may be carried out by taking out the enzyme produced by the microorganism.

<Reaction conditions and separation / purification and quantification of products>
First, the reaction conditions for 2-deoxyribose 5-phosphate aldolase and triose phosphate isomerase will be described. Hereinafter, the reaction until 2-deoxyribose 5-phosphate is produced is also referred to as “first reaction system”.

In the "first reaction system", the initial concentration of glyceraldehyde 3-phosphoric acid or dihydroxyacetone phosphoric acid, which is a starting phosphoric acid compound, is generally 5 to 500.
mM, preferably 25 to 150 mM. The initial concentration of acetaldehyde is generally 15-1000 mM
And preferably 150 to 400 mM. Also,
The higher the concentration of acetaldehyde with respect to the phosphate compound serving as the substrate, the higher the yield of 2-deoxyribose 5-phosphate with respect to the phosphate compound.

The pH of the reaction solution is usually 4.0 to 12.5, preferably 8.5 to 9.5. As the reaction solution, any buffer solution capable of adjusting the above pH can be used. When using glyceraldehyde 3-phosphate as a raw material, a tris-hydrochloric acid buffer solution is preferable. When dihydroxyacetone phosphate is used as the raw material, water may be used without using a buffer solution.

The reaction temperature is usually 20 to 60 ° C, preferably 25 to 40 ° C. Although the reaction time depends on the reaction conditions, it is usually completed in 2 to 6 hours.

The microorganism used in the "first reaction system" is preferably a preserved microorganism which is previously cultured in a nutrient medium for 3 to 25 hours, more preferably 8 to 20 hours. To be done. The bacterial cell concentration used in the reaction is preferably 1.0 to 20% by mass, and the higher the bacterial cell concentration, the more preferable in terms of producing 2-deoxyribose 5-phosphate.

Although the product can be transferred from the reaction solution to the next reaction with or without collection, if it is collected, ultrafiltration, ion exchange separation, adsorption chromatography can be carried out. It can be done by

The quantification of reaction products can be carried out by two methods. The first method is Burton
Law (eg, Tokyo Kagaku Dojin, Biochemistry Dictionary, 3rd
Ed., P. 664, 1998). This method is a highly specific quantitative method for detecting 2-deoxyribose with high sensitivity by the reaction of diphenylamine / acetic acid / sulfuric acid, and the absorption coefficient of 2-deoxyribose 5-phosphate is equal to that of 2-deoxyribose. The second method is cystein-sulfat, which is a colorimetric method for DNA.
e) is an application of the method (eg, Stumpf, P. et al.
K. J .; Biol. Chem. , 169, 367
~ 371, 1947). By this method, 2-deoxyribose 5-phosphate can be quantified.

Next, the reaction conditions using phosphopentomtase and nucleoside phosphorylase will be described. Less than,
2'-from 2-deoxyribose 5-phosphate and nucleobase
The reaction until the deoxynucleoside compound is produced is also referred to as "second reaction system".

When 2-deoxyribose 5-phosphate, which is a raw material of the "second reaction system", is used without being isolated from the "first reaction system", 2-deoxyribose 5-phosphate The initial concentration is the concentration obtained in the "first reaction system". In the "second reaction system", the initial concentration of 2-deoxyribose 5-phosphate as a raw material is preferably 5 to 200 mM, more preferably 5 to 100 mM. The initial concentration of the nucleobase as a raw material is generally 5 to 200 m.
M, preferably 5 to 100 mM. The nucleobase may be added to the reaction solution after a sufficient reaction time such that 2-deoxyribose 5-phosphate is produced in the "first reaction system" may be added. It may be added in advance to the reaction solution of "one reaction system". The initial concentration of nucleobase is preferably higher than the initial concentration of 2-deoxyribose 5-phosphate. In general, the higher the molar concentration of nucleobase relative to 2-deoxyribose 5-phosphate, the higher the yield of nucleoside compound relative to 2-deoxyribose 5-phosphate. However, when a nucleic acid base having a concentration higher than the saturation concentration is added, it may be added in portions while watching the progress of the reaction.

As described above, the "second reaction system" using phosphopentomtase and nucleoside phosphorylase is
It is preferably carried out in the presence of aldehydes. Due to the presence of aldehydes, the equilibrium reaction of the "first reaction system"
It becomes possible to shift to the production side of deoxyribose 5-phosphate. Aldehydes can be added at any time, provided that they are present during the "second reaction system". The initial concentration of aldehydes is generally 5 to 600 mM,
It is preferably 200 to 400 mM.

As described above, the reaction with phosphopentomtase is preferably carried out in the presence of glucose 1,6-diphosphate, which has been reported as an activator of the reaction. Glucose 1,6-diphosphate can be added at any time as long as it is present during the reaction with phosphopentomtase. The initial concentration of glucose 1,6-diphosphate may be a catalytic amount, but is preferably 0.01 to
It is 0.1 mM.

The pH of the reaction solution is usually 6.0 to 11.0, preferably 9.0 to 11.0. As the reaction solution, any buffer solution capable of adjusting the above pH can be used. Preferred is Tris-hydrochloric acid buffer solution.

The reaction temperature is within the optimum temperature range of the thermostable enzyme 40.
It suffices to carry out at ˜65 ° C., preferably 50˜60 ° C. The reaction time depends on the reaction conditions, but usually 4
~ 12 hours to finish.

As the microorganism used in the "second reaction system", preferably used is a preserved microorganism previously cultured in a nutrient medium for 5 to 15 hours, more preferably 7 to 9 hours. To be done. The bacterial cell concentration used in the reaction is preferably 1 to 20% by mass, but the higher the bacterial cell concentration, the more preferable in terms of producing the 2'-deoxynucleoside compound.

Optimum reaction conditions of the "first reaction system" (p
(H, temperature) and the optimum reaction conditions of the "second reaction system" are different from each other in that the reaction time is such that 2-deoxyribose 5-phosphate is produced in the "first reaction system". It is preferable to adjust it later, but during the whole reaction, pH
You may perform at 6.0-11.0 and temperature 40-60 degreeC.

Further, in the present invention, "first reaction system"
It was revealed that the reaction by phosphopentomtase was inhibited by dihydroxyacetone phosphate or glyceraldehyde 3-phosphate by continuously performing the "second reaction system" with. Glyceraldehyde 3-phosphate is the substrate for the reaction by 2-deoxyribose 5-phosphate aldolase, while dihydroxyacetone phosphate is converted to glyceraldehyde 3-phosphate by triosephosphate isomerase. Starting from dihydroxyacetone phosphoric acid or glyceraldehyde 3-phosphate, the product 2-deoxyribose 5-phosphate was not isolated,
When the 2'-deoxynucleoside compound is produced, it is inevitable that a small amount of dihydroxyacetone phosphoric acid or glyceraldehyde 3-phosphate is mixed as an unreacted substrate. Then, as a result of examination for the purpose of releasing the inhibitory effect, it was revealed in the present invention that the enzyme being inhibited can be activated by ammonium acetate or ammonium formate (see Example 3 described later).

It is effective to add ammonium acetate or ammonium formate to the phosphopentomtase reaction system, and the addition concentration is generally 5 to 1000 mM, preferably 200 to 300 mM. The timing of addition is not particularly limited as long as ammonium acetate or ammonium formate is present in the reaction system during the reaction with phosphopentomtase, and may be added in advance.

The 2'-deoxynucleoside 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 accurate quantification can be performed by HPLC equipped with a UV detector and / or a refractometer.

<Culture Conditions and Preparation of Enzymes> The mycological properties of the deposited strains are described in BarJace Manual of Systematic Bacteriology Vol. 1 (1984) and BarJace Manual of Determinant.
It was examined according to the 9th edition of bacteriology (1994). The results are as follows. The experiment was conducted mainly by the method described in the revised edition "Classification and Identification of Microorganisms", edited by Takeharu Hasegawa (Society Press Center, 1985).

Klebsiela Pneumonia (Klebs
iella pneumoniae) B-44 grows well in an ordinary bacterial culture medium and produces deoxyribose 5-phosphate aldolase and triosephosphate isomerase, but 2-deoxyribose, fructose, fructose 1,6-diphosphate is produced in the medium. It is effective to add 0.1 to 2.0% by mass of dihydroxyacetone phosphoric acid or the like to enhance the enzyme activity. Yeast extract, meat extract, peptone, etc. can be used as the carbon source and nitrogen source, and ammonium chloride, potassium nitrate, etc. can be used as the inorganic salt. The cultured cells can be used as they are for this enzymatic reaction, but an enzyme derived from the microorganism can be obtained by an ordinary method (crushing by ultrasonic waves or a mill, centrifugation, ammonium sulfate separation, membrane separation, etc.). Can also be used.

Klebsiela Pneumonia (Klebs
iella pneumoniae) B-44 (IF
O 16579) [hereinafter abbreviated as B-44]. 1. Morphological properties (1) Cell shape and size: bacillus, 0.8 × 0.8-
3.2 μm (2) Gram stainability: Negative (3) Presence or absence of cell polymorphism: None (4) Motility: None (5) Flagella engraftment state: None (6) Presence of spores: None (7 ) Anti-acidity: None 2. Cultural properties (1) Meat broth agar plate culture: circular, smooth on all edges, low convex, surface smooth, milky yellow (2) Slope culture on broth agar: milky yellow, opaque and spread well throughout the medium. (3) Liquid culture of broth: Medium turbidity and no color (4) Meat stab culture of broth: No change (5) Litmus milk: slightly acidic, coagulation, gas generation 3. Physiological properties (1) Nitrate reduction: Positive (2) Denitrification reaction: Negative (3) MR test: Positive (4) VP test: Negative (5) Indole formation: Negative (6) Hydrogen sulfide formation: Negative (7) Hydrolysis of starch: Negative (8) Utilization of citric acid-Koser medium: Positive-Christensen medium:
Positive (9) Utilization of inorganic nitrogen source: -Nitrate: Positive (weak) -Ammonium salt: Positive (weak) (10) Dye production: Negative (11) Urease: Negative (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range ・ pH: 3.5 to 10.2 (optimal 5.0 to 8.0) ・ Temperature range: 10 to 40 ° C (optimal 22 to 30 ° C) (15) Attitude toward oxygen : Uniform growth and gas generation (16) OF test glucose: F 4. Necessary for showing other characteristics (1) Utilization of various carbon sources-Lactose: + -Maltose: + -D-xylose: + -D-mannitol: + -Raffinose: + -D-sorbitol: + Claus: + -Inositol: + -Adonitol: + -L-rhamnose: + -L-arabinose: + -D-mannose: + (2) β-galactosidase: + (3) Arginine decarboxylation:-(4) Lysine desorption Carbonic acid: + (5) Ornithine decarboxylation :-( 6) Esculin hydrolysis: + (7) Utilization of organic acid-Malonic acid: + -Citric acid: + -Gluconic acid: + -n-capric acid: -Adipine Acid: -DL-malic acid: + (8) Use of acetamide :-( 9) Indole-pyruvate production :-( 10) Arginine dehydrolase:- 11) Gelatin hydrolysis: - (12) phenyl acetate assimilation: - 5. Chemical taxonomic properties (1) GC content: 50 to 52 mol% (HPLC method) Based on the above mycological properties, this strain is Klebsiella pneumoni.
ae).

Bacillus coagulans
us coagulans) YGK-6054 (FE
RM P-17852) and Bacillus sp. YGK-6008 (F
ERM P-17851) grows well in an ordinary bacterial medium and produces phosphopentomtase and nucleoside phosphorylase. Add 0.1 to 1% by mass of sugar such as ribose or nucleoside such as inosine to the medium. Is effective in increasing the enzyme activity. As the carbon source and the nitrogen source, inorganic nitrogens such as ammonium chloride and ammonium sulfate, or organic nitrogens such as peptone can be used. Further, it is desirable to add manganese and magnesium to the medium. The cultured cells can be used as they are in this enzymatic reaction, but they are usually processed by ultrasonication or milling, disruption, centrifugation, ammonium sulfate separation, membrane separation, etc.
It is also possible to obtain an enzyme derived from the microorganism and use it.

Bacillus coagulans
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: none (4) motility: yes (5) 1. Flagellation status: perilimbium (6) spores: oval endospore formation, position is terminal (7) anti-acidic: none 2. Culture properties (1) Meat broth agar plate culture: irregular shape, wavy edge, low flat, slightly glossy, cream color (2) Meat broth agar slope culture: cream color, translucent, spreads throughout the medium and grows well (3) Liquid culture of broth: medium turbidity and uniform (4) puncture culture of broth gelatin: whole liquefaction (when cooled) (5) litmus milk: coagulation, pH 8 3. Physiological properties (1) Reduction of nitrate: 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) Dye production: Negative (11) Urease: Negative (12) Oxidase: Positive (13) Catalase: Positive (14) Range of growth-Growth at pH 5 to 6.8: Growing-NaCl concentration: Growing at 1-2%, not growing at 5% -Temperature range : Grown at 42 to 59 ° C (optimal 52 to 55
3. C.), not grown at 30.degree. C. (15) Attitude toward oxygen: facultative anaerobic (16) OF test glucose: F (no gas production). Necessary to show the characteristics of other species (1) Utilization 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 5. Chemical taxonomic properties (1) GC content: 50 to 52 mol% (HPLC method) Based on the above mycological properties, this strain was found to be 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 stainability: positive (3) presence or absence of cell polymorphism: none (4) motility: yes ( 5) Flagellation status: Peripheral hair (6) Spores: Elliptical internal spore formation, position is terminal (7) Anti-acidity: None 2. Cultural properties (1) Meat broth agar plate culture: oval, smooth, low-convex, slightly shiny, cream-colored (2) Meat broth agar slope culture: cream-colored, opaque, grows and grows along streak Is good (3) Meat broth liquid culture: turbidity is moderate and uniform (4) Meat broth gelatin stab culture: whole liquefaction (when cooled) (5) Litmus milk: coagulation, pH 8 3. Physiological properties (1) Reduction of nitrate: 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) Dye production: Negative (11) Urease: Negative (12) Oxidase: Positive (13) Catalase: Positive (14) Range of growth-Growth at pH 5 to 6.8: Not grown at pH 5 to 5.7, grown at pH 6.8-NaCl concentration: 1% Grown, not grown at 2-5%, temperature range: grown at 42-59 ° C (optimal 52-55
3. C.), not grown at 30.degree. C. (15) Attitude toward oxygen: facultative anaerobic (16) OF test glucose: F (no gas production). Necessary to show the characteristics of other species (1) Utilization 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 5. Chemical taxonomic properties (1) GC content: 48 to 50 mol% (HPLC method) Based on the above mycological properties, this strain was found to be Bacillus sp.

[0065]

EXAMPLES The present invention will be described in more detail with reference to production examples and examples.

Deoxyribose 5-phosphate was identified by TL
C analysis was performed. The analysis conditions are as follows. [TLC analysis conditions] TLC plate: Kieselgel 60F ₂₅₄ (manufactured by Merck) Developer: 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: 90-100 ° C

The quantification of the 2'-deoxynucleoside compound was performed by HPLC analysis. The analysis conditions are as follows. [HPLC analysis conditions] Column: Inertsil ODS-2 (manufactured by GL Sciences) φ4.6 mm × 250 mm Eluent: 8% methanol / 0.1M-NH4H2PO4
(PH 7.0) Flow rate: 1 ml / min Column temperature: 40 ° C. Detection: UV 260 nm

Production Example 1: Preparation of cell suspension containing 2-deoxyribose 5-phosphate aldolase and triosephosphate isomerase and confirmation of enzyme activity <Preparation of wet cells> 0.3% 2-deoxyribose, 0 ．
2% ammonium chloride, 0.1% monopotassium phosphate,
Medium containing 0.1% dipotassium phosphate, 0.03% magnesium sulfate heptahydrate, 0.01% yeast extract (pH
5 ml of 7.0) was put into a test tube (16 × 165 mm) and sterilized. One platinum loop of B-44 was inoculated into this and cultured at 28 ° C. for 2 days with shaking at 300 rpm. This culture solution
A 2 L Erlenmeyer flask containing 500 mL of the above medium was inoculated and shake-cultured at 28 ° C. and 120 rpm for 10 to 12 hours. The cells were washed twice with 0.85% saline to obtain wet bacterial cells.
Hereinafter, this wet microbial cell is referred to as B-44 wet microbial cell.

<Confirmation of Enzymatic Activity of Wet Bacteria> 200 mM Tris-HCl buffer (pH 9.0), 200 mM acetaldehyde, 87.5 mM DL-glyceraldehyde 3-
To 60 μL of aqueous solution prepared to be phosphoric acid, 1
2.5% (w / v) B-44 wet bacterial cells were added, and the mixture was stirred at 30 ° C. for 3 hours. The supernatant obtained by centrifugation is TLC
2-deoxyribose 5-phosphate was detected as a result of analysis by.

Similarly, 200 mM acetaldehyde, 1
16.6% (w / v) was added to 60 μL of an aqueous solution prepared to be 16.6 mM dihydroxyacetone phosphate.
B-44 wet cells were added and stirred at 30 ° C. for 3 hours. As a result of TLC analysis of the supernatant obtained by centrifugation, 2
-Deoxyribose 5-phosphate was detected.

Production Example 2: Preparation of cell suspension containing phosphopentomtase and nucleoside phosphorylase and confirmation of enzyme activity <Preparation of wet cells> 0.2% inosine, 0.1% dipotassium phosphate, 0. Medium containing 10 ml of 5% ammonium chloride, 0.01% manganese chloride / tetrahydrate, 0.08% magnesium sulfate / heptahydrate, 0.2% yeast extract (pH
7.0) was placed in a 50 ml Erlenmeyer flask and sterilized. YGK-6054 was inoculated with 1 platinum loop of this, and the temperature was maintained at 52 ° C for 13
The medium was reciprocally shaken at 0 rpm for 9 hours to obtain a preculture liquid. 100 ml of the above medium is put into a 500 ml Erlenmeyer flask and sterilized, and 5 ml of the pre-cultured liquid is added to the mixture at 52 ° C and 100 rpm for 1
It was shaken back and forth for 8.5 hours. The culture solution was centrifuged to obtain quiescent bacterial cells. The cells were suspended in 100 ml of 0.85% saline and centrifuged again to obtain washed cells. Hereinafter, this wet microbial cell is referred to as YGK-6054 wet microbial cell. Wet weight 0.6
At 6 g, it was in the state of sporangia cells.

<Confirmation of enzyme activity of wet cells> 0.3 M Tris-hydrochloric acid buffer (pH 9.0), 50 mM 2-deoxyribose 5-phosphate, 50 mM adenine, 0.1 mM glucose 1,6-diphosphate, 0.3M acetaldehyde,
50 μl of a reaction solution containing 12.5% (w / v) YGK-6054 wet bacterial cells was prepared. The reaction was carried out by shaking at 50 ° C. for 6 hours. The supernatant obtained by centrifugation is subjected to HPLC
As a result of analysis by 1), 11.5 mM of 2'-deoxyadenosine was detected.

Example 1: Coupling of reaction with 2-deoxyribose 5-phosphate aldolase and reaction with phosphopentmutase and nucleoside phosphorylase 200 mM Tris-hydrochloride buffer (pH 9.0), 20
0 mM acetaldehyde, 87.5 mM DL-glyceraldehyde 3-phosphate and 12.5% (w / v) B.
A 50 μL aqueous solution prepared to be −44 wet cells was stirred at 30 ° C. for 5 hours. 600m in the reaction liquid
Contains M Tris-hydrochloric acid buffer (pH 9.0), 600 mM acetaldehyde, 100 mM various nucleobases, 0.1 mM glucose 1,6-diphosphate and 25.0% (w / v) YGK-6054 wet cells. Add 50 μl of solution,
The mixture was stirred at 50 ° C for 12 hours. The supernatant obtained by centrifugation was analyzed by HPLC.

The results are shown in Table 1. When adenine, hypoxanthine and cytosine were added, the corresponding 2'-deoxyadenosine, 2'-deoxyinosine and 2'-deoxycytidine were obtained, respectively.

Example 2: Coupling of reaction with triose phosphate isomerase and 2-deoxyribose 5-phosphate aldolase and reaction with phosphopentmutase and nucleoside phosphorylase 200 mM acetaldehyde, 116.6 mM dihydroxyacetone phosphate and 16.6% (W / v) B-4
50 μL of aqueous solution prepared to give 4 wet cells,
The mixture was stirred at 0 ° C for 5 hours. 600 mM Tris-HCl buffer (pH 9.0), 600 mM acetaldehyde, 100 mM various nucleobases, 0.1 mM glucose 1,6-diphosphate and 25.0% (w / v) YGK were added to the reaction solution.
Add 50 μl of a solution containing -6054 wet bacterial cells and
It was stirred for 12 hours. The supernatant obtained by centrifugation is
It was analyzed by PLC.

The results are shown in Table 1. Adenine, guanine,
When hypoxanthine, cytosine, thymine and uracil were added, the corresponding 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, 2'-deoxycytidine, thymidine and 2'-deoxyuridine were obtained, respectively. Was given.

[0077]

[Table 1]

Example 3 Effect of Various Salts on Inhibition of Phosphopentomtase in Formation of 2'-Deoxynucleoside Compound As described above, the reaction with phosphopentomtase was carried out by dihydroxyacetone phosphate or glyceraldehyde 3-.
Inhibition by phosphoric acid has been raised as a new problem in the present invention. Therefore, the effect of various salts on this inhibition was examined in this example. In this example, a reaction for producing 2'-deoxyadenosine from 2-deoxyribose 5-phosphate and adenine was carried out, and whether or not various salts can alleviate the inhibitory effect of dihydroxyacetone phosphate in the reaction. I checked. The details are given below.

0.3M Tris-HCl buffer (pH 9.
0), 50 mM dihydroxyacetone phosphate (or no addition), 50 mM 2-deoxyribose 5-phosphate, 50
mM adenine, 0.1 mM glucose 1,6-diphosphate, 0.3M acetaldehyde, 0.2M various salts (dipotassium phosphate, sodium chloride, sodium acetate, ammonium acetate, ammonium formate, ammonium phosphate, or ammonium sulfate) , 25% (w / v) YG
50 μl of a reaction solution containing K-6054 wet bacterial cells was prepared.
The reaction was carried out by shaking at 50 ° C. for 6 hours. The supernatant obtained by centrifugation was analyzed by HPLC to detect the produced 2'-deoxyadenosine.

The results are shown in Table 2. In the absence of dihydroxyacetone phosphate, the effect of salt activation on phosphopentmutase was examined, and when ammonium acetate or ammonium formate was added among various salts,
The production activity of 2'-deoxyadenosine is 17
It improved to 0% and 150%.

Further, when the effect of releasing the inhibition by the salt was observed under the condition of inhibiting phosphopentomtase by adding dihydroxyacetone phosphate, it was found that the enzyme activity reduced to 22% by the addition of dihydroxyacetone phosphate showed that various salts Among them, the addition of ammonium acetate or ammonium formate restored the activity to 32% and 49%, respectively.

[0082]

[Table 2]

[0083]

INDUSTRIAL APPLICABILITY According to the present invention, three kinds of enzymes, 2-deoxyribose 5-phosphate aldolase, phosphopentomtase and nucleoside phosphorylase, are produced from acetaldehyde and glyceraldehyde 3-phosphate which are inexpensive and easily available. The desired 2'-deoxynucleoside compound can be selectively obtained by coupling the reactions with each other using.

Further, according to the present invention, four types of triose phosphate isomerase, 2-deoxyribose 5-phosphate aldolase, phosphopentomase and nucleoside phosphorylase are prepared from acetaldehyde and dihydroxyacetone phosphate which are inexpensive and easily available. By coupling reactions to each other using enzymes,
The desired 2'-deoxynucleoside compound can be selectively obtained.

Thus, the present invention has the advantage that the final product can be obtained without isolating the intermediate products of the reaction. Furthermore, in the present invention, it is difficult to shift each equilibrium reaction in a predetermined direction because a plurality of reactions are carried out in a reaction system in which a plurality of enzymes coexist, but the yield is improved by examining additives. In that respect, it has an excellent effect.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Document name] Consignment number change notification

[Submission date] April 16, 2002

[Former name of depositary institution] Ministry of International Trade and Industry, Institute of Industrial Science and Technology

[Old trust number] FERM P-17851

[Former name of depositary institution] Ministry of International Trade and Industry, Institute of Industrial Science and Technology

[Old consignment number] FERM P-17852

[Name of new depositary institution] Japan Patent Institute for Biotechnology, National Institute of Advanced Industrial Science and Technology

[New contract number] FERM BP-7897

[Name of new depositary institution] Japan Patent Institute for Biotechnology, National Institute of Advanced Industrial Science and Technology

[New contract number] FERM BP-7898

Continued front page    (72) Inventor Takashi Sakai             2-2 Ichijoji Jizohonmachi, Sakyo-ku, Kyoto-shi, Kyoto Prefecture               Mansion Yamato 501 (72) Inventor Kyota Saito             584, No. 584, Domonen, Kuromachi, Kanonji, Kagawa Prefecture             Sun Garden Murakuro No. 6 303 (72) Inventor Seiichiro Matsumoto             3-37-1, Sakashita, Itabashi-ku, Tokyo Organic synthetic drug             Shinko Co., Ltd. Tokyo Research Center (72) Inventor Mie Sasaki             3-37-1, Sakashita, Itabashi-ku, Tokyo Organic synthetic drug             Shinko Co., Ltd. Tokyo Research Center (72) Inventor Toyoki Sato             1-22-6 Denenchofu, Ota-ku, Tokyo (72) Inventor Yonosuke Sunaga             2-2-44 Sakurayama, Zushi City, Kanagawa Prefecture (72) Inventor Yoichi Mikami             672-2 Kamizuruma, Sagamihara City, Kanagawa Prefecture F term (reference) 4B064 AF34 CA21 CB30 CD05

Claims (8)

[Claims] 1. Glyceraldehyde 3-phosphate and acetaldehyde are reacted with a microbial cell containing 2-deoxyribose 5-phosphate aldolase or an enzyme derived from the microorganism to give 2-deoxyribose 5-phosphorus. An acid is generated, and then the 2-deoxyribose 5-phosphate and a nucleobase are reacted with a microbial cell containing a phosphopentomtase and a nucleoside phosphorylase or an enzyme derived from the microorganism. -A method for producing a deoxynucleoside compound. 2. Dihydroxyacetone phosphate and acetaldehyde are reacted with a microbial cell containing a 2-deoxyribose 5-phosphate aldolase and a triose phosphate isomerase or an enzyme derived from the microorganism to give 2-
Generating deoxyribose 5-phosphate, and then reacting the 2-deoxyribose 5-phosphate with a nucleobase by a microbial cell containing a phosphopentmutase and a nucleoside phosphorylase or an enzyme derived from the microorganism. A method for producing a characteristic 2'-deoxynucleoside compound. 3. A microorganism containing 2-deoxyribose 5-phosphate aldolase is a microorganism belonging to the genus Klebsiella, the genus Enterobacter, or the genus Escherichia,
The method for producing a 2'-deoxynucleoside compound according to claim 1, wherein the microorganism containing phosphopentomtase and nucleoside phosphorylase is a microorganism belonging to the genus Bacillus. 4. A microorganism containing 2-deoxyribose 5-phosphate aldolase and triosephosphate isomerase is a microorganism belonging to the genus Klebsiella, Enterobacter, or Escherichia, and the microorganism containing phosphopentomase and nucleoside phosphorylase is The method for producing a 2'-deoxynucleoside compound according to claim 2, which is a microorganism belonging to the genus Bacillus. 5. A microorganism containing 2-deoxyribose 5-phosphate aldolase is Klebsiella pneumoniae B-44 (IFO 16579), and a microorganism containing phosphopentomase and nucleoside phosphorylase is Bacillus coagulans YGK-6054.
(FERM P-17852), or Bacillus species YGK-6008 (FERM P-1785)
The method for producing a 2'-deoxynucleoside compound according to claim 1 or 3, which is 1). 6. A microorganism containing 2-deoxyribose 5-phosphate aldolase and triosephosphate isomerase is Klebsiella pneumoniae B-44 (IFO).
16579) and a microorganism containing phosphopentomase and nucleoside phosphorylase is Bacillus coagulans YGK-6054 (FERM P-178).
52), or Bacillus species YGK-600
8 (FERM P-17851).
A method for producing the described 2'-deoxynucleoside compound. 7. Aldehydes when reacting 2-deoxyribose 5-phosphate, which is a reaction intermediate, and a nucleobase with a microbial cell containing a phosphopentomtase and a nucleoside phosphorylase or an enzyme derived from the microorganism Coexistence of any one of claims 1 to 6
Item 2. A method for producing a 2'-deoxynucleoside compound according to Item. 8. Ammonium acetate when reacting 2-deoxyribose 5-phosphate, which is a reaction intermediate, and a nucleobase with a microbial cell containing a phosphopentomase and a nucleoside phosphorylase or an enzyme derived from the microorganism Alternatively, ammonium formate is allowed to coexist, and the method for producing a 2'-deoxynucleoside compound according to any one of claims 1 to 7.