Method for producing guanosine-5'-phosphate

Abstract

To provide a method for producing guanosine-5'-phosphate while dissolving gel.SOLUTION: Provided is a method for producing monosodium guanosine-5'-phosphate by enzymatic phosphorylation of guanosine in a system to which NaCl, NaH2PO4, Na2HPO4, Na2CO3, Na2SO4, Na3PO4, sodium pyrophosphate, sodium tripolyphosphate or a combination of two or more of these has been added, so that the Na ion concentration satisfies the following formula (1): 6>Na ion concentration [mass%]≥0.023×GMP concentration [mass%]+1.8 (1) (wherein GMP means guanylic acid).SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for producing guanosine 5'-phosphate. Guanosine 5'-phosphate is useful as a seasoning, medicine, and a raw material thereof, etc. [Background technology]

[0002] Various methods are known for producing nucleoside-5'-phosphate esters such as guanosine-5'-phosphate (guanylic acid, GMP) by enzymatic phosphorylation of nucleosides such as guanosine. Among them, a method for producing nucleoside-5'-phosphate esters with less by-products and high efficiency has been developed in which acid phosphatase is allowed to act on a nucleoside and a phosphate donor selected from the group consisting of polyphosphate (salt), phenylphosphate (salt) and carbamylphosphate (salt) under conditions of pH 3.0 to 5.5 to produce a nucleoside-5'-phosphate ester (Patent Documents 1 and 2). As another method, a method has been developed in which crystals of a nucleoside or a precursor thereof are crushed by physical treatment and then reacted with a phosphate donor under an enzyme catalyst (Patent Document 3). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO 96 / 37603 [Patent Document 2] WO 01 / 18184 [Patent Document 3] Patent No. 4192408 Summary of the Invention [Problem to be solved by the invention]

[0004] In the method of the above document, when the phosphate donor is used in the form of a sodium salt, there is a problem that the monosodium salt of guanosine-5'-phosphate has high viscosity and spinnability, which reduces the stirring and mixing properties and the reaction yield, as in the case of guanosine-5'-phosphate. Here, the reason why the reaction solution of the monosodium salt of guanosine-5'-phosphate has high viscosity and spinnability is that guanosine-5'-phosphate forms a strong tetramer structure in water through Hoogsteen-type hydrogen bonds (M. Gellert, MN Lipsett, D. R Davies, Proc. Natl Acad. Sci. USA 1962, 48, 2013-2018) and forms a layered gel through sodium ions (TJ Pinnavaia, CL Marshall, CM Mettler, CL Fisk, HT Miles, ED Becker, J. Am. Chem. Soc., 1978, 100, 3625-3627). It is known that this gel also forms in the presence of nucleosides (Y. Yu, D. Nakamura, K. Deboyace, AW Neisius, LB McGown, J. Phys. Chem. B 2008, 112, 1130-1134). Here, spinnability refers to the thread-pulling property of a substance. The present invention has been made in view of the above, and an objective of the present invention is to provide a method for producing guanosine-5'-phosphate while suppressing gelation or eliminating a gel, i.e., while preventing gel formation or turning a gel that has already formed into a slurry. [Means for solving the problem]

[0005] Even in the case of guanosine 5'-phosphate, if the concentration of the phosphate donor used as a substrate, such as tripolyphosphate (salt) or pyrophosphate (salt), is high, or the concentration of guanosine 5'-phosphate produced by the reaction is low, the increase in viscosity due to the sodium salt does not pose a problem (Patent Document 3). However, since the phosphate donor accounts for a large proportion of the cost of each of the above-mentioned patented methods, adding a large amount of phosphate donor to reduce the viscosity increases the production cost compared to the production of other nucleoside 5'-phosphate esters. Furthermore, if the concentration of guanosine 5'-phosphate is reduced in each of the above-mentioned patented methods, the production rate decreases. As a result of various investigations, the inventors have found that by adding an inexpensive salt, such as sodium chloride, in addition to a phosphate donor, it is possible to produce monosodium guanosine 5'-phosphate while eliminating the gel even when a relatively high concentration of guanosine 5'-phosphate is used, without adding an excessive amount of phosphate donor, and thus completed the present invention.

[0006] That is, the present invention provides the following production method. 1. The sodium ion concentration satisfies the following formula (1): 6>Na ion concentration [mass%] ≧ 0.023 × GMP concentration [mass%] + 1.8 (1) (In the formula, GMP means guanylic acid.) In a system to which NaCl, NaH2PO4, Na2HPO4, Na2CO3, Na2SO4, Na3PO4, sodium pyrophosphate, sodium tripolyphosphate, or a combination of two or more of these has been added, A method for producing guanosine-5'-monosodium phosphate, comprising the step of enzymatically phosphorylating guanosine to produce guanosine-5'-monosodium phosphate. 2. A method for producing guanosine 5'-phosphate monosodium, comprising a step of enzymatically phosphorylating guanosine to produce guanosine 5'-phosphate monosodium in a system in which NaCl, NaH2PO4, Na2HPO4, Na2CO3, Na2SO4, Na3PO4, sodium pyrophosphate, sodium tripolyphosphate, or a combination of two or more of these is added to the system so that the Na ion concentration satisfies the following formula (1): 6>Na ion concentration [mass%] ≧ 0.023 × GMP concentration [mass%] + 1.8 (1) (In the formula, GMP means guanylic acid.) 3. The method according to claim 1 or 2, further comprising mixing or stirring.

[0007] 4.a) producing guanosine 5'-phosphate monosodium by the method according to any one of 1 to 3 above; b) preparing the resulting monosodium guanosine 5'-phosphate in an aqueous solution having a pH of 7 to 10; c) isolating the enzyme from the aqueous solution obtained in step b), and d) obtaining a solid of disodium guanosine 5'-phosphate from the aqueous solution from which the enzyme has been separated in step c); The method for producing disodium guanosine-5'-phosphate comprises:

[0008] 5.a) producing guanosine 5'-phosphate monosodium by the method according to any one of 1 to 3 above; b) adjusting the obtained guanosine 5'-phosphate monosodium salt to an aqueous solution having a pH of 7 to 10; c) separating the enzyme from the aqueous solution obtained in step b), e) adding and dissolving inosine-5'-phosphate in an equimolar amount to the aqueous solution from which the enzyme was separated in step c), and adjusting the pH to 7 to 10 to prepare an aqueous solution containing disodium guanosine-5'-phosphate and disodium inosine-5'-phosphate; f) obtaining mixed crystals of disodium guanosine-5'-phosphate and disodium inosine-5'-phosphate from an aqueous solution containing disodium guanosine-5'-phosphate and disodium inosine-5'-phosphate; A method for producing a mixed crystal of disodium guanosine-5'-phosphate and disodium inosine-5'-phosphate, comprising: Effect of the Invention

[0009] According to the present invention, it is possible to produce monosodium guanosine-5'-phosphate, disodium guanosine-5'-phosphate, or a mixed crystal of disodium inosine-5'-phosphate and disodium guanosine-5'-phosphate while suppressing gelation or eliminating gelation. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the relationship between the GMP concentration and the Na ion concentration of the GMP solutions evaluated in the Reference Example (·), Comparative Example (▲), and Example (■). [Diagram 2] Figure 2 shows the appearance of the gel when mixed and when gelled (not mixable). [Diagram 3] The storage modulus and loss modulus were measured using a rheometer for the samples shown in Figure 2. Using a rheometer, it is possible to clearly distinguish between samples not only by appearance but also by dynamic viscoelastic behavior. In other words, for the mixable samples, the loss modulus exceeds the storage modulus in the high strain region (1% to 100%). On the other hand, for the non-mixable samples, the storage modulus exceeds the loss modulus even in the high strain region. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to a method for producing a nucleoside-5'-phosphate ester, which is known from Patent Document 1 and the like, in which a specific sodium salt is added so that the Na ion concentration in the reaction system satisfies the above formula (1), and in the system, guanosine is enzymatically phosphorylated to obtain a target product. Therefore, examples of enzymes that can be used in the present invention include acid phosphatases described in Patent Document 1, which catalyze a reaction to generate guanosine-5'-phosphate by transferring a phosphate group from a specific phosphate donor to guanosine under conditions of pH 3.0 to 5.5. Specifically, although not limited thereto, those derived from microorganisms are preferred, and bacteria belonging to the genera Morganella, Escherichia, Providencia, Enterobacter, Klebsiella, and Serratia have the enzyme activity, and the enzymes are derived from these bacteria. Representative examples of such bacteria include the following strains.

[0012] Morganella morganii NCIMB 10466 Morganella morganii IFO 3168 Morganella morganii IFO 3848 Escherichia blattae JCM 1650 Escherichia blattae ATCC 33429 Escherichia blattae ATCC 33430 Providencia stuartii ATCC 29851 Providencia stuartii ATCC 33672 Enterobacter aerogenes IFO 12010 Enterobacter aerogenes IFO 13534 Klebsiella planticola IFO 14939 Klebsiella planticola IAM 1133 Serratia ficaria IAM 13540 Serratia marcescens IAM 12143

[0013] More preferably, the acid phosphatase has an increased affinity for nucleosides (see JP-A-10-201481). Specific examples of such acid phosphatases include the mutant acid phosphatase derived from Escherichia blattae described in Comparative Example 1 and Example 1 below, the novel mutant acid phosphatase derived from Enterobacter aerogenes, and various mutant acid phosphatases described in JP-A-10-201481. Furthermore, acid phosphatase is an enzyme that originally catalyzes the hydrolysis of phosphate esters under acidic conditions and has nucleotidase activity that degrades nucleoside-5'-phosphate esters produced by phosphoryl transfer reactions. However, mutant acid phosphatases with reduced nucleotidase activity (phosphate ester hydrolysis activity) (see WO96 / 37603) can also be suitably used in the present invention. Furthermore, acid phosphatases with improved temperature stability, or acid phosphatases with increased affinity for nucleosides and improved temperature stability (JP Patent Publication No. 10-201481) can also be suitably used in the present invention.

[0014] The enzyme can be brought into contact with guanosine and a specific phosphate donor to produce guanosine-5'-phosphate in the reaction solution. The concentration of the nucleoside added to the reaction solution is preferably 1 to 20 g / dL. The phosphate donor that can be used in the present invention is pyrophosphate, tripolyphosphate, polyphosphate, or a sodium salt thereof. Among these, tripolyphosphate, which is inexpensive and available in the market, is preferable. The concentration of the phosphate donor used is determined by the concentration of guanosine, which is the phosphate acceptor. Usually, 1 to 5 times the amount of guanosine is desirable. The reaction usually gives good results at a temperature of 20 to 60°C, preferably 30 to 40°C, and at a pH of 3.5 to 6.5, preferably 4.0 to 5.0, which is on the weak acidic side. The reaction may be performed by standing or stirring. Stirring is preferred to efficiently contact the reactants and the enzyme in the enzyme reaction. The reaction time varies depending on conditions such as the activity of the enzyme used and the guanosine concentration, but is typically 1 to 100 hours. The thus produced monosodium guanosine 5'-phosphate can be collected and separated from the reaction mixture by a method using a synthetic adsorption resin, a method using a precipitant, or any other conventional method.

[0015] The amounts of the phosphate donor and other salts to be added can be determined as follows. Step 1: The amount of guanosine to be used as a raw material is determined based on the desired concentration of guanosine-5'-phosphate monosodium in the reaction suspension in the production process. Step 2: The amount of enzyme to be added is determined based on an enzyme whose performance is known in advance and a desired enzymatic phosphorylation reaction time (usually within 24 hours). Step 3: Determine the amount of phosphate donor required to reach a desired concentration of monosodium guanosine 5'-phosphate from the amount of enzyme added and the enzyme performance information. At this time, the amount of phosphate donor required as the enzyme performance information may be expressed as a molar ratio to monosodium guanosine 5'-phosphate.

[0016] Step 4: Determine the amount of salt other than the phosphate donor to compensate for the shortage of Na ions so that the Na ion concentration [mass %] in the reaction suspension system is 0.023 × GMP concentration [mass %] + 1.8 or more. When sodium pyrophosphate, sodium tripolyphosphate, sodium polyphosphate, or a combination of two or more of these is used as the phosphate donor, the amount of these salts may be determined so that the Na ion concentration [mass%] is 0.023 × GMP concentration [mass%] + 1.8 or more (i.e., the phosphate donor is also used as the sodium source). In this case, it is not necessary to add salts other than the phosphate donor. Among them, sodium pyrophosphate, sodium tripolyphosphate, or a combination of these is preferred. On the other hand, the salt other than the phosphate donor that supplements the insufficient Na ions is preferably any one of NaCl, NaH2PO4, Na2HPO4, Na2CO3, Na2SO4, and Na3PO4, or a combination of two or more of them. Among them, NaCl is preferred because it is inexpensive. As the sodium source, a combination of sodium pyrophosphate and sodium tripolyphosphate, or a combination of sodium pyrophosphate, sodium tripolyphosphate and NaCl is preferred.

[0017] Step 5: Guanosine 5'-phosphate monosodium is produced in a reaction system containing the amount of guanylic acid, an enzyme, and a phosphate donor determined as described above, and which may optionally contain a salt other than the phosphate donor. Sub-step 6: If necessary, repeat the experiment to optimize the amount of phosphate donor so as to obtain a desired amount of guanosine 5'-phosphate monosodium at the minimum cost, based on the amount of guanosine 5'-phosphate monosodium obtained after the reaction, the reaction time, and calculation of the cost required for the reaction.

[0018] Whether the Na ion concentration of the system satisfies the above formula (1) can be determined by specifying the concentration from the composition of the system and calculating it. Specifically, the Na ion concentration of the system is calculated assuming that guanosine-5'-phosphate is a monovalent Na salt, phosphate is monovalent, pyrophosphate is divalent, and tripolyphosphate is trivalent, and that they are completely dissociated from each other.

[0019] The reason why the gel is eliminated by the present invention can be explained in principle as follows. That is, the stacking pattern of the tetramer (G-quartet) formed by guanosine-5'-phosphate monosodium greatly affects the local charge repulsion of the phosphate group of guanosine-5'-phosphate monosodium. The microstructure of the gel is determined by this stacking pattern. As the ionic strength increases, the number of G-quartet stacks increases and the rod-like structures become larger. It is known that as the ionic strength increases, the structures accumulate and form liquid crystals (EJ Baldassarri, MG Ortore, F. Spinozzi, A. Round, C. Ferrero, P. Mariani, Nanomaterials, 2020, 10, 629). In order to make it possible to stir, it is important to accumulate the structures. When the Na ion concentration of the system is less than (0.023 × GMP concentration [mass%] + 1.8), the rod-like structures are entangled in the microscopic sense, and the gel has the properties of a storage modulus exceeding the loss modulus and exhibiting spinnability. For example, if a stirring blade is attached to a three-one motor and the gel is stirred at 10 rpm or more, it will stick to the stirring shaft and will not be able to be stirred. However, when the Na ion concentration of the system is (0.023 × GMP concentration [mass %] + 1.8) or more, rod-like structures will accumulate microscopically, and the gel's loss modulus will exceed its storage modulus, and the spinnability will disappear. As a result, by stirring under the same conditions, the gel will become slurry-like and stirring will become possible. Note that "stirring" in this context is a means for verifying whether the gel has been dissolved, and is not a means for dissolving the gel. According to the present invention, the presence of a predetermined amount of Na ions is sufficient in principle to dissolve the gel. Stirring or mixing may be performed in the method of the present invention, thereby making the concentration uniform.

[0020] Here, the storage modulus and loss modulus represent dynamic viscoelasticity, with the storage modulus being the component of energy generated by external forces and strains in an object that is stored inside the object, and the loss modulus being the component that diffuses to the outside. Without being bound by any theory, it is believed that a system that satisfies the Na ion concentration defined by the above formula (1) has a microstructure in which structural bodies are accumulated, thereby preventing the gelation of monosodium salts. In order to obtain the desired product, monosodium guanosine-5'-phosphate, in high yield, the higher the GMP concentration, the better. In order to eliminate gelation according to the present invention, the concentration of Na ions, which are counter ions of GMP, must also be increased according to the GMP concentration. However, as described above, the phosphate donor accounts for a high proportion of the cost, so it is preferable to increase the Na ion concentration with a salt other than the phosphate donor. From an economical point of view, it is lower than the Na ion concentration of 6% derived from the phosphate donor and the like shown in Patent Document 3. For example, a GMP concentration of 4 to 17% by mass and a Na ion concentration of 1.8 to 3.6% by mass are suitable. Furthermore, a GMP concentration of 10 to 16% by mass and a Na ion concentration of 2.1 to 3% by mass are more suitable. In any case, it is preferable to use NaCl as a salt other than the phosphate donor because it is inexpensive.

[0021] The enzymatic phosphorylation reaction can also be carried out while mixing or stirring. Mixing can be carried out, for example, using a general-purpose magnetic stirrer and stirring bar used in laboratories. Stirring can be carried out using a general-purpose stirring device, for example, a paddle mixer, a homodisper, a homogenizer, etc. The mixing or stirring speed is preferably 10 to 200 rpm, more preferably 10 to 100 rpm. Even if the reaction product has gelled, the gel can be made into a slurry by mixing or stirring. The speed and time of mixing or stirring at this time can be appropriately set while observing the state of the gel. The mixing or stirring speed is usually 10 to 200 rpm, preferably 10 to 100 rpm. The mixing or stirring time can be continued continuously while the phosphorylation reaction is continuing, and is usually 3 to 30 hours, preferably 3 to 24 hours. The temperature during mixing or stirring can be set in consideration of the heat of stirring. It is usually 15 to 40°C, preferably 25 to 40°C. For example, it is preferable to carry out the reaction at 20 to 100 rpm for 10 to 24 hours at 35° C. In this case, whether or not it is possible to continue the reaction while dissolving the gel can be judged 3 to 6 hours after the start of the reaction. The pH of the slurry thus obtained is adjusted to an alkaline level (typically about pH 7 to 10), thereby obtaining an aqueous solution of disodium guanosine-5'-phosphate. For example, sodium hydroxide is added to the slurry and stirred to adjust the pH of the slurry to an alkaline level to dissolve the enzyme, and the enzyme is separated. The aqueous solution of disodium guanosine-5'-phosphate can then be concentrated, or an alcohol such as methanol can be added to the aqueous solution to obtain a solid disodium salt. In this case, the enzyme may be removed, and the aqueous solution may then be made strongly alkaline at pH 11 to 13 to remove any remaining phosphoric acid contained in the aqueous solution, before the above-mentioned operation. Alternatively, for example, the pH of the resulting slurry may be adjusted to 1 to 5 to isolate guanosine-5'-phosphate in the form of a free salt or monosodium salt, and phosphoric acid contained in the mother liquor may be removed, followed by preparing an aqueous solution having a pH of 7 to 10 and concentrating the solution, or adding an alcohol such as methanol to the aqueous solution to obtain a solid disodium salt. The disodium salt is useful as a seasoning, medicine, etc. Furthermore, the obtained slurry can be used to obtain a mixed crystal of inosine-5'-phosphate disodium and guanosine-5'-phosphate disodium. For example, the obtained guanosine-5'-phosphate monosodium slurry is adjusted to pH 7-10, the enzyme is separated, and then inosine-5'-phosphate disodium is added and dissolved in an aqueous solution in an approximately equimolar concentration with guanosine-5'-phosphate disodium to prepare an aqueous solution, which is then concentrated and cooled or alcohol is added to obtain a mixed crystal of inosine-5'-phosphate disodium and guanosine-5'-phosphate disodium. The mixed crystal is useful as a seasoning, a medicine, and the like. EXAMPLES

[0022] <Reference Example 1: Elimination of the gel of monosodium guanosine 5'-phosphate by adding sodium chloride> GMP solutions (or slurries) with various GMP and NaCl concentrations, indicated by small black circles in Figure 1, were prepared at room temperature, and the pH was adjusted to 4.5 using 2M hydrochloric acid. The pH was measured at 25°C using a glass electrode. At 25°C, the samples prepared above were placed in a 50 ml beaker, and a 3 cm long stirrer was placed in the beaker, and the mixture was mixed at 50 rpm using a magnetic stirrer (Tokyo Glass Instruments Co., Ltd. F-207). The fluidity was evaluated visually after 30 minutes. As shown in Figure 2, the appearance of the gel was that the gel was transparent and the center was raised, indicating that the gel was spinnable. On the other hand, the gel was not gelatinous, and the stirrer moved throughout the sample, indicating that the gel could be mixed. Using this method, the regions of GMP concentration [mass%] and Na ion concentration [mass%] that could be mixed and not mixed were plotted as shown in Figure 1. The region of mixable regions could be expressed as Na ion concentration [mass%] ≧ 0.023 × GMP concentration [mass%] + 1.8. In addition, the storage modulus and loss modulus of the mixable / immiscible samples shown in Figure 2 were measured using a rheometer. The data are shown in Figure 3.

[0023] <Comparative Example 1> An enzyme reaction was carried out in an enzyme reaction system containing guanosine, sodium tripolyphosphate, and the acid phosphatase described in Patent Document 1 and JP-A-10-201481. The reaction was carried out while mixing at 50 rpm using a magnetic stirrer and a stirrer, as in Reference Example 1. Here, the final GMP concentration [mass%] was 7.2, and the final Na ion concentration [mass%] was 1.2. Separately, a similar experiment was carried out with the final GMP concentration [mass%] being 7.9, and the final Na ion concentration [mass%] being 1.8. In both cases, a gel of GMP was formed as the phosphorylation reaction proceeded, and mixing became impossible. These compositions are indicated by ▲ in FIG. 1.

[0024] <Example 1> An Na ion source necessary to prevent gelation was additionally added to an enzyme reaction system containing guanosine, sodium tripolyphosphate, and the acid phosphatase described in Patent Document 1 and JP-A-10-201481, and the enzyme reaction was carried out under two conditions. As in Reference Example 1, the reaction was carried out while mixing at 50 rpm using a magnetic stirrer and a stirring bar. One was carried out in a system in which NaCl was added as an additional Na ion source, so that the final GMP concentration [mass %] was 14.3 and the final Na ion concentration [mass %] was 2.85. The other experiment was conducted in a similar manner using sodium pyrophosphate as an additional Na ion source, with a final GMP concentration [mass%] of 14.9 and a final Na ion concentration [mass%] of 2.92. In both cases, the GMP produced as the phosphorylation reaction proceeded was observed as a slurry without gelling, and mixing was possible. The mixable compositions are shown in Figure 1 with ■. The mixable compositions satisfied the formula (1), Na ion concentration [mass %] > 0.023 × GMP concentration [mass %] + 1.8.

Claims

1. The Na ion concentration satisfies the following formula (1): 6> Na ion concentration [mass%] ≧ 0.023 × GMP concentration [mass%] + 1.8 (1) (wherein GMP means guanylic acid.) NaCl, NaH 2 P.O. 4 , Na 2 HPO 4 , Na 2 CO 3 , Na 2 SO 4 , Na 3 P.O. 4 In a system to which sodium pyrophosphate, sodium tripolyphosphate, or a combination of two or more thereof is added, A method for producing guanosine-5'-monosodium phosphate, comprising the step of enzymatically phosphorylating guanosine to produce guanosine-5'-monosodium phosphate.

2. NaCl, NaH 2 P.O. 4 , Na 2 HPO 4 , Na 2 CO 3 , Na 2 SO 4 , Na 3 P.O. 4 , sodium pyrophosphate, sodium tripolyphosphate, or a combination of two or more thereof, to a system so that the Na ion concentration satisfies the following formula (1), and then enzymatically phosphorylating guanosine to produce guanosine-5'-phosphate monosodium. 6> Na ion concentration [mass%] ≧ 0.023 × GMP concentration [mass%] + 1.8 (1) (wherein GMP means guanylic acid.)

3. The method of claim 1 or 2, further comprising mixing or stirring.

4. a) producing guanosine 5'-phosphate monosodium salt by the production method according to claim 1 or 2; b) preparing the obtained guanosine-5'-phosphate monosodium salt in an aqueous solution having a pH of 7 to 10; c) separating the enzyme from the aqueous solution obtained in step b), and d) obtaining a solid of guanosine-5'-phosphate disodium salt from the aqueous solution from which the enzyme has been separated in step c); A method for producing disodium guanosine-5'-phosphate, comprising:

5. a) producing guanosine 5'-phosphate monosodium salt by the production method according to claim 1 or 2; b) adjusting the obtained guanosine-5'-phosphate monosodium salt to an aqueous solution having a pH of 7 to 10; c) separating the enzyme from the aqueous solution obtained in step b); e) adding and dissolving inosine-5'-phosphate in an equimolar amount to the obtained guanosine-5'-phosphate monosodium salt to the aqueous solution from which the enzyme was separated in step c), and adjusting the pH to 7 to 10 to prepare an aqueous solution containing guanosine-5'-phosphate disodium and inosine-5'-phosphate disodium; f) obtaining mixed crystals of guanosine-5'-phosphate disodium and inosine-5'-phosphate disodium from an aqueous solution containing guanosine-5'-phosphate disodium and inosine-5'-phosphate disodium; A method for producing a mixed crystal of guanosine-5'-disodium phosphate and inosine-5'-disodium phosphate, comprising:

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