JP2003018997A - Method of production for cytosine nucleoside compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective synthetic method of a cytosine nucleoside compound. SOLUTION: This method producing the cytosine compound from pentose-1- phosphate and cytosine or a cytosine derivative by using a nucleoside phosphorylase having reactivity to the cytosine or a microorganism having the enzymatic activity, the cytosine nucleoside compound is effectively produced by providing a method specifically reducing degrading of activity the matrix or the product. This method produces almost no by-products in the production of the cytosine nucleoside compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a cytosine nucleoside compound useful as a synthetic raw material for pharmaceuticals and the like. More specifically, an enzyme having a cytosine nucleoside phosphorylase activity or a bacterial cell of a microorganism having the enzymatic activity, an enzyme preparation obtained by treating the bacterial cell or a culture solution thereof, or the like is used to prepare pentose-1-phosphate. The present invention relates to a method for producing a cytosine nucleoside compound from cytosine or a cytosine derivative.

[0002]

2. Description of the Related Art Nucleoside phosphorylase is a general term for enzymes that phosphorolytically decompose N-glycoside bonds of nucleosides in the presence of phosphoric acid, and catalyzes a reaction represented by the following formula when ribonucleosides are taken as an example.

Ribonucleoside + phosphoric acid (salt) →
The enzyme, which is roughly classified into nucleobase + ribose 1-phosphate purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, is widely distributed in the living world and is present in tissues such as mammals, birds and fish, yeast, and bacteria. This enzymatic reaction is reversible, and the synthesis of various nucleosides using the reverse reaction has long been known. For example, from 2'-deoxyribose 1-phosphate and a nucleobase (thymine, adenine or guanine), thymidine (JP-A-01-104190), 2'-deoxyadenosine (JP-A-11-137290) or 2 '. -Deoxyguanosine (JP-A-11-137290)
Japanese Patent Publication No.) is known. As described above, the nucleoside compound using nucleoside phosphorylase can be produced positionally and stereospecifically under mild conditions, and the synthesis of many nucleoside compounds has been studied.

Japanese Unexamined Patent Publication (Kokai) No. 1-60396 discloses a method for producing deoxycytidine from deoxyribose-1-phosphate and cytosine by a reaction using the cells themselves as a catalyst. However, the method of the publication is a reaction using the cells themselves as a catalyst, and the presence of cytosine nucleoside phosphorylase itself has not been confirmed.
For example, deoxycytidine accumulated in the cells was eluted from the cells during the reaction, or the nucleoside deoxyribosyl transferase in the cells transferred the cytosine added as a substrate to the base of the deoxynucleosides in the cells. It is possible that deoxycytidine was detected. In order to clarify these points, the present inventors ordered 7 strains deposited in the examples of the publication and performed deoxyribose-1-in the same manner as the examples.
It was tested whether deoxycytidine was produced from phosphoric acid and cytosine. As a result, no deoxycytidine was detected in the reaction solution in any reaction using any strain, and it was not confirmed that the strain itself had an activity corresponding to cytosine nucleoside phosphorylase.

On the other hand, JP-A-3-12786 discloses a novel nucleoside phosphorylase that acts on both purine bases and pyrimidine bases, and describes that it acts on deoxyuridine, deoxycytidine and deoxythymidine as pyrimidine bases. However, the substrate specificity is not disclosed in the specification. Also,
Although the publication has already been withdrawn and its activity cannot be confirmed, the inventors of the publication purified the nucleoside phosphorylase from a strain having the same name as the microorganism disclosed in the publication and characterized it by Applied and E
nvironmental Microbilog
y, Vol. 56, PP3830-3834, (199
0). The document states that this enzyme has no activity on cytosine, cytidine and deoxycytidine.

Other than the above, it has not been reported that cytosine or a cytosine derivative serves as a substrate as a nucleobase in any of purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase. For example, Method. Enzymology, Vol. 5
1, PP437-442 (1978) salmonella
Typhimurium (Salmonera typhimu)
It is described that thymidine phosphorylase, which is a kind of pyrimidine nucleoside phosphorylase of Rium), has no reactivity with deoxycytidine. Also, J. Biol. Chem. , Vol. 248, N
o. 6, PP 2040 to 2043 (1973), Salmonella typhimurium (Salmonera type)
purine nucleoside phosphorylase of (Himurium) is described as having no activity on pyrimidine nucleosides, and uridine, cytidine, deoxyuridine,
Deoxycytidine is exemplified.

[0007] Considering these reports comprehensively, although there was a finding to predict the existence of an enzyme corresponding to the cytosine nucleoside phosphorylase according to the present invention, its existence was confirmed in any of the prior art documents, There is no report that this enzyme was actually obtained.

[0008]

The production of nucleoside compounds using nucleoside phosphorylase allows positionally and stereospecifically producing nucleoside compounds under mild conditions, and synthetic research of various nucleoside compounds has been actively conducted. However, an isolated and purified nucleoside phosphorylase capable of synthesizing a cytosine nucleoside compound using cytosine or a cytosine derivative and a phosphorylated sugar as a substrate has not been reported so far.

Further, according to the study by the present inventors, since microorganisms generally have cytosine deaminase activity and cytidine deaminase activity, the microorganisms themselves are used, or the enzyme prepared from the microorganisms or the culture solution thereof is used. When these deaminase activities remain in the preparation, cytosine or a cytosine derivative as a substrate and a deamino compound of a cytosine nucleoside compound as a reaction product are synthesized, and the cytosine nucleoside compound is efficiently accumulated. Things will be difficult.

That is, an object of the present invention is to provide an amino acid sequence of a nucleoside phosphorylase capable of synthesizing a cytosine nucleoside compound. Further provided are a recombinant plasmid containing the gene, a transformant containing the recombinant plasmid, a method for producing the enzyme using the transformant strain, and a method for producing a cytosine nucleoside compound using the transformant strain. That is. Furthermore, by reducing the activity of cytosine deaminase and cytidine deaminase while maintaining the activity of nucleoside phosphorylase, or by efficiently using a microorganism expressing cytosine nucleoside phosphorylase in a microorganism lacking both enzymes, cytosine nucleoside phosphorylase can be efficiently used. It is to provide a method for producing a compound.

In addition, it is possible to reduce the activity of the dephosphorylating enzyme of the phosphorylated sugar as a raw material, or to efficiently use a microorganism expressing cytosine nucleoside phosphorylase in a microorganism lacking the enzyme. It is intended to provide a method for producing a cytosine nucleoside compound.

[0012] When producing a cytosine nucleoside compound, especially when it is used as a raw material for a drug, the inclusion of a trace amount of a by-product is a serious problem. Separation of by-product nucleosides in the purification step not only increases the purification load but also reduces the recovery rate of cytosine nucleoside compounds, which is a major problem in industrial production. When it is used for such purpose, it is necessary to almost completely inactivate the cytosine deaminase activity and the cytidine deaminase activity from the enzyme preparation.

[0013]

Means for Solving the Problems As a result of intensive studies for solving these problems, the present inventors have found that purine nucleoside phosphorylase, which originally catalyzes a reaction using a purine base as a substrate, surprisingly reacts with a pyrimidine base. It has been found to catalyze the reaction of certain cytosines or cytosine derivatives with pentose-1-phosphate to form cytosine nucleoside compounds.

The presence of this purine nucleoside phosphorylase found by the present inventors indicates the presence of cytosine nucleoside phosphorylase.

On the other hand, when the present inventors attempted a reaction for producing a cytosine nucleoside compound from cytosine or a cytosine derivative and pentose-1-phosphate using a microorganism having this enzyme activity, most of the product was It is a deamino form of cytosine or a cytosine derivative and a cytosine nucleoside compound, and the target cytosine nucleoside compound could not be efficiently accumulated. Therefore, as a result of extensive studies, the formation of this deamino body is due to the action of deaminase, and the cells having the deaminase activity are stirred or allowed to stand in an organic solvent, or the cells are heated. The activity of cytosine deaminase and cytidine deaminase can be inactivated by the addition of these compounds, and further, these treatments both inactivate the cytosine nucleoside phosphorylase to be maintained by the presence of phosphate or phosphorylated sugar in the treatment solution. It has been found that this can be suppressed more effectively.

According to the above method, it is possible to specifically reduce both the substrate and the decomposition activity of the product while maintaining the activity of the nucleoside phosphorylase, and the inactivation or reduction treatment of the deaminase activity as described above. It was found that a cytosine nucleoside compound can be produced from a cytosine or a cytosine derivative and a phosphorylated sugar by using a microbial cell that has been subjected to the above-mentioned method, and thus completed the present invention.

According to the deaminase inactivation or reduction treatment of the present invention, it is possible to almost completely inactivate the degrading activity while maintaining the cytosine nucleoside phosphorylase activity almost completely. The present inventors have also found that a cytosine nucleoside compound can be efficiently produced by expressing the enzyme in a microorganism deficient in both cytosine deaminase and cytidine deaminase enzyme activity, and completed the present invention.

Further, it was found that the reaction yield is lowered in the presence of the enzyme activity for dephosphorylating the phosphorylated sugar as the raw material. It was found that the enzyme activity can be selectively removed by adding a polar solvent to the cell lysate of the bacterial cells having the enzyme activity. Further, the enzymatic activity can be removed by fractionating by salting out or purifying cytosine nucleoside phosphorylase with a carrier such as an ion exchange resin. The inventors have also found that a cytosine nucleoside compound can be efficiently produced by using such a treated product, and have completed the present invention. Furthermore, the present inventors have also found that a cytosine nucleoside compound can be efficiently produced by expressing cytosine nucleoside phosphorylase in a microorganism lacking the enzyme activity, and completed the present invention.

According to the present invention, it is possible to provide an efficient method for synthesizing a cytosine nucleoside compound by a cytosine nucleoside phosphorylase, which has not been achieved so far.

That is, the present invention is as follows.

The method for producing a cytosine nucleoside compound according to the present invention comprises a step of reacting a phosphorylated saccharide with a cytosine or a cytosine derivative in the presence of an enzyme having a cytosine nucleoside phosphorylase activity to obtain a cytosine nucleoside compound. It is characterized by

As the above-mentioned enzyme, an enzyme having a purine nucleoside phosphorylase activity can be used,
An enzyme preparation containing an enzyme having a purine nucleoside phosphorylase activity can be suitably used in the method for producing a cytosine nucleoside compound according to the present invention. Examples of such an enzyme having a purine nucleoside phosphorylase activity include an enzyme derived from Escherichia coli.

The above cytosine derivative is represented by the following formula (I):

[0024]

[Chemical 2] (In the formula (I), X represents a carbon atom or a nitrogen atom, and Y represents
Represents a hydrogen atom, a halogen atom or a lower alkyl group. The compound represented by these can be used. Specific examples thereof include azacytosine and 5-fluorocytosine.

Further, the above-mentioned enzyme having cytosine nucleoside phosphorylase activity is a bacterial cell of a microorganism having this enzyme or a culture solution thereof, or a crude enzyme extract, crude enzyme solution, crude enzyme preparation and purification obtained from them. It can be supplied into the reaction system in the form of an enzyme preparation such as an enzyme preparation.

[0026] These bacterial cells or enzyme preparations have no cytosine deaminase activity and cytidine deaminase activity, or have cytosine nucleoside phosphorylase activity even if they are present, and are capable of producing cytosine nucleoside compounds to a certain extent. Is preferred. For example, bacterial cells and enzyme preparations having higher cytosine deaminase activity and cytosine nucleoside phosphorylase activity than cytidine deaminase activity can be preferably used.

Examples of such bacterial cells and enzyme preparations include those obtained by reducing their cytosine deaminase activity and cytidine deaminase activity by a reduction treatment.

The microbial cells that have been subjected to the treatment for deactivating or reducing the deaminase activity are, for example, contacted with water containing an organic solvent to bring the microbial cells having cytosine nucleoside phosphorylase activity into contact with cytosine. It can be obtained by selectively reducing the deaminase activity and the cytidine deaminase activity. As this organic solvent,
Polar solvents such as alcohols can be used.
As the organic solvent, for example, one or more kinds of organic solvents selected from methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, dioxane and acetone can be used.

Further, when the cytosine deaminase activity and the cytidine deaminase activity are inactivated or reduced by the organic solvent, the concentration of the organic solvent in water is preferably 20% by volume or more.

On the other hand, the microbial cells that have been treated to inactivate or reduce the deaminase activity have the cytosine deaminase activity and the cytidine deaminase activity in the aqueous medium of the microbial cells having the cytosine nucleoside phosphorylase activity. It can also be obtained by heat treatment at a temperature at which it is selectively reduced. The conditions for this heat treatment include:
Conditions of 10 minutes or more and 40 hours or less at 50 ° C. or more can be adopted.

On the other hand, the presence of pentose-1-phosphate in the treatment for deactivating or reducing the deaminase activity makes it possible to more effectively maintain the cytosine nucleoside phosphorylase activity to be maintained. It will be possible. The concentration of pentose-1-phosphate at that time was 1 mM.
It is preferably 100 mM or less.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a cytosine nucleoside phosphorylase refers to an enzyme having an activity of producing a cytosine nucleoside compound using cytosine or a cytosine derivative as a substrate, and any animal, plant or microorganism can be used as long as this requirement is satisfied. Can be the origin of.
The enzyme cytosine nucleoside phosphorylase is not generally known in the art. Therefore, only in the present invention, such terms are defined and used as described above. Specific examples of such a nucleoside phosphorylase include Escherichia coli (Escherichia coli).
Escherichia such as E. coli
Suitable examples include enzymes known as conventional purine nucleoside phosphorylases of microorganisms belonging to the genus a and also having cytosine nucleoside phosphorylase activity. As a specific example, the DNA base sequence of Escherichia coli purine nucleoside phosphorylase is shown in SEQ ID NO: 3, and the amino acid sequence translated from the base sequence is shown in SEQ ID NO: 4. In addition, recent advances in genetic engineering have made it easy to modify the amino acid sequence by inactivating, inserting, or substituting a part of the base sequence. In view of the state of the art, a part of the nucleotide sequence is modified within a range that does not affect the desired enzyme activity, for example, within a range where the enzyme activity can be maintained or improved, and the amino acid sequence is also modified. It is intended to be included in the cytosine nucleoside phosphorylase of the invention. For example, the amino acid sequence shown in SEQ ID NO: 4 with a deletion, substitution or addition of 2 to 3 amino acids within a range that does not affect the intended enzyme activity, or SEQ ID NO: 3 Nucleotide sequence that does not affect the intended enzyme activity of the nucleotide sequence of, and has a mutation such as deletion, substitution or addition within the range in which its complementary sequence can hybridize under stringent conditions. Those having an amino acid sequence encoded by can be used.

As described above, having purine nucleoside suphorylase activity makes it possible to produce a purine nucleoside compound and a cytosine nucleoside compound with one kind of enzyme, which is very advantageous for industrial production of nucleosides. is there.

The enzyme having cytosine nucleoside phosphorylase activity used in the method of the present invention is supplied to the reaction system in the form of a microbial cell having the enzyme, an enzyme preparation prepared from the microbial cell or a culture solution thereof, and the like. be able to. The enzyme preparation includes various processed products of bacterial cells or culture solutions thereof, enzyme extracts, crude purified products and isolated purified products using the enzyme extracts.

As the bacterial cells and enzyme preparations of these microorganisms, commercially available products and those prepared by utilizing various methods can be used. For example, as the preparation having this enzyme activity, a commercially available enzyme, a microbial cell having the enzyme activity, a treated microbial cell or an immobilized product thereof can be used. The treated cells are, for example, acetone-dried cells and mechanical disruption, ultrasonic disruption, freeze-thaw treatment, pressure reduction treatment,
Examples include crushed cells prepared by osmotic pressure treatment, autolysis, cell wall decomposition treatment, detergent treatment, etc., and if necessary, those that have been purified by ammonium sulfate precipitation, acetone precipitation, or column chromatography. May be.

The microorganism having cytosine nucleoside phosphorylase activity is not particularly limited as long as it expresses a nucleoside phosphorylase which produces a cytosine nucleoside compound using cytosine or a cytosine derivative as a substrate. Such microorganisms are, for example,
It can be selected from those expressing common nucleoside phosphorylase. Specific examples of the microorganisms producing such nucleoside phosphorylase include Escherichia coli.
Examples of suitable microorganisms include microorganisms contained in the genus Escherichia.

Due to recent advances in molecular biology and genetic engineering, the gene of the protein is obtained from the microbial strain by analyzing the molecular biological properties and amino acid sequence of purine nucleoside phosphorylase of the above-mentioned microbial strain. Then, it becomes possible to construct a recombinant plasmid in which the gene and a control region necessary for expression are inserted, and to introduce this into an arbitrary host to produce a recombinant bacterium expressing the protein, and , Became relatively easy. In view of such a state of the art, a genetically modified bacterium in which such a nucleoside phosphorylase gene is introduced into an arbitrary host is also included in the microorganism expressing the nucleoside phosphorylase of the present invention.

The term "regulatory region required for expression" as used herein refers to a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence and the like. Specific examples of the promoter sequence include:
E. coli-derived tryptophan operon trp promoter, lactose operon lac promoter, lambda phage-derived PL promoter and PR promoter, and Bacillus subtilis-derived gluconate synthase promoter (gnt) / alkaline protease promoter (ap
r) ・ Neutral protease promoter (npr) ・ α-
Examples thereof include an amylase promoter (amy). In addition, uniquely modified / designed sequences such as the tac promoter can also be used. Examples of the ribosome binding sequence include sequences derived from Escherichia coli or Bacillus subtilis, but are not particularly limited as long as the sequences function in a desired host such as Escherichia coli and Bacillus subtilis. For example, a consensus sequence having 4 or more consecutive bases complementary to the 3'terminal region of 16S ribosomal RNA may be prepared by DNA synthesis and used. The transcription termination sequence is not always necessary, but those independent of ρ factor, such as lipoprotein terminator and trp operon terminator, can be used. Regarding the sequence order of these control regions on the recombinant plasmid, the promoter sequence, the ribosome binding sequence, the gene encoding the nucleoside phosphorylase, and the transcription termination sequence are preferably arranged in this order from the 5′-terminal side upstream.

As a concrete example of the plasmid mentioned here,
PBR3 having a region capable of autonomous replication in Escherichia coli
22, pUC18, Bluescript II SK
(+), PKK223-3, pSC101, and pUB110, p having a region capable of autonomous replication in Bacillus subtilis
TZ4, pC194, ρ11, φ1, φ105, etc. can be used as a vector. In addition, as an example of a plasmid capable of autonomous replication in two or more types of hosts, p
HV14, TRp7, YEp7 and pBS7 can be used as vectors.

The arbitrary host referred to herein may be Escherichia coli as described in the examples below.
However, the Bacillus subtilis (Bacillus subt) is not limited to E. coli.
and other microbial strains such as yeast and actinomycetes.

The cytosine nucleoside compound in the present invention is a compound in which a phosphorylated saccharide and cytosine or a cytosine derivative as a nucleic acid base are bound by an N-glycoside bond. Typical examples thereof include cytidine, deoxycytidine, dideoxycytidine, azacitidine,
Deoxyazacytidine, 5-fluorocytidine and 5-
Examples thereof include, but are not limited to, fluorodeoxycytidine and the like.

The cytosine derivative in the present invention has a cytosine structure portion, and this cytosine structure portion can be converted into a cytosine nucleoside structure by the action of cytosine nucleoside phosphorylase activity. Preferred examples of the cytosine derivative include the cytosine derivative represented by the general formula (I). The lower alkyl group as Y in the general formula (I) is an alkyl group having 1 to 4 carbon atoms, for example, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group,
Mention may be made of sec-butyl and tert-butyl groups. Among these cytosine derivatives, for example, azacytosine, 5-fluorocytosine, 5-methylcytosine and the like are particularly preferable.

The phosphorylated saccharide in the present invention is a polyhydroxyaldehyde or polyhydroxyketone and its derivative in which phosphoric acid is ester-bonded to the 1-position. Preferred representative examples thereof include ribose 1-phosphate, 2'-deoxyribose 1-phosphate, and 2'-deoxyribose 1-phosphate.
Examples include ′, 3′-dideoxyribose 1-phosphate, arabinose 1-phosphate, dioxolane sugar-phosphate.

The term "polyhydroxyaldehyde or polyhydroxyketone" as used herein means a natural product derived from an aldopentose such as D-arabinose, L-arabinose, D-xylose, L-lyxose or D-ribose, or D. -Xylulose, L-xylulose, D-
Examples thereof include, but are not limited to, ketopentose such as ribulose, and deoxy sugars such as D-2-deoxyribose and D-2,3-dideoxyribose.

These phosphorylated sugars are produced by a phosphorolysis reaction of a nucleoside compound by the action of a nucleoside phosphorylase (J. Biol. Che.
m. Vol. 184, 437, 1950), or anomer-selective chemical synthesis method. It can also be adjusted by the enzymatic method for synthesizing deoxyribose-1-phosphate described in WO 01/14566. (The dRP synthesis route of Roche's patent is also described.) In the present invention, the treatment for reducing cytosine deaminase activity and cytidine deaminase activity means inactivating or reducing cytosine deaminase activity and cytidine deaminase activity without deactivating nucleoside phosphorylase. It is not particularly limited as long as it can be made into a form, but preferable examples include exposing the microorganisms, the culture solution or the treated products of the microorganisms to an organic solvent, or performing heat treatment.

In the present invention, the organic solvent is not particularly limited as long as it is a solvent capable of deactivating cytosine deaminase activity and cytidine deaminase activity.
Specifically, polar solvents such as methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, dioxane, tetrahydrofuran, methyl ethyl ketone and acetone, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethyl-
Alcohols such as 1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-octanol, 2-octanol, 1-nonanol, propyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate,
Esters such as pentyl acetate, isopentyl acetate, cyclohexyl acetate, benzyl acetate, pentane, hexane, 2-methylhexane, 2,2-dimethylbutane,
2,3-Dimethylbutane, cyclohexane, methylcyclohexane, heptane, cycloheptane, octane, cyclooctane, isooctane, nonane, decane, dodecane, petroleum ether, petroleum benzine, ligroine, industrial gasoline, kerosene, benzene, toluene, xylene, ethylbenzene. , Hydrocarbons such as propylbenzene, cumene, mesitylene and naphthalene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene, phenols such as cresol and xylenol, methyl isobutyl ketone, 2 -Ketones such as hexanone, dipropyl ether, diisopropyl ether, diphenyl ether,
Examples of ethers such as dibenzyl ether include pentane, hexane, heptane, octane, cyclopentane,
Hydrocarbons such as cyclohexane, cycloheptane, cyclooctane, toluene and ethylbenzene can be mentioned.
In addition, N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylbenzamide, N-
Methyl-2-pyrrolidone, N-methylformamide, N
Amide compounds such as -ethylformamide, N-methylacetamide, formamide, acetylamide, benzoic acid amide and urea compounds such as urea, N, N'-dimethylurea, tetramethylurea and N, N-dimethylimidazolidinone. Examples thereof include sulfoxide compounds such as dimethyl sulfoxide, diethyl sulfoxide and diphenyl sulfoxide.

The organic solvent treatment for inactivating or reducing the deaminase activity referred to in the present invention means especially if the cytosine deaminase activity and the cytidine deaminase activity can be inactivated or decreased without deactivating the nucleoside phosphorylase. Although not limited, for example, the cells of the microorganism, the culture solution, or the processed product thereof are usually adjusted to pH 4.
0 or more and 10.0 or less, preferably 6.0 or more and 9.
At 0 or less, the concentration of the organic solvent in water is, for example, 10% by volume or more, preferably 20% by volume or more, more preferably 3% by volume.
At a concentration of 0% by volume or more, usually at a temperature of 0 ° C or higher, preferably 20 ° C, more preferably 50 ° C or higher and 80 ° C or lower,
A method in which it is allowed to stand or suspend for 10 minutes or more and 40 hours or less, more preferably 1 hour or more and 20 hours or less can be mentioned.

By adding 1 mM or more, preferably 100 mM or less of phosphorylated saccharide to the treatment liquid, nucleoside phosphorylase can be further stabilized.

Similar effects can be obtained by adding these organic solvents to the reaction solution.

The heat treatment as referred to in the present invention is not particularly limited as long as it can inactivate or reduce cytosine deaminase activity and cytidine deaminase activity without deactivating nucleoside phosphorylase. Alternatively, the pH of these treated products in an aqueous medium is usually 4.0 or more and 10.0 or less, preferably 6.0 or more and 9.0 or less, and the temperature is usually 50 ° C. or more, preferably 60 ° C. or more 80 ° C. In the following, a method of standing or suspending for 10 minutes or more, more preferably 30 minutes or more can be mentioned. The heating time is more preferably 40 hours or less. In addition, nucleoside phosphorylase can be further stabilized by adding 1 mM or more, preferably 10 mM or more and 100 mM or less of phosphorylated saccharide to the treatment liquid.

The microorganisms which do not have both cytosine deaminase and cytidine deaminase enzymatic activities in the present invention include, for example, only cytidine deaminase in Bacillus bacteria, and the presence of cytosine deaminase is not known. Thus, a cytidine deaminase-deficient strain of Bacillus bacterium can be used. For example, Bacillus Genetic Stock C
Bacill available from enter (BGCS)
Us subtilus 1A479 etc. can be illustrated. It is also possible to use cells expressing nucleoside phosphorylase in such strains.

Examples of the phosphatase-deficient strain used in the present invention include Escherichia coli K-12 DH5α as an alkaline phosphatase-deficient strain. For such strains, it is also possible to use cells expressing cytosine nucleoside phosphorylase.

By heat-treating such cells, acid phosphatase can be inactivated or reduced, and a higher reaction yield can be achieved. For example, the bacterial cells of the microorganism, the culture solution or a treated product thereof in an aqueous medium have a pH of usually 4.0 or more and 10.0 or less,
It is preferably 6.0 or more and 9.0 or less, and the temperature is usually 50 ° C.
As mentioned above, a method of standing at 60 ° C. or more and 80 ° C. or less for 10 minutes or more, more preferably for 30 minutes or more or suspending can be mentioned.

The term “reducing or removing a substance that inhibits cytosine nucleoside phosphorylase activity” as used in the present invention is not particularly limited as long as the substance that inhibits the reaction can be removed or reduced. For example, the bacterial cells may be disrupted by ultrasonication. Of the cytosine nucleoside phosphorylase activity by a method of obtaining a cytosine nucleoside phosphorylase as a precipitate by adding a polar solvent to the enzyme solution, a method of collecting the precipitate by salting out by adding ammonium sulfate, or a method of purifying with an ion exchange resin or the like. It is possible to remove or reduce the substance that inhibits.

The synthetic reaction of a cytosine nucleoside compound in the present invention is a microorganism expressing a nucleoside phosphorylase capable of synthesizing a cytosine nucleoside compound using a cytosine or a cytosine derivative and a phosphorylated sugar as a substrate, and the cytosine or cytosine derivative or cytosine nucleoside is synthesized. The reaction conditions such as appropriate pH and temperature may be selected by using cells of the microorganisms, culture solutions or processed products thereof in which the activity of decomposing the compound is inactivated or deleted, but usually p
It can be performed in the range of H4 to 10 and temperature of 10 to 80 ° C. The concentration of the phosphorylated saccharide and the cytosine or cytosine derivative used in the reaction is appropriately about 0.1 to 1000 mM, and the molar ratio of both is such that the ratio of the cytosine or the cytosine derivative to be added to the phosphorylated saccharide or its salt is 0. 1 ~
It can be performed in a 10-fold molar amount. 0.95 considering the reaction conversion rate
A double molar amount is preferable.

Further, the reaction yield can be increased by allowing the presence of a phosphoric acid and a sparingly soluble metal salt for the purpose of trapping the phosphoric acid formed in the reaction solution, or the presence of a carrier such as an ion exchange resin. Is.

The cytosine nucleoside compound can be collected from the reaction solution by utilizing the difference in solubility in a solvent such as water, by ion exchange or by using an adsorption resin.

A microorganism in which a small amount of cytosine remaining in the reaction solution is treated with a cytosine deaminase-expressing microorganism to reduce or deactivate the cytidine deaminase, or a cytidine deaminase-deficient strain is expressed in the cytosine deaminase. And a small amount of cytosine can be converted to uracil by allowing the presence of it in the reaction solution.
By passing the reaction solution through a cation exchange resin, unreacted cytosine is converted to uracil or a uracil nucleoside compound, which is a decomposition product of a cytosine nucleoside compound, is not adsorbed, and only the cytosine nucleoside compound is adsorbed and easily purified. can do.

[0059]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Analysis Method] All the produced nucleoside compounds were quantified by high performance liquid chromatography. The analysis conditions are as follows. Column; Develosil ODS-MG-5 4.
6 × 250 mm (Nomura Chemical) Column temperature: 40 ° C. Pump flow rate: 1.0 ml / min. Detection: UV254 nm, eluent: 50 mM potassium potassium phosphate: methanol = 8:
1 (V / V) Reference Example 1 (Reproducibility test of JP-A 1-60396) Synthesis of deoxycytidine was examined according to Example 1 of JP-A 1-60396. That is, among the 24 strains described in Example 1, 7 deposited strains shown in Table 1 were ordered. 50 ml of a medium (pH 7.0) containing 0.5 g / dl of yeast extract, 1.0 g / dl of peptone, 1.0 g / dl of meat extract and 0.5 g / dl of NaCl was dispensed into a 500 ml shoulder flask and sterilized. To this medium, the microorganisms shown in Table 1 which had been precultured in broth agar medium at 30 ° C. for 16 hours were used.
Each platinum loop was inoculated and shake cultured at 30 ° C. for 16 hours. The cells were separated from the obtained culture solution by centrifugation, washed with 0.05M phosphate buffer (pH 7.0), and further centrifuged to prepare washed cells.

The washed cells were treated with 20 mM of 2'-deoxyribose-1-phosphate and 20 mM of cytosine.
5 g in 10 ml of 05M Tris buffer (pH 7.2)
/ Dl, and reacted at 60 ° C. for 24 hours. As a result of diluting the reaction solution and analyzing it by HPLC, cytosine as a substrate was decomposed and almost disappeared, but deoxycytidine was not detected.

[0061]

[Table 1]

Reference Example 2 (PNP cloning and preparation of expression strain, preparation of control cells) Escherichia coli chromosomal DNA was prepared as follows.

Escherichia coli K-12 / XL-10
The strain (Stratagene) was inoculated into 50 ml of LB medium, cultured overnight at 37 ° C., and then collected to collect lysozyme 1.
Lysis was performed with a lysate containing mg / ml. After the lysate was treated with phenol, DNA was precipitated by ethanol precipitation by a usual method. The generated DNA precipitate was wound around a glass rod, collected, washed, and used for PCR.

As the primer for PCR, the nucleotide sequence of the deoD gene encoding the known purine nucleoside phosphorylase of Escherichia coli (hereinafter referred to as PNP) (GenBank accession no.
AE000508 (coding region is base number 11531)
-12250) based on the nucleotide sequence shown in SEQ ID NOS: 1 and 2 (synthesized by Hokkaido System Science Co., Ltd.) was used. These primers have EcoRI and HindIII restriction enzyme recognition sequences near the 5'end and the 3'end, respectively.

Denaturation: 96 ° C., 1 minute, annealing: 55 ° C., 1 minute using 0.1 ml of PCR reaction solution containing 6 ng / μl of the Escherichia coli chromosomal DNA completely digested with the restriction enzyme HindIII and 3 μM of each primer. ,
Extension reaction: 3 reaction cycles consisting of 1 minute at 74 ° C
PCR was performed under the condition of 0 cycle.

The above reaction product and plasmid pUC18
(Takara Shuzo Co., Ltd.) was digested with EcoRI and HindIII, ligated with Ligation High (Toyobo Co., Ltd.), and Escherichia coli DH5α was transformed with the obtained recombinant plasmid. The transformant was treated with ampicillin (Am) 50 μg / ml and X-Ga.
l (5-bromo-4-chloro-3-indolyl-β-D
-Galactoside) -containing LB agar medium was cultured to obtain a transformant strain resistant to Am and forming white colonies.

A plasmid was extracted from the transformant thus obtained, and the plasmid into which the desired DNA fragment was inserted was designated as pUC-PNP73. pUC-P
The base sequence of the DNA fragment introduced into NP73 was confirmed by a usual base sequence determination method. The obtained base sequence is shown in SEQ ID NO: 3, and the amino acid sequence translated from the base sequence is shown in SEQ ID NO: 4. The molecular weight of the subunit of this enzyme is about 26000, and it is known that it is actively expressed as a hexamer. The optimum temperature of this enzyme is about 70
C, and the optimum pH is about 7.0 to 7.5. The transformant thus obtained was used as Escherichia coli MT.
It was named -10905.

The Escherichia coli MT-10905 strain was used in 100 mL of LB medium containing 50 μg / ml of Am for 37 times.
The cells were shaken and cultured at 0 ° C overnight. The culture solution was centrifuged at 13000 rpm for 10 minutes, and the obtained bacterial cells were mixed with 20 mL of 10
The cells were suspended in 0 mM Tris-HCl buffer (pH 8.0). The suspension was centrifuged again at 13000 rpm for 10 min,
2 mL of 100 mM Tris-HCl buffer (pH 8.0) and 10 mM of 2'-deoxyribose 1
-Phosphoric acid di (monocyclohexyl ammonium) salt (S
It was suspended in IGMA) and stored frozen at -20 ° C.

Escherichia coli DH5α was transformed with the plasmid pUC18 (Takara Shuzo Co., Ltd.).
The transformant was named pUC18 / DH5α. pUC
The 18 / DH5α strain was shake-cultured in 100 mL of LB medium containing 50 μg / mL of Am at 37 ° C. overnight. 1 culture
Centrifugation was performed at 3000 rpm for 10 minutes, and the obtained cells were 20 mL of 100 mM Tris-HCl buffer (pH 8.
0). Suspension again at 13000 rpm for 10
After centrifuging for min, the obtained bacterial cells were added to 2 mL of 100 mM.
It was suspended in Tris-hydrochloric acid buffer solution (pH 8.0), 10 mM 2'-deoxyribose 1-phosphate di (monocyclohexyl ammonium) salt (manufactured by SIGMA), and frozen and stored at -20 ° C.

Comparative Example 1 (Synthesis of deoxycytidine in Escherichia coli recombinant with PNP) 20 mM 2'-deoxyribose 1-phosphate di (monocyclohexyl ammonium) salt (manufactured by SIGMA), 2
A reaction solution consisting of 0 mM cytosine (manufactured by Wako Pure Chemical Industries, special grade), 100 mM Tris-HCl buffer (pH 8.0), and 0.1 mL of the pUC18 / DH5α strain bacterial cell suspension obtained in Reference Example 1. 0 ml was reacted at 50 ° C. for 20 hours. A comparative example was prepared by keeping the same temperature without adding a substrate. When the reaction solution was diluted and then analyzed, deoxycytidine was not detected. A comparative example is shown in FIG. 1 (A), and an HPLC analysis chart of the reaction solution is shown in FIG. 1 (B).

Comparative Example 2 (Synthesis of Escherichia coli deoxycytidine without PNP recombination) 20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 2
A reaction solution consisting of 0 mM cytosine (manufactured by Wako Pure Chemical Industries, special grade), 100 mM Tris-HCl buffer (pH 8.0), and 0.1 mL of the MT-10905 strain bacterial cell suspension obtained in Reference Example 1. 0 ml was reacted at 50 ° C. for 20 hours. A comparative example was prepared by keeping the same temperature without adding a substrate. When the reaction solution was diluted and then analyzed, 10 mM deoxyuridine and 3 mM uracil were produced, but deoxycytidine was not detected. FIG. 2A shows a comparative example and FIG.
The HPLC analysis chart of the reaction solution is shown in (B).

Example 1 (Treatment with Organic Solvent) Methanol (MeOH) was added to the suspension of MT-10905 strain of Reference Example 2 at a concentration shown in Table 2 to obtain 30 ° C.
It was kept warm for 1 hour. Hereinafter, cytidine deaminase is cd
Notated as d. The cdd activity was measured by adding a bacterial cell suspension to 1.0 ml of a reaction solution of 100 mM Tris-HCl buffer (pH 8.0) and 20 mM cytidine, and reacting at 50 ° C. for 2 hours. Add MeOH to the above-mentioned cell suspension.
100% of the product that was kept at 30 ℃ for 1 hour without adding
It was shown as a relative value as%.

The activity of PNP is 20 mM 2'-deoxyribose 1-phosphate di (monocyclohexyl ammonium) salt (manufactured by SIGMA), 5.4 mM adenine (manufactured by Wako Pure Chemical Industries, Ltd.), 100 mM Tris-HCl buffer. (PH
8.0) Add the cells to 1.0 ml of the reaction solution, and add 15 ml at 50 ° C.
It was measured by reacting for minutes. The reaction was carried out using the amount of cells having a production amount of deoxyadenosine of 2 mM or less. 3 without adding MeOH to the above cell suspension
The value was shown as a relative value with 100% kept at 0 ° C. for 1 hour. As shown in the results in Table 2, the PNP activity was hardly reduced, but the cdd activity was almost inactivated.

[0074]

[Table 2]

Example 2 (Treatment with Organic Solvent) The organic solvent shown in Table 3 was added to the suspension of MT-10905 strain of Reference Example 2, and the mixture was kept at 30 ° C. for 1 hour. As shown in Table 3, the PNP activity was hardly reduced, but the cdd activity was almost inactivated.

[0076]

[Table 3]

Example 3 (Heat Treatment) The suspension of MT-10905 strain of Reference Example 2 was kept at 60 ° C. Hereinafter, cytosine deaminase is referred to as cod. The cod activity is 100 mM Tris-HCl buffer (pH
8.0), adding the cell suspension to 20 mM cytosine
It was measured by reacting at 0 ° C. for 2 hours. When the amount of cytosine decomposed when untreated cells were added is 100%,
The residual activity was expressed as a relative value. The results are shown in Table 4,
The activity of PNP was hardly reduced, but the activity of cod was almost inactivated.

[0078]

[Table 4]

Example 4 (heat treatment + organic solvent treatment) The heat-treated 20 hr cells obtained in Example 3 were treated with MeOH in the same manner as in Example 1. It was displayed as a relative value when the bacterial cell activity of 20 hr treatment of Example 3 was 100%. As shown in Table 5, the PNP activity was hardly reduced, but the cod and cdd activities were almost inactive.

[0080]

[Table 5]

Example 5 (Synthesis of Deoxycytidine by Recombinant PNP Expressing Bacterium Treated with Example 4) 20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 2
1.0 ml of a reaction solution consisting of 0 mM cytosine (manufactured by Wako Pure Chemical Industries, special grade), 100 mM Tris-HCl buffer (pH 8.0), and 0.1 ml of the microbial cell solution treated with 50% MeOH obtained in Example 4 was added. The reaction was carried out at 50 ° C for 20 hours. When the reaction solution was diluted and then analyzed, 10.7 mM deoxycytidine, 0.2 mM deoxyuridine, and 0.1 mM uracil were formed. The reaction yield at that time was 53%. The HPLC analysis chart of the reaction solution is shown in FIG. 2 (C).

Example 6 (Synthesis of deoxycytidine by Escherichia coli treated in Example 4) 20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 2
50 mM cytosine (manufactured by Wako Pure Chemical Industries, special grade), 100 mM Tris-HCl buffer (pH 8.0), and the pUC18 / DH5α bacterial cell suspension obtained in Reference Example 2 were treated in the same manner as in Example 4. 0.1 ml of cell suspension treated with% MeOH
Was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, it was found to be 0.14 mM.
Deoxycytidine of 0.17 mM deoxyuridine was produced. The reaction yield at that time was 0.7%. FIG. 1C shows an HPLC analysis chart of the reaction solution.

Example 7 (Synthesis of Cytidine by Recombinant PNP Expressing Bacterium Treated with Example 4) 20 mM ribose 1-phosphate di (cyclohexyl ammonium) salt (manufactured by SIGMA), 20 mM cytosine (Wako Pure Chemical Industries, Ltd.) (Manufactured, special grade), 100 mM Tris-HCl buffer (pH 8.0), and a reaction solution 1.0 ml consisting of 0.1 ml of the 50% MeOH-treated cell suspension obtained in Example 4 at 50 ° C. for 20 hours. It was made to react. When the reaction solution was diluted and then analyzed, 5 mM cytidine, 0.1 mM uridine, and 0.1 mM uracil were produced. The reaction yield at that time was 25%.

Example 8 (Purification of PNP and synthesis of deoxycytidine by purified PNP) The cells obtained in Example 4 were suspended in 10 mL of 10 mM Tris-hydrochloric acid buffer (pH 7.5) and sonicated with an ultrasonic crusher. The cells were crushed. Then, the disrupted solution was centrifuged to prepare a crude enzyme solution, which was equilibrated with 50 mM Tris-HCl buffer (pH 7.5), DEAE-Toyopearl (TOSO) 3 cm × 1.
Add to 0 cm column, 50 mM NaCl to 500 m
The active fraction was collected by elution with a linear gradient of MNaCl. The eluate was collected as a precipitate with 70% ammonium sulfate saturation and dialyzed against 10 mM Tris-HCl buffer (pH 7.5). Hydroxyapatite column (3 cm x 1) equilibrated with 10 mM Tris-HCl buffer (pH 7.5)
The dialysate is added to 5 cm) and 10 mM Tris-HCl buffer (pH 7.5) to 50 mM Tris-HCl buffer (pH 7.5)
Elution at 7.5) and the active fraction was collected. 70 enzyme solution
The precipitate was recovered as a precipitate with saturated ammonium sulfate, dissolved in 1 mL of 10 mM Tris-HCl buffer (pH 7.5), and dialyzed against 10 mM Tris-HCl buffer (pH 7.5) to obtain 2 mL of purified PNP. The purified PNP thus obtained was confirmed to have a single band by SDS-polyacrylamide gel electrophoresis. The results are shown in Fig. 3. 20 mM 2'-
Reaction consisting of deoxyribose 1-phosphate di (monocyclohexyl ammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemical Industries, special grade), 100 mM Tris-HCl buffer (pH 8.0), and 0.1 ml of purified enzyme The liquid (1.0 ml) was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 11.5 mM deoxycytidine was produced, and the reaction yield at that time was 57.5%.

Example 9 (Synthesis of azacitidine by recombinant PNP-expressing bacteria treated in Example 4) 20 mM ribose 1-phosphate di (cyclohexyl ammonium) salt (manufactured by SIGMA), 20 mM azacytosine (manufactured by SIGMA) A reaction solution 1.0 ml consisting of 100 mM Tris-HCl buffer (pH 8.0) and 0.1 ml of the 50% MeOH-treated cell suspension obtained in Example 4 was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 3.2 mM of azacitidine was produced. The reaction yield at that time was 16%.

Example 10 (Synthesis of fluorocytidine by recombinant PNP-expressing bacterium subjected to the treatment of Example 4) 20 mM ribose 1-phosphate di (cyclohexyl ammonium) salt (manufactured by SIGMA), 20 mM 5-fluorocytosine (Manufactured by SIGMA), 100 mM Tris-HCl buffer (pH 8.0), 50% Me obtained in Example 4.
Reaction solution 1.0 consisting of 0.1 ml of OH-treated cell suspension
ml was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 2.4 mM 5-fluorocytidine was produced. The reaction yield at that time was 12%.

Example 11 (Synthesis of methylcytidine by recombinant PNP-expressing bacteria treated in Example 4) 20 mM ribose 1-phosphate di (cyclohexylammonium) salt (manufactured by SIGMA), 20 mM 5-methylcytosine (Manufactured by SIGMA), 100 mM Tris-HCl buffer (pH 8.0), 50% MeO obtained in Example 4
Reaction solution 1.0m consisting of 0.1 ml of H-treated cell suspension
1 was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 2.1 mM 5-methylcytidine was produced. The reaction yield at that time was 10.5%.

Example 12 (Synthesis of deoxycytidine by cells in which PNP was incorporated into a degrading activity-deficient strain) (1) Preparation of Escherichia coli-Bacillus subtilis shuttle vectors pPNP04 and pPNP05 for expressing Escherichia coli PNP in Bacillus subtilis pUC- PC using PNP73 as a template and synthetic oligonucleotide primers of SEQ ID NO: 5 and SEQ ID NO: 6
The E. coli purine nucleoside phosphorylase gene was amplified by the R method to obtain a 0.8 kb DNA fragment A. Next, by using the plasmid pNP150 (FERM BP-425) described in JP-A-60-210986 as a template and the PCR method using the synthetic oligonucleotide primers of SEQ ID NO: 7 and SEQ ID NO: 8, Bacillus amyloliquefaciens ( Bacillus amylolique
efaciens), a transcription promoter derived from a neutral protease, and a region containing a translational regulatory site are amplified, and
A 0 kb DNA fragment B was obtained. Then, annealing is performed using the overlapping regions of the DNA fragment A and the DNA fragment B,
SEQ ID NO: 6 and SEQ ID NO: 7 using this generated fragment as a template
Amplification was carried out by the PCR method using the synthetic oligonucleotide primer of Example 1 to obtain a DNA fragment C of about 1.8 kb. This DNA fragment C is treated with restriction enzymes EcoRI and HindIII.
Was digested with and used as an insert fragment. Vector D
NA is Bacillus subtilis / E. Coli shuttle vector pRB37
3, and pRB374 with Bacillus Genet
Obtained from ic Stock Center (BGSC; OH), each of which has restriction enzymes EcoRI and HindI.
The vector fragment was prepared by digesting with II. This vector fragment was ligated in the presence of an excess amount of the above insert fragment, and E. coli purine nucleoside phosphorylase expression shuttle vector pPNP04,
And pPNP05 were made. (2) Introduction of pPNP04 and pPNP05 into Bacillus subtilis, and synthesis of deoxycytidine using transformant As Bacillus subtilis, Bacillus subtilis having no cytidine deaminase activity is used.
lis) 1A479 strain and Bacillus subtilis (Ba)
Cillus subtilis) 1A480 strain as Ba
cillus Genetic Stock Center
Used from r (BGSC; OH).

The plasmid pPNP0 obtained in (1)
4 and pPNP05 transformation was performed by the Chang protoplast method (Chang. Sand and Cohe.
n, S. N. Mol. Gen. Genet. 16
8, 111 (1978)), transformants 1A480 (pPNP04), 1A480 (pPNP).
05) and 1A479 (pPNP04) and 1A479
(PPNP05) was obtained.

Next, the deoxycytidine synthesizing activity of the 1A480 (pPNP04) strain was evaluated as follows.

1A480 (pPNP04) was cultured in 20 mL of double concentration L medium at 35 ° C. for 17 hours. The cells obtained by centrifuging 1.5 mL of this culture solution were frozen and stored at -20 ° C. 24 mM 2'-deoxyribose 1-phosphate di (monocyclohexyl ammonium) salt (SIGM
A), 20 mM cytosine (Wako Pure Chemical Industries, special grade), 1
1 ml of a reaction solution consisting of 00 mM Tris-HCl buffer (pH 8.0) was added to frozen cells and shaken at 50 ° C.
Sampling was performed after 1.5 hours and 18 hours, diluted 20-fold with water, centrifuged to remove bacterial cells, and the supernatant was diluted with H.
When analyzed by PLC, 1.4 to 1.
5 mM, 6.4 to 7.0 mM of deoxycytidine was accumulated in 18 hours, and no decomposition products of the substrate and the product were observed.

1A480 (pPNP05), 1A479
(PPNP04) and 1A479 (pPNP05) were also measured for deoxycytidine synthesis, and the result was 1A48.
It has been revealed that it retains the deoxycytidine synthesizing ability almost equal to that of the 0 (pPNP04) strain.

Example 13 (Acquisition of amino acid substitution product retaining cytosine nucleoside phosphorylase activity) Plasmid D of pUC-PNP73 obtained in Reference Example 2
Quick mold from STRATAGENE using NA as a template
Mutations were introduced using the kChange Site-Directed Mutagenesis Kit. Hereinafter referred to simply as a kit. In the following examples, the principle and operating method of the kit were basically followed.

10 ml of LB liquid medium was prepared in a 30 ml test tube and sterilized by autoclaving at 121 ° C. for 20 minutes. Ampicillin was added to this medium so that the final concentration was 100 μg / ml, and the MT-10905 strain obtained in Reference Example 2 was inoculated with a single bacterium ear to 37 ° C./30° C.
It was cultured at 0 rpm for about 20 hours. 1 ml of the culture solution was collected in an appropriate centrifuge tube and then centrifuged (15000).
The cells were separated by (rpm × 5 minutes). Then, a plasmid DNA of pUC-PNP73 was prepared from the cells by the alkaline SDS extraction method.

SEQ ID NOs: 9 and 10, 11 and 12, 13 and 1 using pUC-PNP73 plasmid DNA as a template.
4, 15 and 16, 17 and 18, 19 and 20, 21 and 2
2, 23 and 24, 25 and 26, 27 and 28, 29 and 3
0, 31 and 32, 33 and 34, 35 and 36, 37 and 3
8, 39 and 40, 41 and 42, 43 and 44, 45 and 46
PCR was carried out using the DNA of 1. PCR
The composition of the reaction solution is shown in Table 6. The amplification conditions are shown in Table 7.

The restriction enzyme DpnI was added to the PCR amplification reaction solution.
Was added for 4 hours and kept at 37 ° C. for 1 hour. 1 μL of this reaction solution was added to 100 μL of competent cells of Escherichia coli K-12DH5α (Toyobo), and the mixture was kept in ice for 30 minutes. After soaking in a constant temperature water bath at 42 ° C for 30 seconds, add 0.9 mL of NZY + medium attached to the competent cell and add 1 at 37 ° C.
Shake for time. The above culture solution was smeared on a medium prepared by adding ampicillin to LB agar medium at 100 μg / mL, and incubated at 37 ° C. for 20 hours to form colonies.

Five clones arbitrarily selected from the colony were inoculated into each of the single white ears and L containing Am 50 μg / ml was inoculated.
It was shake-cultured in 100 mL of B medium at 37 ° C. overnight. The culture solution was centrifuged at 13000 rpm for 10 minutes, and the obtained bacterial cells were added to 20 mL of 100 mM Tris-HCl buffer solution (pH
8.0). The suspension was centrifuged again at 13000 rpm for 10 min, and the obtained bacterial cells were mixed with 2 mL of 100
mM Tris-HCl buffer (pH 8.0), 10 mM 2 '
-Deoxyribose 1-phosphoric acid di (monocyclohexyl ammonium) salt (manufactured by SIGMA) was suspended in Example 4
The microbial cells were treated in the same manner as in, and the synthesis reaction of deoxycytidine was performed in the same manner as in Example 5. As a result of measuring deoxycytidine by HPLC analysis, 4 out of 5 clones
The production of deoxycytidine was detected in the clones, and it was confirmed that they retain cytosine nucleoside phosphorylase activity.

The cells of the 4 clones were separated from the remaining 1 ml of the above culture solution used for the measurement of cytosine nucleoside phosphorylase activity, and the plasmid DNA of each clone was prepared by the alkaline SDS extraction method. The base sequence of the DNA fragment was confirmed by the usual method for determining the base sequence. The activity was measured by the same method as in Example 1.
A list of the results is shown in Table 8.

[0099]

[Table 6]

[0100]

[Table 7]

[0101]

[Table 8]

Reference Example 3 (Transformation into Escherichia coli K-12 W3110 Strain) The plasmid pUC-PNP73 obtained in Reference Example 2 was transformed into E. coli K-12 W3110 strain (ATCC) according to a conventional method.
27325). The transformant obtained is M
It was named T-10948. The activity of the transformant cultured in the same manner as in Reference Example 2 was twice as high as that of the transformant for E. coli K-12 DH5α strain.

Example 14 (Removal of Reaction Inhibiting Substance: Precipitation with Polar Solvent) MT-10948 cells cultured in the same manner as in Reference Example 2 were disrupted by an ultrasonic disruptor. The same volume of acetone as the disrupted liquid was added, and the precipitate was removed by centrifugation. Next, half of the above acetone was added to the above-mentioned centrifugal supernatant liquid, and the precipitate was recovered by centrifugation. The collected precipitate was dried by a vacuum dryer. 2 mL of 100 mM Tris-HCl buffer (pH 8.0) and 10 mM 2'-deoxyribose 1 were added to the dried precipitate.
-Phosphoric acid di (monocyclohexyl ammonium) salt (S
IGMA) and treated in the same manner as in Example 4. 2 mL of the enzyme solution fractionated with acetone was added to cytosine (6.
96 g), 2'-deoxyribose 1-phosphate diammonium salt (18.7 g), magnesium hydroxide (6.2
g) was added to water (105.3 g) and reacted at 50 ° C. for 24 hours. After completion of the reaction, analysis by HPLC gave 21.4-deoxycytidine (11.4 g, 80%). As a comparative example, 2 mL of bacterial cell liquid before acetone treatment was reacted in the same manner as above, and 8.5 g (60%) of 2'-deoxycytidine was obtained.
Obtained.

Example 15 (Removal of Reaction Inhibiting Substance: Precipitation with Ammonium Sulfate) MT-10948 cells cultured in the same manner as in Reference Example 2 were disrupted by an ultrasonic disruptor. The crushed ammonium sulphate was slowly added to the crushed liquid, and ammonium sulfate was saturated to 40%. The mixture was slowly stirred in ice water for 1 hour, and the precipitate was removed by centrifugation. Ammonium sulfate crushed by centrifugation was added to ammonium sulfate up to 70% saturation, and similarly slowly stirred in ice water for 1 hour, and a precipitate was obtained by centrifugation. Precipitation 2
mL of 100 mM Tris-HCl buffer (pH 8.0), 1
It was dissolved in 0 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA). When treated in the same manner as in Example 4 and reacted in the same manner as in Example 14, 2'-deoxycytidine was converted to 1
1.0 g (80%) was obtained.

Example 16 (Removal of reaction inhibitor: purified enzyme is the enzyme of Example 8) When 2 mL of the enzyme solution obtained by the same method as in Example 8 was used to carry out the same reaction as in Example 14, 11.0 g (80%) of 2'-deoxycytidine was obtained.

Example 17 (Effect of phoA deficient strain) When 2 ml of the bacterial cell obtained in Example 4 was used and the same reaction as in Example 14 was carried out, 11.0 g of 2'-deoxycytidine was obtained.
(80%) obtained. On the other hand, MT-10948 strain was used in Example 4
Although the activity was detected twice as much as MT-10905 when treated in the same manner as in the above, the same reaction as in Example 14 was performed, and only 2'-deoxycytidine was obtained in an amount of 8.5 g (60%).

Example 18 Cytosine 6.96 g (62.6 mmol), 2'-deoxyribose 1-phosphate diammonium salt 18.7 g
(75.4 mmol), magnesium hydroxide 7.58 g
(132 mmol), frozen bacterial cells (2.0 g) prepared in Reference Example 3 were added to water (96.4 g) and cyclohexane 4.
The mixture was added to 8 g of the mixed liquid, and the reaction liquid was reacted at 45 ° C. for 18 hours while controlling the pH of the reaction liquid to 8.8. After the reaction was completed, the product was analyzed by HPLC and was found to be 2′-
10.03 g (70.5 mol% / cytosine) of deoxycytidine was obtained. At this time, the by-product uracil is 0.7
3 g (10.4 mol% / cytosine) and 2'-deoxyuridine were 1.80 g (12.6 mol% / cytosine).

Example 19 Table 9 and Table 10 show the results when 2'-deoxycytidine was synthesized under the same conditions as in Example 18 except that the solvent used for treating the bacterial cells and the solvent added during the reaction were changed. Show.

[0109]

[Table 9]

[0110]

[Table 10]

Example 20 Synthesis of 2'-deoxycytidine A strong basic anion exchange resin (MP500) was added to 20 g of pure water.
Levatide: exchange capacity 1.1 eq / L) 20 ml (total exchange capacity 24 mmol) and 2-deoxyribose 1-phosphate di (ammonium) salt (4.96 g, 20 mmo).
1) was added, and the mixture was stirred at room temperature for 30 minutes. After 30 minutes, the enzyme solution (0.5 ml) prepared in the same manner as in Example 4 and cytosine (2.11 g, 19 mmol) were added, and the mixture was stirred for 5
The reaction was carried out at 0 ° C for 10 hours. HP after 10 hours
As a result of LC analysis, the desired 2'-deoxycytidine was obtained with a reaction yield of 80%.

[0112]

INDUSTRIAL APPLICABILITY The production of a nucleoside compound using a nucleoside phosphorylase can positionally and stereospecifically produce a nucleoside compound under mild conditions, and it is desired to establish an industrial production method. Until now, no industrial method for synthesizing cytosine nucleoside compounds by nucleoside phosphorylase was known.

According to the present invention, a nucleoside phosphorylase having reactivity with cytosine is used, and pentose-1-
It becomes possible to produce a cytosine nucleoside compound from phosphoric acid and cytosine or a cytosine derivative. At that time, a cytosine nucleoside compound can be efficiently produced by providing a method for specifically reducing the decomposition activity of a substrate or a product.

[0114]

[Sequence list] SEQUENCE LISTING <110> MITSUI CHEMICALS, INC. <120> A method of producing a cytosine nucleside compound <130> P0001150 <160> 46 <210> 1 <211> 30 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer for cloning of purine nucle oside phophrylase of E. coli K-12 <400> 1 gtgaattcac aaaaaggata aaacaatggc 30 <210> 2 <211> 29 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer for cloning of purine nucle oside phophrylase of E. coli K-12 <400> 2 tcgaagcttg cgaaacacaa ttactcttt 29 <210> 3 <211> 720 <212> DNA <213> Escherichia coli <400> 3 atggctaccc cacacattaa tgcagaaatg ggcgatttcg ctgacgtagt tttgatgcca 60 ggcgacccgc tgcgtgcgaa gtatattgct gaaactttcc ttgaagatgc ccgtgaagtg 120 aacaacgttc gcggtatgct gggcttcacc ggtacttaca aaggccgcaa aatttccgta 180 atgggtcacg gtatgggtat cccgtcctgc tccatctaca ccaaagaact gatcaccgat 240 ttcggcgtga agaaaattat ccgcgtgggt tcctgtggcg cagttctgcc gcacgtaaaa 300 ctgcgcgacg tcgttatcgg tatgggtgcc tgcaccgatt ccaaagttaa ccgcatccgt 360 tttaaagacc atgactttgc cgctatcgct gacttcgaca tggtgcgtaa cgcagtagat 420 gcagctaaag cactgggtat tgatgctcgc gtgggtaacc tgttctccgc tgacctgttc 480 tactctccgg acggcgaaat gttcgacgtg atggaaaaat acggcattct cggcgtggaa 540 atggaagcgg ctggtatcta cggcgtcgct gcagaatttg gcgcgaaagc cctgaccatc 600 tgcaccgtat ctgaccacat ccgcactcac gagcagacca ctgccgctga gcgtcagact 660 accttcaacg acatgatcaa aatcgcactg gaatccgttc tgctgggcga taaagagtaa 720 <210> 4 <211> 239 <212> PRT <213> Escherichia coli <300> <400> 4 Met Ala Thr Pro His Ile Asn Ala Glu Met Gly Asp Phe Ala Asp Val 1 5 10 15 Val Leu Met Pro Gly Asp Pro Leu Arg Ala Lys Tyr Ile Ala Glu Thr             20 25 30 Phe Leu Glu Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met Leu Gly         35 40 45 Phe Thr Gly Thr Tyr Lys Gly Arg Lys Ile Ser Val Met Gly His Gly     50 55 60 Met Gly Ile Pro Ser Cys Ser Ile Tyr Thr Lys Glu Leu Ile Thr Asp 65 70 75 80 Phe Gly Val Lys Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Leu                 85 90 95 Pro His Val Lys Leu Arg Asp Val Val Ile Gly Met Gly Ala Cys Thr             100 105 110 Asp Ser Lys Val Asn Arg Ile Arg Phe Lys Asp His Asp Phe Ala Ala         115 120 125 Ile Ala Asp Phe Asp Met Val Arg Asn Ala Val Asp Ala Ala Lys Ala     130 135 140 Leu Gly Ile Asp Ala Arg Val Gly Asn Leu Phe Ser Ala Asp Leu Phe 145 150 155 160 Tyr Ser Pro Asp Gly Glu Met Phe Asp Val Met Glu Lys Tyr Gly Ile                 165 170 175 Leu Gly Val Glu Met Glu Ala Ala Gly Ile Tyr Gly Val Ala Ala Glu             180 185 190 Phe Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser Asp His Ile Arg         195 200 205 Thr His Glu Gln Thr Thr Ala Ala Glu Arg Gln Thr Thr Phe Asn Asp     210 215 220 Met Ile Lys Ile Ala Leu Glu Ser Val Leu Leu Gly Asp Lys Glu 225 230 235 <210> 5 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 5 cattattttc aaaaaggggg atttattatg gctaccccac ac 42 <210> 6 <211> 24 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 6 cagaagcttg cgaaacacaa ttac 24 <210> 7 <211> 33 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 7 tttgaattcc ttagtgcttt catagattaa act 33 <210> 8 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 8 ttctgcatta atgtgtgggg tagccataat aaatccccct tt 42 <210> 9 <211> 33 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 9 ccacacatta atgcagaagc aggcgatttc gct 33 <210> 10 <211> 33 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 10 agcgaaatcg cctgcttctg cattaatgtg tgg 33 <210> 11 <211> 41 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 11 cttgaagatg cccgtgaagt gaacctggtt cgcggtatgc t 41 <210> 12 <211> 41 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 12 agcataccgc gaaccaggtt cacttcacgg gcatcttcaa g 41 <210> 13 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 13 ctgggcttca ccggtactta ctccggccgc aaaatttccg ta 42 <210> 14 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 14 tacggaaatt ttgcggccgg agtaagtacc ggtgaagccc ag 42 <210> 15 <211> 40 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 15 atgggtcacg gtatgggtct gccgtcctgc tccatctaca 40 <210> 16 <211> 40 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 16 tgtagatgga gcaggacggc agacccatac cgtgacccat 40 <210> 17 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 17 cgcgtgggta acctgttctc ctccgacctg ttctactctc cg 42 <210> 18 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 18 cggagagtag aacaggtcgg aggagaacag gttacccacg cg 42 <210> 19 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 19 atggaaaaat acggcattct cgcagtggaa atggaagcgg ct 42 <210> 20 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 20 agccgcttcc atttccactg cgagaatgcc gtatttttcc at 42 <210> 21 <211> 41 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 21 aaatacggca ttctcggcac cgaaatggaa gcggctggta t 41 <210> 22 <211> 41 <212> DNA <213> Artificial Sequece <220> <223> <400> 22 ataccagccg cttccatttc ggtgccgaga atgccgtatt t 41 <210> 23 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 23 gtatctgacc acatccgcac tctggagcag accactgccg ct 42 <210> 24 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 24 agcggcagtg gtctgctcca gagtgcggat gtggtcagat ac 42 <210> 25 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 25 accttcaacg acatgatcaa atccgcactg gaatccgttc tg 42 <210> 26 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 26 cagaacggat tccagtgcgg atttgatcat gtcgttgaag gt 42 <210> 27 <211> 41 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 27 atcaaaatcg cactggaatc cctgctgctg ggcgataaag a 41 <210> 28 <211> 41 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 28 tctttatcgc ccagcagcag ggattccagt gcgattttga t 41 <210> 29 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 29 attctcggcg tggaaatgga atccgctggt atctacggcg tc 42 <210> 30 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 30 gacgccgtag ataccagcgg attccatttc cacgccgaga at 42 <210> 31 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 31 gaatttggcg cgaaagccct ggcaatctgc accgtatctg ac 42 <210> 32 <211> 42 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 32 gtcagatacg gtgcagattg ccagggcttt cgcgccaaat tc 42 <210> 33 <211> 39 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 33 tactctccgg acggcgaaac gttcgacgtg atggaaaaa 39 <210> 34 <211> 39 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 34 tttttccatc acgtcgaacg tttcgccgtc cggagagta 39 <210> 35 <211> 39 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 35 tctccggacg gcgaaatgtc cgacgtgatg gaaaaatac 39 <210> 36 <211> 39 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 36 gtatttttcc atcacgtcgg acatttcgcc gtccggaga 39 <210> 37 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 37 ggcgatttcg ctgacgcagt tttgatgcca ggcgac 36 <210> 38 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 38 gtcgcctggc atcaaaactg cgtcagcgaa atcgcc 36 <210> 39 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 39 accatctgca ccgtatttga ccacatccgc actcac 36 <210> 40 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 40 gtgagtgcgg atgtggtcaa atacggtgca gatggt 36 <210> 41 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 41 ccgtcctgct ccatctacgc caaagaactg atcacc 36 <210> 42 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 42 ggtgatcagt tctttggcgt agatggagca ggacgg 36 <210> 43 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 43 atcgctgact tcgacatgat gcgtaacgca gtagat 36 <210> 44 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 44 atctactgcg ttacgcatca tgtcgaagtc agcgat 36 <210> 45 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 45 gtaaaactgc gcgacatcgt tatcggtatg ggtgcc 36 <210> 46 <211> 36 <212> DNA <213> Artificial Sequece <220> <223> Oligonucleotide to act as a PCR primer <400> 46 ggcacccata ccgataacga tgtcgcgcag ttttac 36

[Brief description of drawings]

1A is an HPLC analysis chart of Comparative Example 1, FIG. 1B is an HPLC analysis chart of a reaction solution of Comparative Example 1, and FIG. 1C is an HPLC analysis chart of Example 6. is there.

FIG. 2 (A) is the HP of Comparative Example 2 in Comparative Example.
6 is an LC analysis chart, (B) is an HPLC analysis chart of the reaction liquid of Comparative Example 2, and (C) is an HPLC analysis chart of Example 5.

FIG. 3 shows the results of SDS-polyacrylamide electrophoresis in Example 8.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/10 C12N 15/00 ZNAA C12P 19/40 5/00 A (72) Inventor Kaori Kato Chiba 1144 Togo, Mobara-shi Mitsui Chemicals, Inc. (72) Inventor Reiko Abe, 1144 Togo, Mobara-shi, Chiba (72) Inventor, Toshihiro Oikawa 1144, Togo, Mobara-shi, Chiba (72) Inventor, Mitsui Chemicals, Inc. Yasuko Matsuba 30 Asamu-cho, Omuta-shi, Fukuoka Prefecture Mitsui Chemicals Co., Ltd. (72) Inventor Kiyoteru Nagahara Kiyoteru Nagahara 30 Asamu-cho, Omuta-shi, Fukuoka Prefecture In-house Mitsui Chemicals Co., Ltd. (72) Fukuiri Yasushi 30 Mitsui Chemicals Co., Ltd. (72) Inventor Taiki Ishibashi 30 Asmuta-cho, Omuta-shi, Fukuoka 30 Mitsui Chemicals Co., Ltd. F-term (reference) 4B024 AA01 BA10 BA80 CA04 DA01 DA02 DA05 DA06 DA11 EA04 GA11 HA08 HA09 4B050 CC03 DD02 LL05 4B064 AF33 CA02 CA05 CA10 CA11 CA19 CB27 CC24 CD01 CD12 DA01 4B065 AA01X AA26X AA26Y AA57X AA87X AB01 BD26 BD28 BD34 CA23 CA44

Claims (16)

[Claims] 1. A method for producing a cytosine nucleoside compound, which comprises a step of reacting a phosphorylated saccharide with a cytosine or a cytosine derivative in the presence of an enzyme having a cytosine nucleoside phosphorylase activity to obtain a cytosine nucleoside compound. A method for producing a cytosine nucleoside compound, which is characterized. 2. The method according to claim 1, wherein the enzyme has purine nucleoside phosphorylase activity. 3. The method according to claim 1, wherein the enzyme is derived from Escherichia coli. 4. The cytosine derivative has the following formula (I): (In the formula (I), X represents a carbon atom or a nitrogen atom, and Y represents
Represents a hydrogen atom, a halogen atom or a lower alkyl group. The manufacturing method in any one of Claims 1-3 which is a compound represented by these. 5. The phosphorylated sugar is ribose-1-phosphate,
2-deoxyribose-1-phosphate or 2 ', 3'-
The production method according to claim 1, which is dideoxyribose 1-phosphate or dioxolane sugar phosphate. 6. The method according to claim 1, wherein the enzyme is supplied as a bacterial cell of a microorganism having cytosine nucleoside phosphorylase activity, or an enzyme preparation obtained from the bacterial cell or a culture solution thereof. Production method. 7. The method according to claim 6, wherein the microorganism has a cytosine nucleoside phosphorylase activity and no cytosine deaminase activity and / or cytidine deaminase activity and / or a phosphatase activity. 8. The production method according to claim 7, wherein the cytosine deaminase activity and / or the cytidine deaminase activity of the bacterial cells or the enzyme preparation are inactivated or decreased by a reduction treatment. 9. The bacterial cell of the reduced microorganism is
9. The method according to claim 8, wherein the cells of a microorganism having a cytosine nucleoside phosphorylase activity are brought into contact with water containing an organic solvent to selectively inactivate or reduce the cytosine deaminase activity and the cytidine deaminase activity.
The manufacturing method described in. 10. The microbial cell or enzyme preparation thereof, comprising:
The production method according to claim 7, wherein the substance that inhibits the cytosine nucleoside phosphorylase activity is removed by any of the following a) to c). a) obtaining cytosine nucleoside phosphorylase as a precipitate from the enzyme preparation in a polar solvent, b) obtaining cytosine nucleoside phosphorylase as a precipitate from the enzyme preparation by salting out, c) using a suitable carrier such as a resin from the enzyme preparation Isolate cytosine nucleoside phosphorylase. 11. 1 of the amino acid sequences of SEQ ID NO: 4
0th, 42nd, 54th, 67th, 157th,
178th, 179th, 183rd, 199th, 2
A cytosine nucleoside phosphorylase obtained by substituting any one or more of the 10th, 228th, and 233rd amino acids with another amino acid. 12. The amino acid sequence of SEQ ID NO: 4, 1
Cytosine nucleoside phosphorylase in which the 60th position is phenylalanine and the 205th position is aspartic acid. 13. A recombinant plasmid containing a gene encoding the cytosine nucleoside phosphorylase according to claim 11 or 12. 14. A transformant strain carrying the recombinant plasmid according to claim 13. 15. A cytosine nucleoside phosphorylase, which comprises recovering cytosine nucleoside phosphorylase from the transformant obtained by culturing, the transformant obtained by culturing, the culture broth, and a treated product thereof. Production method. 16. The enzyme preparation according to any one of claims 7 to 9, which comprises an enzyme having purine nucleoside phosphorylase activity.