Method for producing ribose-1-phosphate

Abstract

The present invention provides a method for producing ribose-1-phosphate that is simple and yields improved efficiency. [Solution] A method for producing ribose-1-phosphate, comprising a contact step to produce ribose-1-phosphate and hypoxanthine by contacting inosine, a phosphate donor, an enzyme having purine nucleoside phosphorylase activity, and a medium in a reaction solution containing inosine, a phosphate donor, an enzyme having purine nucleoside phosphorylase activity, and a medium, wherein in the contact step, the hypoxanthine is present in an amount exceeding the saturation concentration relative to the medium.

Description

Technical Field

[0001] The present invention relates to a method for producing ribose-1-phosphate.

Background Art

[0002] Some nucleoside compounds are useful as raw materials for pharmaceuticals, functional chemicals, etc. For example, regarding nicotinamide riboside, it has been reported that it can increase the amount of nicotinamide adenine dinucleotide in cells, and thereby can exhibit effects such as improvement of metabolic disorders and reduction of production of β-amyloid that causes Alzheimer's disease. An example of a method for synthesizing nucleoside compounds is a method of stereoselectively enzymatically synthesizing nucleoside compounds using a compound of pentose-1-phosphate and purine bases or pyrimidine bases as substrates and nucleoside phosphorylases such as purine nucleoside phosphorylase (EC2.4.2.1), pyrimidine nucleoside phosphorylase (EC2.4.2.2), uridine phosphorylase (EC2.4.2.3). Since pentose-1-phosphate is useful as a starting material for synthesizing nucleoside compounds, efficient production thereof is required. As a method for stereoselectively synthesizing pentose-1-phosphate, an enzymatic synthesis method is useful.

[0003] For example, Patent Document 1 describes obtaining ribose-1-phosphates by allowing a microbial cell containing heat-resistant phosphopentomutase (EC5.4.2.7) and substantially free of phosphatase or an enzyme derived from the microorganism to act on ribose-5-phosphates.

[0004] Patent Document 2 describes using S-methylthioribose (MTR) kinase and adenosine triphosphate (ATP) in a method for synthesizing ribose-1-phosphate by reacting ribose or a ribose derivative with a phosphate source.

[0005] Patent Document 3 describes the decomposition of nucleosides and / or nucleotides with the cells or processed products of Corynebacterium or Aerobacter species to produce ribose-1-phosphate.

[0006] Patent Document 4 describes a method for the biosynthesis of nicotinamide mononucleotide, nicotinamide mononucleotide derivatives, or mixtures thereof, and states that ribose-1-phosphate can be produced from nucleosides such as inosine using a catalyst such as purine nucleoside phosphorylase.

[0007] Patent document 5 describes a method for obtaining pentose-1-phosphate by phosphorolytic decomposition of a compound in which the base of a nucleoside raw material has been methylated.

[0008] Patent Document 6 describes (1) a nucleoside selected from the group consisting of inosine and deoxyinosine and a pyrimidine base being subjected to a base exchange reaction by purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase in an aqueous solution in the presence of phosphoric acid or phosphate, and further converting the hypoxanthine produced by this base exchange reaction to uric acid by xanthine oxidase, or (2) a nucleoside selected from the group consisting of inosine and deoxyinosine and a purine base being subjected to a base exchange reaction by purine nucleoside phosphorylase in an aqueous solution in the presence of phosphoric acid or phosphate, and further converting the hypoxanthine produced by this base exchange reaction to uric acid by xanthine oxidase.

[0009] Patent Document 7 describes a method for producing nicotinamide ribozide, nicotinamide ribozide derivatives, or mixtures thereof, and describes how, when contacting a ribonucleoside with nucleoside phosphorylase to produce a free nitrogen base and α-D-ribose 1-phosphate, the conversion of ribonucleoside to α-D-ribose 1-phosphate is enhanced by oxidizing and removing hypoxanthine with xanthine oxidase and oxygen.

[0010] Patent document 8 describes the production of ribose-1-phosphate by adding a nucleoside and nucleoside phosphorylase to water, adjusting the pH and temperature of the reaction system, stirring the reaction solution, passing the reaction solution through a strongly acidic cationic resin, and collecting the effluent to obtain the product. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Japanese Patent Publication No. 2002-95494 [Patent Document 2] Special Publication No. 2023-553990 [Patent Document 3] Special Publication No. 44-024308 [Patent Document 4] U.S. Patent Application Publication No. 2021 / 246476 [Patent Document 5] Russian Patent Application Publication No. 2708971 [Patent Document 6] Japanese Patent Application Publication No. 4-197193 [Patent Document 7] U.S. Patent Application Publication No. 2017 / 0121746 [Patent Document 8] Chinese Patent Application Publication No. 113403356 Specification [Overview of the project] [Problems that the invention aims to solve]

[0012] The reaction described in Patent Document 1 is a phosphate rearrangement reaction from ribose-5-phosphate, but in this reaction, the reaction equilibrium is heavily skewed towards the ribose-5-phosphate side, i.e., the starting material side. Therefore, in order to efficiently synthesize the desired ribose-1-phosphate using this method, a coupling reaction between phosphopentomtase and nucleoside phosphorylase is necessary, which presents the problem of a complicated process.

[0013] The method described in Patent Document 2 requires the use of adenosine triphosphate (ATP), which is expensive and therefore disadvantageous in terms of cost.

[0014] In the methods described in Patent Documents 3 and 4, the amount of α-pentose-1-phosphate produced depends on the reaction equilibrium point, so there was room for improvement in the yield.

[0015] The method described in Patent Document 5 requires an additional step of methylation of the base of the nucleoside raw material, and the methods described in Patent Documents 6 and 7 require an additional enzyme to remove the by-product hypoxanthine. Therefore, these methods have the problem of being complicated processes.

[0016] Patent Document 8 describes an example of producing ribose-1-phosphate from inosine, in which the reaction is stopped when the conversion rate reaches 60% at 37°C. However, in the method described in Patent Document 8, the amount of ribose-1-phosphate produced depends on the reaction equilibrium point, making it practically difficult to achieve a conversion rate of 60%.

[0017] As described above, currently, there is no established method for producing ribose-1-phosphate that is simple and improves the reaction yield using conventional technology.

[0018] One aspect of the present invention aims to solve the above problems and provide a method for producing ribose-1-phosphate that can be produced simply and in improved yield (i.e., efficiently). [Means for solving the problem]

[0019] This invention encompasses the following items. [1] In a reaction solution containing inosine, a phosphate donor, an enzyme having purine nucleoside phosphorylase activity, and a medium, by contacting the inosine, the phosphate donor, and the enzyme having purine nucleoside phosphorylase activity, a method for producing ribose-1-phosphate, comprising a contacting step of producing ribose-1-phosphate and hypoxanthine, A method for producing ribose-1-phosphate, wherein in the contacting step, the hypoxanthine is present in an amount exceeding the saturation concentration with respect to the medium. [2] The method for producing ribose-1-phosphate according to item 1, wherein in the contacting step, the hypoxanthine concentration is 0.33% by mass to 5.00% by mass with respect to 100% by mass of the total amount of the reaction solution. [3] The method for producing ribose-1-phosphate according to item 1 or 2, wherein the concentration of inosine subjected to the contacting step is 2.1% by mass to 22% by mass with respect to 100% by mass of the total mass of the reaction solution. [4] The method for producing ribose-1-phosphate according to any one of items 1 to 3, wherein in the contacting step, the pH of the reaction solution is less than 9.00. [5] The method for producing ribose-1-phosphate according to any one of items 1 to 4, wherein in the contacting step, the temperature of the reaction solution is less than 33°C. [6] The method for producing ribose-1-phosphate according to item 5, wherein the temperature of the reaction solution is 10°C to 25°C. [7] The method for producing ribose-1-phosphate according to any one of items 1 to 6, wherein the enzyme having purine nucleoside phosphorylase activity is a purine nucleoside phosphorylase enzyme having an amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. [Effects of the Invention]

[0020] According to one aspect of the present invention, a method for producing ribose-1-phosphate can be provided, which can produce ribose-1-phosphate simply and with an improved yield (that is, efficiently). [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows the relationship between inosine concentration, hypoxanthine concentration, and reaction yield. [Figure 2] This figure shows the relationship between temperature and reaction yield. [Figure 3] This figure shows the relationship between pH and reaction yield. [Figure 4] This figure shows the relationship between hypoxanthine concentration and reaction yield. [Modes for carrying out the invention]

[0022] The following describes exemplary embodiments of the present invention (hereinafter also referred to as "these embodiments"), but the present invention is not limited to these embodiments. Each component referred to in this disclosure may consist of one or more substances. In the case of multiple substances, the various values ​​for each component refer to the values ​​for all components together unless otherwise specified. Two or more combinations of the various embodiments referred to in this disclosure are also included in this disclosure.

[0023] ≪Method for producing ribose-1-phosphate≫ One aspect of the present invention provides a method for producing ribose-1-phosphate, comprising a contact step in which inosine, a phosphate donor, and an enzyme having purine nucleoside phosphorylase activity are brought into contact in a reaction solution containing inosine, a phosphate donor, an enzyme having purine nucleoside phosphorylase activity, and a medium. In the contact step, ribose-1-phosphate is produced by a phosphorolytic decomposition reaction catalyzed by purine nucleoside phosphorylase, and hypoxanthine, in equimolar amounts with ribose-1-phosphate, is produced as a byproduct. Since the above phosphorolytic decomposition reaction is a reversible reaction, the reaction rates of the forward and reverse reactions match at a specific composition ratio, reaching a reaction equilibrium. Therefore, the reaction yield depends on the reaction equilibrium point. The inventors have found that the equilibrium point can be shifted to the product side by controlling the reaction conditions so that the byproduct hypoxanthine precipitates as an insoluble component. By achieving such an equilibrium shift while maintaining the desired enzyme activity, it becomes possible to produce ribose-1-phosphate simply and in improved yield (i.e., efficiently).

[0024] The reaction conditions for enzyme-catalyzed reactions are usually designed with the aim of maximizing enzyme activity. However, in this embodiment, while assuming that enzyme activity is guaranteed to a desired degree, the reaction conditions are designed particularly with the aim of facilitating the precipitation of hypoxanthine. Therefore, the reaction conditions for the contact step in this embodiment may differ from the optimal conditions from the viewpoint of enzyme activity. According to the inventors' studies, factors influencing the precipitation of hypoxanthine include the solubility (saturation concentration) of hypoxanthine in the medium, the amount of hypoxanthine present in the reaction solution, the temperature of the reaction solution, and the pH of the reaction solution. The term "reaction solution" in this disclosure is intended to include any insoluble components present. In one embodiment, employing one or more methods selected from (1) using a medium with low hypoxanthine solubility, (2) increasing the concentration of the raw materials inosine and phosphate donor in the reaction solution, (3) lowering the temperature of the reaction solution, and (4) lowering the pH of the reaction solution is advantageous for promoting the precipitation of hypoxanthine.

[0025] A method for precipitating hypoxanthine, such as one or more methods selected from (1) to (4) above, can shift the equilibrium point of the phosphoric acid decomposition reaction to the product side without requiring additional operations such as the removal of hypoxanthine from the system or the conversion of hypoxanthine to another substance, and especially without requiring additives to react with hypoxanthine. This is because the hypoxanthine precipitate remains in the reaction solution but is away from the reaction site of the phosphoric acid decomposition reaction. The fact that no additional operations are required is advantageous in that it simplifies the process, and in particular, the fact that no additives to react with hypoxanthine are required is advantageous in that there is no concern about reaction inhibition due to the use of such additives.

[0026] <Contact process> In the contact step, hypoxanthine is present in an amount exceeding the saturation concentration of the medium. That is, in the contact step, an insoluble component is present in the reaction solution, and this insoluble component contains hypoxanthine. The reaction solution is typically a slurry. The presence or absence of hypoxanthine in the insoluble component is confirmed by the method described in the [Examples] section of this disclosure, or by a method that is understood to those skilled in the art to be equivalent thereto. The insoluble component may include hypoxanthine and other components (such as inosine). At concentrations above the solubility of inosine, the insoluble component in the reaction solution is hypoxanthine and unreacted inosine. In a typical embodiment, the solubility of ribose-1-phosphate in the medium used in the contact step is extremely high. Therefore, in a typical embodiment, the entire amount of ribose-1-phosphate is present as a soluble component.

[0027] The contact method is not particularly limited. For example, inosine, a phosphate donor, and an enzyme having purine nucleoside phosphorylase activity may be dissolved or dispersed in a medium, respectively. In one embodiment, inosine and a phosphate donor may be present in a buffer adjusted to a desired pH, and the enzyme may be added thereto. The reaction mode is typically batch, but is not limited thereto. The reaction conditions may remain constant throughout the contact step, but may be changed as needed. For example, the temperature may be set to a relatively high value in the first half of the step, and then shifted to a relatively low value when the reaction approaches equilibrium in the second half of the step. In the exemplary embodiment, the temperature may be set to 30°C to 55°C (e.g., 30°C) in the first half of the contact step, and then shifted to 5°C to 20°C (e.g., 20°C, 15°C, 10°C, or 5°C) in the second half of the step.

[0028] [Inosine] The inosine used in the contact step may be in the form of a free form or a salt thereof, and examples of salt forms include sodium salt, potassium salt, and ammonium salt. The free form of inosine or a salt thereof may be a commercially available product, for example, inosine from Tokyo Chemical Industry Co., Ltd. The inosine may also be obtained by recycling (e.g., isolation and purification) the unreacted portion after use in the reaction. For example, if it is a sodium salt, examples include inosine isolated and purified by the method described in Japanese Patent Publication No. 42-12432 and Japanese Patent Publication No. 46-29790.

[0029] [Phosphate donor] The phosphate donor used in the contact process can be in the form of orthophosphoric acid in aqueous solution, and examples include phosphoric acid or its salts; one or more types may be used. Preferred examples of phosphates include diammonium hydrogen phosphate, dipotassium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hydrogen phosphate, and disodium hydrogen phosphate.

[0030] [Enzymes possessing purine nucleoside phosphorylase activity] The enzyme having purine nucleoside phosphorylase activity used in the contact step only needs to have the ability to recognize the substrate inosine and catalyze the phosphorolytic decomposition reaction to ribose-1-phosphate, and its origin is not limited. Purine nucleoside phosphorylase is widely distributed in the biological world and can be found in the tissues of mammals, birds, fish, etc., as well as in yeast, bacteria, etc. In one embodiment, the enzyme having purine nucleoside phosphorylase activity may be used in the contact step in the form of an enzyme solution (e.g., an aqueous solution or an aqueous suspension) obtained by dissolving or dispersing the enzyme, bacterial cells, culture medium, or a mixture thereof in water or a water-containing medium. The bacterial cells may be treated products or immobilized products of bacterial cells. The treated products may be, for example, acetone-dried bacterial cells, or bacterial cell lysates prepared by mechanical destruction, ultrasonic disruption, freeze-thaw treatment, pressurization / vacuum treatment, osmotic treatment, autolysis, cell wall decomposition treatment, surfactant treatment, etc. If necessary, ammonium sulfate precipitation, acetone precipitation, purification by column chromatography, etc. may be performed. The immobilized product may be produced by immobilizing the enzyme or bacterial cell with materials commonly used for immobilizing enzymes or bacterial cells, such as polyacrylamide gel or carrageenan.

[0031] Microorganisms that express enzymes possessing purine nucleoside phosphorylase activity include microorganisms belonging to the Escherichia genus, such as Escherichia coli; microorganisms belonging to the Klebsiella genus, such as Klebsiella pneumoniae; microorganisms belonging to the Morganella genus, such as Morganella morganii; and microorganisms belonging to the Moritella genus, such as Moritella sp. However, microorganisms belonging to the Escherichia genus, such as Escherichia coli, are preferred. Transformed organisms obtained by introducing the genes of such microorganisms into any host can also be used.

[0032] Transformants can be prepared, for example, by creating an expression vector for purine nucleoside phosphorylase and then introducing this expression vector into a host. The expression vector contains a polynucleotide (DNA, RNA, etc.) encoding the amino acid sequence of purine nucleoside phosphorylase. In addition to the polynucleotide, the expression vector may further contain regions such as a promoter, terminator, and a region encoding a drug resistance gene. The expression vector may be a plasmid or an integrative vector. The expression vector may be a viral vector or a cell-free vector. Examples of hosts for introducing the expression vector include prokaryotic cells such as Escherichia coli and other Escherichia species, Corynebacterium species, and Bacillus species, and eukaryotic cells such as Saccharomyces species, Pichia species, and Aspergillus species. Among these, Escherichia species, especially Escherichia coli, are typical. By transforming a host using an expression vector and culturing the transformed host, transformants expressing purine nucleoside phosphorylase can be obtained. The culture medium for the host can be selected from commonly used media such as LB medium.

[0033] The transformants may be subjected to the contact process as they are, or the purine nucleoside phosphorylase recovered from the transformants may be subjected to the contact process. The purine nucleoside phosphorylase may be recovered from the transformants by methods commonly used in this industry, such as freeze-thaw cycles, sonication, or dissolution.

[0034] The phosphoric acid decomposition reaction in this embodiment is an enzyme-catalyzed reaction, and since the equilibrium point does not substantially shift even if the enzyme species (molecular species) is different, there are no particular limitations on the molecular structure of the purine nucleoside phosphorylase. In one embodiment, the enzyme species may be appropriately selected according to the desires of availability, reaction rate, optimal pH, etc.

[0035] In one embodiment, the purine nucleoside phosphorylase may be a purine nucleoside phosphorylase having an amino acid sequence that has 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. Sequence ID 1: MATPHINAEMGDFADVVLMPGDPLRAKYIAETFLEDAREVNNVRGMLGFTGTYKGRKISVMGHGMGIPSCSIYTKELITDFGVKKIIRVGSCGAVLPHVKLRDVVIGMGACTDSKVNRI RFKDHDFAAIADFDMVRNAVDAAKALGIDARVGNLFSADLFYSPDGEMFDVMEKYGILGVEMEAAGIYGVAAEFGAKALTICTVSDHIRTHEQTTAAERQTTFNDMIKIALESVLLGDKE

[0036] The purine nucleoside phosphorylase having the amino acid sequence of Sequence ID No. 1 is a wild-type purine nucleoside phosphorylase that can be obtained from the GenBank database provided by the National Center for Biotechnology Information (NCBI) as Genbank Accession No. WP_000224877.1 (Proteobacteria, Gammaproteobacteria, Escherichia coli).

[0037] The sequence identity with the amino acid sequence of Sequence ID No. 1 may, in one embodiment, be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more. In this disclosure, the value (%) of sequence identity of the amino acid sequence of the enzyme refers to the value (Identity) expressed as a percentage when the number of matching amino acid residues is divided by the number of aligned amino acid residues, after aligning the reference amino acid sequence with the amino acid sequence of the enzyme to be evaluated to maximize the agreement. When aligning the reference amino acid sequence with the amino acid sequence of the enzyme to be evaluated, deletions (gaps) may be included. The sequence identity of the amino acid sequence can be calculated by methods well known to those skilled in the art, but the sequence identity of the amino acid sequence shown in this disclosure is a value calculated using Blasp (protein-protein BLAST), a search program of BLAST (Basic Local Alignment Search Tool).

[0038] Examples of sequences with amino acid sequence identity of 70% or more with SEQ ID NO: 1 include SEQ ID NOs: 2 to 4, but in this embodiment, purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 1 is preferred. Sequence ID 2: MATPHINAEMGDFADVVLMPGDPLRAKHIAETFLEDVREVNNVRGMLGFTGTYKGRKISVMGHGMGIPSCSIYTKELITDFGVKKIIRVGSCGAVREDVKLRDVVIGMGACTDSKVNRL RFKDHDFAAIADFGMVRNAVDATKALGVDARVGNIFSADLFYTPDPSMFDVMEKYGILGVEMEAAGIYGVAAEFGAKALTICTVSDHIRTHEQTTAAERQTTFNDMIKIALESVLLGDKE Sequence ID 3: MATPHINAERGDFADVVLMPGDPLRAKYIAENFLEDIKQVNNVRGMLGFTGTYKGRRISVMGHGMGIPSCSIYAKELITEFDVKTIIRVGSCGAISRDVKLRDVVIGMGASTDSKVNRL RFKDNDFAAIADFGLVRNAVEAAETAGIPARVGNIFSTDLFYTPDPQMFDVMEKYGILGVEMEAAGIYGVAAEFGAKALTICTVSDHIRTGENLPAEERQTTFNEMMTIALESVLLGDKK Sequence ID 4: MATPHINAEKGDFAETVLFPGDPLRAKYIAETFLEDVKQVNDVRNMGFGFTGTYKGKRVSVMGSGMGIPSCSIYAKELITEYGVKNLIRVGSCGAISTDVKVRDVVIGMGACTDSAVNR ARFDGYDFAAIASWELLSKVTRAAKACNIDAKVGNIFSADLFYTPKPELFDTMEKLGILGVEMEAAGLYGVAAEFGANAICICTVSDHIRTGEVTTAEERQLTFNDMIIMALESILIED

[0039] The amino acid sequence of Sequence ID No. 2 is that of a wild-type purine nucleoside phosphorylase, obtainable from the GenBank database as Genbank Accession No. WP_195807586.1 (Proteobacteria, Gammaproteobacteria, derived from Klebsiella pneumoniae), and has a sequence identity of 95% with the amino acid sequence of Sequence ID No. 1. The amino acid sequence of Sequence ID No. 3 is that of a wild-type purine nucleoside phosphorylase, obtainable from the GenBank database as Genbank Accession No. WP_126324207.1 (Proteobacteria, Gammaproteobacteria, derived from Morganella morganii), and has an 84% sequence identity with the amino acid sequence of Sequence ID No. 1. The amino acid sequence of Sequence ID No. 4 is that of a wild-type purine nucleoside phosphorylase, obtainable from the GenBank database as Genbank Accession No. WP_293148083.1 (Proteobacteria, Gammaproteobacteria, Moritella sp.), and has a sequence identity of 74% with the amino acid sequence of Sequence ID No. 1.

[0040] The enzyme possessing purine nucleoside phosphorylase activity may be a commercially available product. Examples of commercially available products include PNP-311 from Toyobo Co., Ltd.

[0041] Enzymes having purine nucleoside phosphorylase activity may be pretreated at 100°C or below, preferably 70°C or below, and / or with organic solvents such as methanol, ethanol, isopropanol, dimethylformamide, and dimethyl sulfoxide, as necessary.

[0042] [Medium] The medium used in the contact step is selected to obtain the desired precipitation of hypoxanthine. The saturation concentration varies depending on the type of medium and temperature. For example, if the medium is water at 20°C (pH 7), the saturation concentration is approximately 0.07% by mass for hypoxanthine and approximately 2.1% by mass for inosine. In the contact step, hypoxanthine is present in the medium in an amount greater than the saturation concentration. On the other hand, in the contact step, inosine and ribose-1-phosphate may be present in the medium in an amount greater than the saturation concentration or in an amount less than or equal to the saturation concentration. In one embodiment, the saturation concentration of the medium in the contact step is hypoxanthine < inosine < ribose-1-phosphate, and hypoxanthine and inosine are present in amounts greater than the saturation concentration of the medium, while ribose-1-phosphate is present in an amount less than or equal to the saturation concentration of the medium.

[0043] From the viewpoint of shifting the equilibrium point of the phosphoric acid decomposition reaction more toward the product side, it is preferable that the saturation concentration of hypoxanthine be significantly low. For example, when the medium is water at 50°C (pH 7), the saturation concentration is approximately 8.0% by mass for inosine and approximately 0.17% by mass for hypoxanthine. For example, when the medium is water at 20°C (pH 7), the saturation concentration is approximately 2.1% by mass for inosine and approximately 0.07% by mass for hypoxanthine. For example, when the medium is water at 5°C (pH 7), the saturation concentration is approximately 1.0% by mass for inosine and approximately 0.004% by mass for hypoxanthine.

[0044] For example, the saturation concentration of hypoxanthine in the medium during the actual contact process may be 0.004% by mass or less, or 0.08% by mass or less, and the saturation concentration of inosine in the medium may be 0.5% by mass or more, or 1.0% by mass or more.

[0045] In terms of ease with which the enzyme activity can be increased to a desired level while adjusting the saturation concentration of hypoxanthine to a desired range, a preferred medium is water, or a combination of water and an organic solvent. These mediums may be pH-adjusted with a buffer as needed. The organic solvent is preferably miscible with water at the reaction temperature. Examples of organic solvents include alcohols such as methanol, ethanol, and isopropanol; ketones such as acetone; amides such as dimethylformamide; sulfoxides such as dimethyl sulfoxide; and hydrocarbons such as hexane, heptane, and toluene. One or more of these may be combined with water in any ratio. In one embodiment, the content of the organic solvent in the total 100% by mass of water and the organic solvent may be 1% by mass or 5% by mass or more, and in another embodiment, it may be 30% by mass or less, or 20% by mass or less.

[0046] [Additional ingredients] The reaction solution may contain additional components as needed. These additional components may be one or more selected from, for example, buffers, pH adjusters, surfactants, inorganic salts, etc.

[0047] Examples of buffering agents include sodium acetate buffer, ammonium acetate buffer, Tris (trishydroxymethylaminomethane) buffer, and MES (2-morpholinoethanesulfonic acid) buffer.

[0048] Examples of pH adjusters include acids such as hydrochloric acid, sulfuric acid, nitric acid, formic acid, acetic acid, and propionic acid, and alkalis such as sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, lithium hydroxide, lithium carbonate, and ammonia. Examples of surfactants include sodium dodecyl sulfate. Examples of inorganic salts include magnesium hydroxide, magnesium chloride, magnesium sulfate, calcium chloride, barium chloride, calcium sulfate, and barium sulfate.

[0049] [Composition of the reaction solution] The hypoxanthine concentration in the reaction solution (i.e., the total concentration of dissolved and insoluble components) tends to increase as the concentration of inosine, the starting material, increases. Since hypoxanthine precipitates easily, improving the reaction yield is straightforward. Therefore, the hypoxanthine concentration in the reaction solution is preferably 0.33% by mass or more, or 0.35% by mass or more, or 0.47% by mass or more, or 1.00% by mass or more, or 2.00% by mass or more. If the amount of insoluble components in the reaction solution becomes excessive due to precipitation, the fluidity of the reaction solution tends to decrease, reducing operability. Therefore, from the viewpoint of obtaining good operability, the hypoxanthine concentration in the reaction solution is preferably 5.00% by mass or less, or 4.50% by mass or less, or 4.40% by mass or less, or 3.50% by mass or less, or 2.80% by mass or less. The hypoxanthine concentration is a value measured by the method described in the [Examples] section of this disclosure, or by a method that is understood to those skilled in the art to be equivalent thereto. In one embodiment, the hypoxanthine concentration exemplified above is a value measured at the end of the contact process.

[0050] At the start of the contact process, the inosine concentration in the reaction solution is preferably 2.1% by mass or more, or 3% by mass or more, or 3.5% by mass or more, or 4% by mass or more, in terms of the effect of improving the yield by precipitating hypoxanthine. From the viewpoint of maintaining good fluidity of the reaction solution by preventing excessive precipitation of hypoxanthine, it is preferably 22% by mass or less, or 20% by mass or less, or 19% by mass or less, or 18% by mass or less, or 17% by mass or less, or 16% by mass or less, or 15% by mass or less, or 14% by mass or less, or 13% by mass or less.

[0051] In the reaction solution at the start of the contact process, the molar ratio of the phosphate donor to inosine is preferably 0.40 or higher, or 0.60 or higher, from the viewpoint of ensuring that the phosphoric acid decomposition reaction proceeds smoothly, and preferably 1.5 or lower, or 1.2 or lower, from the viewpoint of reducing the amount of unreacted residual phosphate donor discharged.

[0052] In the reaction solution at the start of the contact process, the amount of enzyme having purine nucleoside phosphorylase activity is preferably 0.10 to 0.001 g-dried bacterial cells / g-inosine relative to inosine. For example, it may be 0.001 parts by mass or more, or 0.005 parts by mass or more, per 100 parts by mass of inosine, and may be 1.0 part by mass or less, or 0.05 parts by mass or less, from the viewpoint of the bacterial cell and enzyme removal process.

[0053] In the reaction solution at the end of the contact process, the inosine may be entirely dissolved, or some of it may have precipitated.

[0054] In one embodiment, the yield of ribose-1-phosphate four hours after the start of the reaction in the contact step may be 20% or more, 30% or more, or 40% or more. A higher yield is advantageous, but from the viewpoint of ease of process control, in one embodiment, it may be 90% or less, 80% or less, 70% or less, or 60% or less. Since ribose-1-phosphate and hypoxanthine are produced in equimolar amounts, the above yield can be determined as (amount of hypoxanthine produced) / (amount of hypoxanthine produced + amount of residual inosine). Details of the measurement method are described in the [Examples] section.

[0055] [pH] In the contact step, if the pH of the reaction solution is 5.00 or higher and less than 9.00, practical problems such as inactivation of enzymes having purine nucleoside phosphorylase activity usually do not occur. However, in the alkaline range, especially above pH 9.50, hypoxanthine tends not to precipitate easily. Therefore, the pH of the reaction solution is preferably 9.50 or lower, or 9.00 or lower, or 8.60 or lower, or 8.00 or lower, or 7.30 or lower. In particular, from the viewpoint of suppressing the decomposition of ribose-1-phosphate, a preferred pH is 7.30 or lower. The lower limit of the pH is preferably 5.00 or higher, or 5.50 or higher, or 6.10 or higher, or 6.30 or higher, or 6.60 or higher, in terms of good enzyme reaction rate. pH is a value measured by the glass electrode method. In one embodiment, the pH in the above example is a value measured at the start of the contact step.

[0056] [temperature] In the contact step, the temperature of the reaction solution may be, for example, 5°C to 55°C. If the temperature of the reaction solution is 10°C to 55°C, practical problems such as inactivation of enzymes having purine nucleoside phosphorylase activity usually do not occur. However, under high temperature conditions, especially above 55°C, hypoxanthine tends to precipitate less easily. Therefore, the temperature of the reaction solution is preferably 55°C or lower, or 50°C or lower, or 45°C or lower, or 40°C or lower, or 35°C or lower, or 33°C or lower, or less than 33°C, or 30°C or lower, or 25°C or lower, or 20°C or lower. The reaction yield tends to improve when the temperature of the reaction solution is lower, such as below 33°C. However, under low temperature conditions, for example below 10°C, the rate of the enzyme reaction tends to decrease, which may result in a lower reaction yield within a given time. From this viewpoint, the temperature of the reaction solution is preferably 10°C or higher, or 12°C or higher, or 15°C or higher. The preferred temperature range for the reaction solution is 10°C to 25°C, more preferably 10°C to 20°C, and even more preferably 15°C to 20°C. The temperature of the reaction solution is preferably maintained within the above exemplary range throughout the entire contact process, but it may be maintained within the above exemplary range only if the desired reaction proceeds, for example, excluding a portion of the contact process.

[0057] [time] The contact time is preferably sufficient for the phosphoric acid decomposition reaction to reach equilibrium. The time to reach equilibrium varies depending on the type and amount of enzyme, but in one example it is about 4 hours. The contact time is preferably 1 hour or more, or 2 hours or more, and preferably 24 hours or less, or 20 hours or less.

[0058] <Post-process> After the contact step, ribose-1-phosphate in the reaction solution may be isolated using conventional methods such as filtration, concentration, crystallization, dissolution, electrodialysis, or adsorption / desorption (ion exchange resin, activated carbon, silica gel column chromatography, etc.). In one embodiment, the reaction solution may be cooled (to below 5°C in one embodiment) before isolation. For example, ribose-1-phosphate can be isolated by immobilization using a metal salt. Examples of metal salts that can be used include barium hydroxide, barium chloride, magnesium hydroxide, and magnesium chloride. The amount of metal salt added may be 0.1 moles or more, or 0.5 moles or more, or 10 moles or less, or 5 moles or less, per mole of ribose-1-phosphate in one embodiment. The metal salt may be added to the reaction solution all at once or sequentially.

[0059] After the contact step, unreacted phosphate donors in the reaction solution may be removed by adding an inorganic salt and precipitating it, or by removing them with an anion exchange resin, etc. Examples of inorganic salts include magnesium hydroxide, calcium hydroxide, barium hydroxide, iron hydroxide, copper hydroxide, manganese hydroxide, cobalt hydroxide, zinc hydroxide, nickel hydroxide, magnesium chloride, magnesium acetate, magnesium sulfate, iron chloride, iron sulfate, calcium chloride, and barium chloride. Preferably, magnesium hydroxide, calcium hydroxide, magnesium chloride, magnesium acetate, and magnesium sulfate are used, and more preferably, magnesium hydroxide and magnesium chloride. When phosphate is precipitated as a poorly water-soluble salt and removed by filtration, the absence of sodium ions also has the effect of increasing the filtration rate.

[0060] After the contact step, the enzymes in the reaction solution may be removed by adsorption using, for example, activated carbon. In this process, inosine can also be adsorbed and removed by the activated carbon. The removed inosine can be reused as a raw material after purification by an appropriate method.

[0061] Uses of ribose-1-phosphate Nucleoside compounds such as nicotinamide ribozide, nicotinamide mononucleotide, ribavirin, and tecadenoson can be produced using ribose-1-phosphate as a raw material. Particularly useful applications include nicotinamide ribozide and nicotinamide mononucleotide. [Examples]

[0062] The following describes illustrative embodiments of the present invention with reference to examples, but the present invention is not limited to these examples. Note that each of the specific numerical values ​​described below may be replaced with the upper or lower limit of the numerical range described in the [Modes for Carrying Out the Invention] section of this disclosure.

[0063] ≪Analysis method≫ <Reaction yield> In the synthesis reaction of ribose-1-phosphate, after a predetermined time had elapsed since the start of the reaction, the reaction solution was sampled while stirred and diluted 400 times by mass with a 0.4% by mass sodium hydroxide aqueous solution to obtain a sample solution. At this time, in reaction solutions containing insoluble components, the insoluble components were completely dissolved. High-performance liquid chromatography (HPLC) was used on the obtained sample solution to determine the amount of hypoxanthine produced and the amount of inosine remaining by calculating the area under the peak curve of the chromatograph corresponding to hypoxanthine and inosine. The reaction yield was calculated as (amount of hypoxanthine produced) / (amount of hypoxanthine produced + amount of inosine remaining). Measurements using high-performance liquid chromatography (HPLC) were performed according to the following conditions. Equipment: Manufactured by JASCO Corporation, model number Flow rate: 1.0ml / min Eluent: 10 mM phosphoric acid, methanol:water = 1:9 Analytical column: Develosil ODS-MG-5 (4.6 μm × 250 mm) Column temperature: 40℃ Detection method: UV 254nm

[0064] <Presence or absence of insoluble components and their types> At the start of the reaction and 4 hours after the start of the reaction, the amount of insoluble components precipitated in the reaction solution was measured by HPLC after separating the supernatant from the insoluble components by centrifugation or filtration. The insoluble components were evaluated according to the following criteria. Precipitation "Present": Four hours after the start of the reaction, insoluble components were present, and the amount of insoluble components had increased compared to the start of the reaction. Precipitation "None": Four hours after the start of the reaction, no insoluble components were present, or the amount of insoluble components had not increased compared to the start of the reaction. Furthermore, the types of substances contained in the insoluble components were identified using the following method. For example, the reaction solutions of Examples 8 and 9 were separated into supernatant (soluble components) and precipitate (insoluble components) by filtration or centrifugation, and each was subjected to HPLC analysis. The presence of hypoxanthine and inosine was confirmed in the precipitate. The amount of hypoxanthine in the precipitate could be calculated from the difference between the amount of hypoxanthine in the reaction solution and the amount of hypoxanthine in the supernatant, which were determined by HPLC analysis. The percentage of precipitated hypoxanthine was calculated to be 98-99%.

[0065] <<Preparation of enzyme solution>> The Escherichia coli strain MT-10905 was obtained following the procedure described in Example 9 of Japanese Patent Publication No. 2008-029358. The specific procedure is as follows:

[0066] Escherichia coli chromosomal DNA was prepared as follows: 50 ml of Escherichia coli K-12 / XL-10 strain (Stratagene) was inoculated into LB medium and incubated overnight at 37°C. The cells were then collected and lysed with a lysozyme 1 mg / ml lysozyme lysate. After treating the lysozyme lysate with phenol, the DNA was precipitated by ethanol precipitation using a standard method. The resulting DNA precipitate was collected by wrapping it around a glass rod, washed, and used for PCR.

[0067] For the PCR primers, we used oligonucleotides (synthesized by Hokkaido System Science Co., Ltd.) having the nucleotide sequences shown in Sequence ID Nos. 5 and 6 (corresponding to Sequence ID Nos. 1 and 2 in Japanese Patent Publication No. 2008-029358, respectively), which were designed based on the nucleotide sequence of the known deoD gene of Escherichia coli (GenBank accession No. AE000508 (code region is nucleotide number 11531-12250)). Sequence ID 5: gtgaattcac aaaaaggata aaacaatggc Sequence ID 6: tcgaagcttg cgaaacacaa ttactcttt These primers have restriction enzyme recognition sequences for EcoRI and HindIII near their 5' and 3' ends, respectively.

[0068] PCR was performed using 0.1 ml of PCR reaction solution containing 6 ng / μl of E. coli chromosomal DNA completely digested with the restriction enzyme HindIII and 3 μM each of primers, under a reaction cycle consisting of denaturation: 96°C for 1 minute, annealing: 55°C for 1 minute, and extension reaction: 74°C for 1 minute, for 30 cycles.

[0069] The above reaction products and plasmid pUC18 (Takara Shuzo Co., Ltd.) were digested with EcoRI and HindIII, ligated using Ligation Hi (Toyobo Co., Ltd.), and then Escherichia coli DH5α was transformed using the resulting recombinant plasmid. The transformed strains were cultured on LB agar medium containing ampicillin (Am) 50 μg / ml and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) to obtain Am-resistant transformed strains with white colonies.

[0070] Plasmids were extracted from the transformed strains obtained in this manner to obtain a plasmid (pUC-PNP73) in which the target DNA fragment was inserted. Following the above procedure, a transformant similar to Escherichia coli MT-10905 described in Example 9 of Japanese Patent Publication No. 2008-029358 was obtained.

[0071] These transformants were cultured in 100 mL of LB medium containing 100 mg / L ampicillin sodium at 33°C for 16 hours with shaking. The resulting culture was centrifuged at 8000 rpm for 20 minutes to collect the cells. The collected cells were suspended in pure water and then frozen and stored at -20°C.

[0072] The above bacterial cell suspension was freeze-thawed to prepare a purine nucleoside phosphorylase-containing bacterial cell suspension, which was used as an enzyme solution in the synthesis reaction of ribose-1-phosphate. The solid content of the suspension, calculated from the mass of the bacterial cells in the purine nucleoside phosphorylase-containing bacterial cell suspension, was approximately 12-14% by mass.

[0073] <<Production of ribose-1-phosphate>> <Investigation of the effect of raw material concentration on reaction yield> [Comparative Example 1] 0.40 mmol of inosine (commercially available from Tokyo Chemical Industries, Ltd., 98% purity; the same applies to inosine hereafter) and 0.51 mmol of diammonium hydrogen phosphate (commercially available from Fujifilm Wako Pure Chemical Industries, Ltd., 98.5% purity; the same applies to diammonium hydrogen phosphate hereafter) were dissolved in 9.77 g of pure water, and 0.04 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis reaction of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the raw materials. The final concentration of inosine was 1.06% by mass, the reaction pH (measured at the start of the reaction using a glass electrode pH meter (Horiba, Ltd.); the same measurement was performed in other examples) was 7.80, and the amount of hypoxanthine produced at 4 hours and 24 hours of reaction time was quantitatively analyzed by high-performance liquid chromatography to calculate the reaction yield. The results showed a yield of 14% after 4 hours of reaction and 13% after 24 hours of reaction. Since the yields at 4 and 24 hours were almost the same, it was determined that the reaction reached equilibrium within 4 hours, and the yield stopped at 14%. No precipitation was observed in the reaction solution. The mass concentration of hypoxanthine was 0.08% by mass.

[0074] [Comparative Example 2] 0.77 mmol of inosine and 0.79 mmol of diammonium hydrogen phosphate were dissolved in 9.51 g of pure water, and 0.16 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 2.05% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 13% at 4 hours of reaction. No precipitation was observed in the reaction solution. The mass concentration of hypoxanthine was 0.13% by mass.

[0075] [Example 1] 1.17 mmol of inosine and 1.30 mmol of diammonium hydrogen phosphate were mixed with 9.41 g of pure water, and 0.24 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 3.14% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 23% at 4 hours of reaction. Precipitation was observed at 4 hours from the start of the reaction. The mass concentration of hypoxanthine was 0.36% by mass.

[0076] [Example 2] 1.53 mmol of inosine and 1.65 mmol of diammonium hydrogen phosphate were mixed with 9.22 g of pure water, and 0.29 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 4.11% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 32% at 4 hours of reaction. Precipitation was observed at 4 hours from the start of the reaction. The mass concentration of hypoxanthine was 0.64% by mass.

[0077] [Example 3] 1.87 mmol of inosine and 1.87 mmol of diammonium hydrogen phosphate were mixed with 9.09 g of pure water, and 0.34 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 5.02% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 33% at 4 hours of reaction. Precipitation was observed at 4 hours from the start of the reaction. At this time, the mass concentration of hypoxanthine was 0.81% by mass.

[0078] [Example 4] 3.87 mmol of inosine and 4.11 mmol of diammonium hydrogen phosphate were mixed with 7.80 g of pure water, and 0.71 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 10.40% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 40% at 4 hours of reaction. Precipitation was observed at 4 hours from the start of the reaction. At this time, the mass concentration of hypoxanthine was 2.32% by mass.

[0079] [Example 5] 4.76 mmol of inosine and 4.82 mmol of diammonium hydrogen phosphate were mixed with 7.17 g of pure water, and 0.81 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 12.78% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 41% at 4 hours of reaction. Precipitation was observed at 4 hours from the start of the reaction. At this time, the mass concentration of hypoxanthine was 2.66% by mass.

[0080] [Example 6] 5.55 mmol of inosine and 5.57 mmol of diammonium hydrogen phosphate were mixed with 6.87 g of pure water, and 1.02 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The final concentration of inosine was 14.90% by mass, and the reaction pH was 7.80. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction. The result was 41% at 4 hours of reaction. Precipitation was observed at 4 hours from the start of the reaction, and an increase in the viscosity of the reaction solution was confirmed as the reaction progressed. At this time, the mass concentration of hypoxanthine was 3.68% by mass.

[0081] [Example 7] 7.44 mmol of inosine and 7.63 mmol of diammonium hydrogen phosphate were dissolved in 5.64 g of pure water, and 1.33 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added to carry out the synthesis of ribose-1-phosphate at 30°C. The final concentration of inosine was 19.96% by mass, and the reaction pH was 7.80. The amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.1 g-dried bacterial cells / g-inosine relative to the starting material. The reaction yield was calculated from the amount of hypoxanthine produced at 4 hours of reaction time. The result was 44% at 4 hours of reaction time. As the reaction progressed, the viscosity of the reaction solution increased significantly, making it impossible to stir the reaction solution. At this time, the mass concentration of hypoxanthine was 4.51% by mass. The results are summarized in Table 1 and Figure 1.

[0082] [Table 1]

[0083] In Comparative Examples 1 and 2, where the inosine concentration was low, hypoxanthine did not precipitate due to the small amount produced. Furthermore, the increase in hypoxanthine concentration when the inosine concentration was increased from Comparative Example 1 to Comparative Example 2 was slight, and the reaction yield of Comparative Example 2 was lower than that of Comparative Example 1. On the other hand, in Examples 1 to 7, where the inosine concentration was above the predetermined level, hypoxanthine precipitated. Moreover, the increase in hypoxanthine concentration when the inosine concentration was increased was significant, and the reaction yield improved with increasing inosine concentration. From these results, it can be seen that under reaction conditions in which hypoxanthine precipitates, the equilibrium point shifted to the hypoxanthine side (i.e., the product side).

[0084] <Investigation of the effect of reaction temperature on reaction yield> [Example 8] 3.67 mmol of inosine and 3.82 mmol of diammonium hydrogen phosphate were mixed with 8.41 g of pure water, and 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was 9.81% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.01 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out at a reaction temperature of 10°C for 4 hours. The amount of hypoxanthine produced 4 hours after the start of the reaction was quantified, and the reaction yield was 44%. At this time, the mass concentration of hypoxanthine was 2.00% by mass.

[0085] [Example 9] 3.68 mmol of inosine and 3.82 mmol of ammonium hydrogen phosphate were mixed with 8.41 g of pure water, and 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was 9.86% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.01 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out at a reaction temperature of 15°C for 4 hours. The amount of hypoxanthine produced 4 hours after the start of the reaction was quantified, and the reaction yield was calculated. At this time, the mass concentration of hypoxanthine was 2.41% by mass.

[0086] [Example 10] 3.65 mmol of inosine and 3.78 mmol of ammonium hydrogen phosphate were mixed with 8.40 g of pure water, and 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was 9.80% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.01 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out at a reaction temperature of 20°C for 4 hours. The amount of hypoxanthine produced 4 hours after the start of the reaction was quantified, and the reaction yield was 48%. At this time, the mass concentration of hypoxanthine was 2.52% by mass.

[0087] [Example 11] 3.65 mmol of inosine and 3.71 mmol of ammonium hydrogen phosphate were mixed with 8.55 g of pure water, and 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was 9.50% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.01 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out at a reaction temperature of 30°C for 4 hours. The amount of hypoxanthine produced 4 hours after the start of the reaction was quantified, and the reaction yield was 40%. At this time, the mass concentration of hypoxanthine was 1.96% by mass.

[0088] [Example 12] 0.029 mol of inosine and 0.029 mol of diammonium hydrogen phosphate were mixed with 87.00 g of pure water, and 1.00 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was approximately 7.84% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.02 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out for 4 hours at a reaction temperature of 45°C. The amount of hypoxanthine produced after 4 hours from the start of the reaction was quantified, and the reaction yield was 27%. At this time, the mass concentration of hypoxanthine was 1.04% by mass.

[0089] [Example 13] 0.047 mol of inosine and 0.048 mol of diammonium hydrogen phosphate were mixed with 79.58 g of pure water, and 1.00 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was approximately 12.73% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.01 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out for 4 hours at a reaction temperature of 50°C. The amount of hypoxanthine produced after 4 hours from the start of the reaction was quantified, and the reaction yield was 37%. At this time, the mass concentration of hypoxanthine was 2.21% by mass.

[0090] [Example 14] 0.036 mol of inosine and 0.036 mol of diammonium hydrogen phosphate were mixed with 84.27 g of pure water, and 1.00 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added. The final concentration of inosine was approximately 9.80% by mass, and the amount of dried bacterial cells expressing purine nucleoside phosphorylase used was 0.01 g-dried cells / g-inosine relative to the starting material. The pH was 7.80. The reaction was carried out for 4 hours at a reaction temperature of 55°C. The amount of hypoxanthine produced 4 hours after the start of the reaction was quantified, and the reaction yield was 32%. At this time, the mass concentration of hypoxanthine was 1.91% by mass. The results are summarized in Table 2 and Figure 2.

[0091] [Table 2]

[0092] The results from Examples 9-11 and 14 showed a tendency for the reaction yield to improve as the reaction temperature decreased. Note that the inosine concentration in Examples 8-11 and 14 was approximately 10% by mass, while the inosine concentration in Example 12 was slightly lower at 7.84% by mass, and the inosine concentration in Example 13 was slightly higher at 12.73% by mass. Therefore, it should be noted that the reaction yield values ​​were lower in Example 12 and higher in Example 13. When the reaction temperature was lowered to 10°C, as in Example 8, the reaction yield tended to decrease. This is presumed to be because the low temperature led to a decrease in enzyme activity (i.e., a decrease in the reaction rate), and equilibrium was not reached after a reaction time of 4 hours.

[0093] <Investigation of the effect of reaction solution pH on reaction yield> [Example 15] Inosine 1.90 mmol, diammonium hydrogen phosphate 1.96 mmol, and 0.5 M MES Buffer 9.16 g (pH 5.50) were mixed. 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added, and the ribose-1-phosphate synthesis reaction was carried out at 20°C. The pH of the reaction solution was 6.10, and the inosine mass concentration was 4.99% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 39%. At this time, the hypoxanthine mass concentration was 0.74% by mass.

[0094] [Example 16] Inosine 1.87 mmol, diammonium hydrogen phosphate 1.92 mmol, and 0.5 M MES Buffer 9.16 g (pH 6.00) were mixed. 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added, and the synthesis of ribose-1-phosphate was carried out at 20°C. The pH of the reaction solution was 6.38, and the inosine mass concentration was 4.89% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 39%. At this time, the hypoxanthine mass concentration was 0.90% by mass.

[0095] [Example 17] Inosine 1.93 mmol, diammonium hydrogen phosphate 1.92 mmol, and 0.5 M MES Buffer 9.16 g (pH 6.50) were mixed. 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added, and the ribose-1-phosphate synthesis reaction was carried out at 20°C. The pH of the reaction solution was 6.80, and the inosine mass concentration was 5.05% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 42%. At this time, the hypoxanthine mass concentration was 1.06% by mass.

[0096] [Example 18] Inosine 1.89 mmol, diammonium hydrogen phosphate 1.92 mmol, and 9.16 g of 1 M Tris-HCl Buffer (pH 7.00) were mixed. 0.10 g of a suspension of purine nucleoside phosphorylase-containing bacterial cells was added, and the ribose-1-phosphate synthesis reaction was carried out at 20°C. The pH of the reaction solution was 7.15, and the inosine mass concentration was 4.96% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 43%. At this time, the hypoxanthine mass concentration was 1.00% by mass.

[0097] [Example 19] 1.88 mmol of inosine and 1.86 mmol of diammonium hydrogen phosphate were mixed with 9.04 g of pure water. 0.34 g of purine nucleoside phosphorylase solution was added, and the synthesis of ribose-1-phosphate was carried out at 30°C. The pH was adjusted to 8.58 with a caustic aqueous solution. The mass concentration of inosine was 5.04% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 30%. At this time, the mass concentration of hypoxanthine was 0.75% by mass.

[0098] [Comparative Example 3] 1.91 mmol of inosine and 1.90 mmol of diammonium hydrogen phosphate were mixed with 8.97 g of pure water. 0.34 g of purine nucleoside phosphorylase solution was added, and the synthesis of ribose-1-phosphate was carried out at 30°C. The pH was adjusted to 9.00 with a caustic aqueous solution. The mass concentration of inosine was 5.13% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 13%. At this time, the mass concentration of hypoxanthine was 0.47% by mass.

[0099] [Comparative Example 4] 1.88 mmol of inosine and 1.87 mmol of diammonium hydrogen phosphate were mixed with 8.98 g of pure water. 0.38 g of purine nucleoside phosphorylase solution was added, and the synthesis of ribose-1-phosphate was carried out at 30°C. The pH was adjusted to 9.50 with a caustic aqueous solution. The mass concentration of inosine was 5.05% by mass. The reaction yield was calculated from the amount of hypoxanthine produced after 4 hours of reaction, and was found to be 12%. At this time, the mass concentration of hypoxanthine was 0.30% by mass. The results are summarized in Table 3 and Figure 3.

[0100] [Table 3]

[0101] In Comparative Examples 3 and 4, where the pH was 9.00 or higher, hypoxanthine did not precipitate and the reaction yield was low. In contrast, in Examples 3, 15-19, where the pH was less than 9.00, hypoxanthine precipitated and a good reaction yield was observed.

[0102] Furthermore, regarding the reaction solution at the start of the reaction, no insoluble components were detected in Comparative Examples 1-4 and Examples 1, 13, and 14, while the presence of insoluble components was detected in Examples 2-12 and 15-19.

[0103] Figure 4 summarizes the relationship between hypoxanthine concentration and reaction yield shown in Tables 1-3. Figure 4 also shows that the reaction yield was low under reaction conditions where precipitation did not occur, and high under reaction conditions where precipitation occurred. In particular, focusing on the reaction yield around a hypoxanthine concentration of 0.3% to 0.5% by mass, it is noteworthy that even with equivalent hypoxanthine concentrations, the reaction yield was low when no precipitation occurred (white triangles in Figure 4), and high when precipitation occurred (black circles in Figure 4). These results indicate that, rather than simply setting the hypoxanthine concentration within a predetermined range, precipitating at least a portion of the hypoxanthine is effective in improving the reaction yield.

Claims

1. A method for producing ribose-1-phosphate, comprising a contact step to produce ribose-1-phosphate and hypoxanthine by contacting inosine, a phosphate donor, an enzyme having purine nucleoside phosphorylase activity, and a medium in a reaction solution containing inosine, a phosphate donor, an enzyme having purine nucleoside phosphorylase activity, and a medium, A method for producing ribose-1-phosphate, wherein in the contact step, hypoxanthine is present in an amount exceeding the saturation concentration in the medium.

2. The method for producing ribose-1-phosphate according to claim 1, wherein in the contact step, the hypoxanthine concentration is 0.33% to 5.00% by mass based on 100% by mass of the total amount of the reaction solution.

3. The method for producing ribose-1-phosphate according to claim 1 or 2, wherein the concentration of inosine subjected to the contact step is 2.1% to 22% by mass based on 100% by mass of the total mass of the reaction solution.

4. A method for producing ribose-1-phosphate according to claim 1 or 2, wherein in the contact step, the pH of the reaction solution is less than 9.

00.

5. A method for producing ribose-1-phosphate according to claim 1 or 2, wherein in the contact step, the temperature of the reaction solution is less than 33°C.

6. The method for producing ribose-1-phosphate according to claim 5, wherein the temperature of the reaction solution is 10°C to 25°C.

7. The method for producing ribose-1-phosphate according to claim 1 or 2, wherein the enzyme having purine nucleoside phosphorylase activity is a purine nucleoside phosphorylase enzyme having an amino acid sequence that has 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

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