Application of inorganic pyrophosphatase and method for synthesizing nucleoside diphosphate by using inorganic pyrophosphatase

Abstract

The invention provides a catalytic system. The catalytic system is used for catalyzing nucleoside monophosphate into nucleoside diphosphate; the catalytic system comprises nucleotide kinase, acetate kinase, acetyl phosphate or salts thereof, a phosphoric acid donor, inorganic pyrophosphatase and a solvent. The method has the beneficial effects that a catalytic system of nucleotide kinase, acetate kinase and inorganic pyrophosphatase is simple and efficient, nucleoside monophosphate can be converted into nucleoside diphosphate in one step, and the method is particularly suitable for converting 3, 7-dimethyl guanosine monophosphate into 3, 7-dimethyl guanosine diphosphate. The catalytic system provided by the invention is high in conversion efficiency and can be used for large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and in particular to an application of inorganic pyrophosphatase and a method for synthesizing nucleoside diphosphates. Background Art

[0002] In recent years, mRNA vaccines have been increasingly valued as a therapeutic method in the field of gene therapy. 3,7-Dimethylguanosine diphosphate (3,7-MGDP, Formula 1) is one of the important intermediates for the synthesis of various cap compounds; and the 5' end cap structure of eukaryotic mRNA is a very critical component, playing an extremely important role in mRNA stability and transport, and protein synthesis.

[0003]

[0004] There are two main methods for preparing nucleotides: chemical and enzymatic. Unlike traditional chemical synthesis of nucleotides, bioenzymatic synthesis has unique advantages such as mild reaction conditions and good stereoselectivity. However, the process of synthesizing nucleotides by the kinase method must supply a large amount of phosphate donors, such as adenosine triphosphate (ATP) and other nucleoside triphosphates. Yao Yefeng's master's thesis "Enzymatic Synthesis of Deoxyguanosine Triphosphate and Guanosine Triphosphate" reported that because acetyl phosphate (Acetyl phosphate, ACP) is easy to synthesize, it is widely used in the regeneration of nucleoside triphosphate (NTP) in combination with acetate kinase (Acetate kinase, ACKase). This method is a relatively economical way to regenerate NTP on a large scale. Specifically, nucleoside monophosphate (NMP) is converted into nucleoside triphosphate (NTP) by a nucleotide kinase (Nucleotidekinase), ACKase, and ACP catalytic system. Among them, ACKase and ACP form an NTP cycle, for example, adenosine diphosphate (ADP) after the reaction can be regenerated into adenosine triphosphate (ATP) and used as a phosphate donor again. However, this catalytic system cannot directly produce nucleoside diphosphate (NDP). Summary of the invention

[0005] In order to achieve low-cost and high-efficiency preparation of nucleoside diphosphates, the present invention provides an application of an inorganic pyrophosphatase to hydrolyze nucleoside triphosphates into nucleoside diphosphates.

[0006] Preferably, 3,7-dimethylguanosine triphosphate is hydrolyzed to 3,7-dimethylguanosine diphosphate.

[0007] The present invention also provides a catalytic system comprising auxiliary metal ions, nucleotide kinase, acetate kinase, acetyl phosphate or its salt, phosphate donor, and inorganic pyrophosphatase.

[0008] Among them, auxiliary metal ions are used to assist inorganic pyrophosphatase in its function.

[0009] The catalytic system refers to a reaction system for catalyzing the production of nucleoside diphosphates. It can be stored independently of the raw materials and added to the solution of the corresponding raw materials (such as 3,7-dimethylguanosine monophosphate) when it is used. The storage form of the catalytic system can be solid, and the reagents contained therein can be stored separately or sealed together. In addition, it can also refer to the part of the reaction solution that is responsible for catalyzing the reaction of the raw materials during the reaction. It can also refer to the combination of the part to be added and the part that has been added to the reaction solution, regardless of the order of feeding, which can ultimately catalyze the raw materials (such as nucleoside monophosphates) to obtain nucleoside diphosphates.

[0010] Preferably, the catalytic system is used to catalyze nucleoside monophosphates to nucleoside diphosphates.

[0011] Preferably, the catalytic system is used to catalyze guanosine monophosphate to guanosine diphosphate.

[0012] Preferably, the metal ion is a transition metal ion.

[0013] Preferably, the metal ion is a divalent metal ion.

[0014] Preferably, the metal ion is selected from one of magnesium ion, zinc ion, manganese ion, cobalt ion or a combination thereof.

[0015] Preferably, the metal ion is a magnesium ion. Compared with other metal ions, in the magnesium chloride system, the content of 3,7-dimethylguanosine triphosphate in the final product is the lowest, and the content of 3,7-dimethylguanosine diphosphate is the highest.

[0016] Preferably, the catalytic system is used to catalyze 3,7-dimethylguanosine monophosphate to 3,7-dimethylguanosine diphosphate.

[0017] Preferably, the inorganic pyrophosphatase is selected from membrane-bound pyrophosphatase and / or soluble pyrophosphatase.

[0018] Preferably, the soluble pyrophosphatase is selected from type I and / or type II and / or type III.

[0019] Preferably, the inorganic pyrophosphatase is derived from one or more of Escherichia coli, yeast and thermotolerant bacteria.

[0020] As an exemplary explanation, the inorganic pyrophosphatase is derived from yeast, which means that the inorganic pyrophosphatase may be a wild type naturally existing in yeast or a mutant type after amino acid mutation.

[0021] The present invention also provides a method for synthesizing nucleoside diphosphates by enzymatic method, wherein at least inorganic pyrophosphatase and auxiliary metal ions are added to a solution containing nucleoside triphosphates.

[0022] The present invention also provides a method for synthesizing nucleoside diphosphates by enzymatic method, wherein at least auxiliary metal ions, nucleotide kinase, acetate kinase, acetyl phosphate or its salt, phosphate donor, and inorganic pyrophosphatase are added to a solution containing nucleoside monophosphates.

[0023] Preferably, the nucleoside monophosphate is 3,7-dimethylguanosine monophosphate, the nucleoside diphosphate is 3,7-dimethylguanosine diphosphate, and the nucleoside triphosphate is 3,7-dimethylguanosine triphosphate.

[0024] The present invention also provides a method for synthesizing 3,7-dimethylguanosine diphosphate by enzymatic method, in which 3,7-dimethylguanosine monophosphate is subjected to a catalytic reaction in a liquid containing at least 3,7-dimethylguanosine monophosphate, nucleotide kinase, acetate kinase, acetyl phosphate or its salt, a phosphate donor, and inorganic pyrophosphatase to obtain 3,7-dimethylguanosine diphosphate.

[0025] Preferably, the order of adding the raw materials and reagents in the method is not specific.

[0026] The nucleotide kinase, acetate kinase and inorganic pyrophosphatase added to the liquid can be purchased from the market or obtained by bacterial fermentation. If obtained by bacterial fermentation, the enzymes can be added to the liquid after extraction and separation. Alternatively, in order to simplify the purification steps and reduce production costs, the fermentation liquid containing the enzymes or the crude extract of the fermentation liquid containing the enzymes or the bacterial cells containing the enzymes can be directly added to the liquid.

[0027] Preferably, metal ions are required to assist the inorganic pyrophosphatase in performing its function, and thus metal ions need to be added to the liquid.

[0028] Preferably, the liquid is a buffer solution system.

[0029] Preferably, the buffer solution is a phosphate buffer solution or a Tris-HCl buffer solution.

[0030] Preferably, the reaction temperature is not less than 10°C.

[0031] Preferably, the reaction temperature is not less than 20°C.

[0032] Preferably, the reaction temperature is not less than 25°C.

[0033] Preferably, the reaction temperature does not exceed 70°C.

[0034] Preferably, the reaction temperature does not exceed 65°C.

[0035] Preferably, the reaction temperature does not exceed 60°C.

[0036] Preferably, the reaction temperature is 30-70°C or 35-65°C or 35-60°C or 40-60°C or 45-55°C or 30-40°C or 30-50°C or 30-60°C or 35°C or 40°C or 45°C or 50°C or 55°C or 60°C.

[0037] Preferably, the pH of the reaction solution is 5.0 to 8.5, or 5.5 to 8.0, or 6.0 to 8.0, or 6.5 to 7.5, or 5.5, or 6.0, or 6.5, or 7.0, or 7.5, or 8.0.

[0038] Preferably, pH control is included during the reaction.

[0039] Preferably, the pH of the reaction solution is adjusted to 7.0 with alkali every 4 to 6 hours.

[0040] Preferably, the acetyl phosphate salt is acetyl phosphate sodium salt.

[0041] Preferably, the base is a Lewis base.

[0042] Preferably, the base is selected from one of sodium hydroxide, potassium hydroxide, calcium hydroxide or a combination thereof.

[0043] Preferably, the nucleotide kinase is guanylate kinase and / or deoxyguanylate kinase.

[0044] Preferably, the nucleotide kinase is deoxyguanylate kinase.

[0045] Preferably, the phosphate donor is adenosine triphosphate or a salt thereof.

[0046] Preferably, the phosphate donor is sodium adenosine triphosphate.

[0047] Preferably, the concentration of magnesium chloride is 0.03-0.3 g / L.

[0048] Preferably, the concentration of 3,7-dimethylguanosine monophosphate is 0.05 to 0.2 mol / L.

[0049] Preferably, the concentration of adenosine triphosphate is 0.001 to 0.005 mol / L.

[0050] Preferably, the concentration of sodium acetyl phosphate is 0.1 to 0.8 mol / L.

[0051] Preferably, the sequence of the nucleotide kinase is shown as SEQ ID NO.1.

[0052] Preferably, the sequence of acetate kinase is shown as SEQ ID NO.2.

[0053] Preferably, the sequence of the inorganic pyrophosphatase is shown as SEQ ID NO.3.

[0054] In the present invention, the nucleosides in the terms nucleotide, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, etc., unless otherwise specified, are ribonucleosides and / or deoxyribonucleosides, which are formed by the condensation of bases and ribose and / or deoxyribose, wherein the bases, ribose, and deoxyribose may be chemically unmodified (the form existing in nature) or chemically modified.

[0055] The beneficial effects of the present invention are: the catalytic system composed of nucleotide kinase, acetate kinase and inorganic pyrophosphatase has a simple composition and high catalytic efficiency, and can realize the conversion of nucleoside monophosphate into nucleoside diphosphate in one step, and is particularly suitable for converting 3,7-dimethylguanosine monophosphate into 3,7-dimethylguanosine diphosphate. Compared with chemical methods, the conversion efficiency is high, and there are few by-products. In addition to unreacted raw materials, there are no other by-products, thereby effectively reducing the purification steps, reducing production costs, and can be used for large-scale production. DETAILED DESCRIPTION

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0058] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.

[0059] The following examples exemplarily provide methods for constructing engineered bacteria that produce nucleotide kinase, acetate kinase and inorganic pyrophosphatase. It should be noted that the enzymes involved in the examples are known and have not been mutated or modified. The present invention merely utilizes known genetic engineering means to construct engineered bacteria to obtain these enzymes. For those skilled in the art, the genetic engineering used in the following examples belongs to the known prior art, and its use does not require creative work.

[0060] Example 1

[0061] The gene sequences of deoxyguanylate kinase (Gene ID: 937037), acetate kinase (Gene ID: 75205658), and inorganic pyrophosphatase (Gene ID: 852296) were obtained from the NCBI database (National Center for Biotechnology Information).

[0062] S1: The gene sequences of deoxyguanylate kinase (Gene ID: 937037), acetate kinase (Gene ID: 75205658) and inorganic pyrophosphatase (Gene ID: 852296) were artificially synthesized (commissioned by Nanjing Qingke Biotechnology Co., Ltd.) to obtain the gene sequences of the three enzymes;

[0063] S2: The genes of the three enzymes and the pET28a plasmid vector (the pET28a plasmid vector was purchased from Nanjing Qingke Biotechnology Co., Ltd.) were double-digested with NcoI and XhoI, and the digestion products were recovered by gel recovery;

[0064] S3: Use T4 ligase to connect the three enzyme digestion products respectively, and transform the three enzyme digestion and ligation products into BL21 (DE3) host Escherichia coli BL21 (DE3) (host Escherichia coli was purchased from Nanjing Qingke Biotechnology Co., Ltd.);

[0065] S4: Pick out the transformed single colonies of the three enzymes and inoculate them into test tubes containing LB liquid culture medium, and culture them at 37°C and 250rpm for 6-10h; extract the recombinant plasmids of the three enzymes respectively;

[0066] S5: Perform enzyme digestion verification, and perform sequencing verification on the recombinant plasmids containing the deoxyguanylate kinase gene, acetate kinase gene, and inorganic pyrophosphatase gene, respectively (sequencing is sent to Nanjing Qingke Biotechnology Co., Ltd., and the sequencing results show that the strain with complete recombinant gene sequence is the strain carrying the recombinant plasmid of the deoxyguanylate kinase gene, acetate kinase gene, and inorganic pyrophosphatase gene, respectively), and select the strains with correct deoxyguanylate kinase gene, acetate kinase gene, and inorganic pyrophosphatase gene, respectively, which are the genetically engineered bacteria expressing deoxyguanylate kinase, acetate kinase, and inorganic pyrophosphatase.

[0067] The following are the expressed enzyme sequences.

[0068] Deoxyguanylate kinase (NCBI Reference Sequence: NP_387896.1) SEQ ID NO.1:

[0069] MNTAPFIAIEGPIGAGKTTLATMLSQKFGFPMINEIVEDNPYLDKFYDNIKEWSFQLEMFFLCHRYKQLEDTSDHFLKKGQPVIADYHIYKNVIFAERTLSPHQLEKYKKIYHLLTDDLPKPNFIIYIKASLPTLLHRIEKRGRPFEKKIETSYLEQLISDYEVAIKQLQEADPELTVLTVDGDSKDFVLNKSDFERIAAHVKELIV

[0070] Acetate kinase (GenBank: QNC62623.1) SEQ ID NO.2:

[0071] MSSKLVLVLNCGSSSLKFAIIDAVNGEEYLSGLAECFHLPEARIKWKMDGNKQEAALGAGAAHSEALNFIVNTILAQKPELSAQLTAIGHRIVHGGEKYTSSVVIDESVIQGIKDAASFAPLHNPAHLIGIEEALKSFPQLKDKNVAVFDTAFHQTMPEESYLYALPYNLYKEHGIRRYGAHGTSHFYVTQEAAKMLNKPVEELNIITCHLGNGGSVSAIRNGKCVDTSMGLTPLEGLVMGTRSGDIDPAIIFHLHDTLGMSVDAINKLLTKESGLLGLTEVTSDCRYVEDNYTTKEDAKRAMDVYCHRLAKYIGAYTALMDGRLDAVVFTGGIGENAAMVRELSLGKLGVLGFEVDHERNLAARFGKSGFINKEGTRPAVVIPTNEELVIAQDASRLTA

[0072] Inorganic pyrophosphatase (NCBI Reference Sequence: NP_009565.1) SEQ ID NO.3:

[0073] MTYTTRQIGAKNTLEYKVYIEKDGKPVSAFHDIPLYADKENNIFNMVVEIPRWTNAKLEITKEETLNPIIQDTKKGKLRFVRNCFPHHGYIHNYGAFPQTWEDPNVSHPETKAVGDNDPIDVLEIGETIAYTGQVKQVKALGI MALLDEGETDWKVIAIDINDPLAPKLNDIEDVEKYFPGLLRATNEWFRIYKIPDGKPENQFAFSGEAKNKKYALDIIKETHDSWKQLIAGKSSDSKGIDLTNVTLPDTPTYSKAASDAIPPASPKADAPIDKSIDKWFFISGSV

[0074] Example 2

[0075] The genetically engineered bacteria of the three enzymes screened in Example 1 were inoculated into LB plates containing 100 mg / L kanamycin and cultured at 37° C. for 12-16 h.

[0076] Single colonies of the three enzymes were picked out respectively and placed in LB medium containing 100 mg / L kanamycin, and cultured at 37°C and 250 rpm for 10-15 h to form seed solution.

[0077] The seed solutions of the three enzymes were transferred to LB medium containing 100 mg / L kanamycin at an inoculation rate of 8% and cultured at 37° C. and 250 rpm until OD600 reached 1.2.

[0078] IPTG with a final concentration of 0.4 mmol / L was added to the culture medium of the three enzyme engineering bacteria respectively, and fermentation was induced at 22°C. After 7 hours of induction, the fermentation was terminated, and the fermentation broth was centrifuged in a low-temperature refrigerated high-speed centrifuge at 4°C, 10,000g for 20 minutes to obtain the bacterial cells for catalysis, which were frozen at -20°C for later use.

[0079] Example 3

[0080] The cells expressing the recombinant bacteria of deoxyguanylate kinase prepared in Example 2 were added to a Tris-HCl buffer system containing a magnesium chloride concentration of 0.6 g / L at a concentration of 100 g / L, and the cells expressing the recombinant bacteria of acetate kinase were added at a concentration of 5 g / L to prepare a reaction system, wherein the concentration of the buffer salt Tris-HCl was 0.5 mol / L, and the catalytic substrates 3,7-dimethylguanosine monophosphate, adenosine triphosphate (ATP) sodium salt and acetyl phosphate sodium salt were added to the reaction system, wherein the concentration of 3,7-dimethylguanosine monophosphate was 0.2 mol / L, the concentration of adenosine triphosphate (ATP) sodium salt was 0.005 mol / L, and the concentration of acetyl phosphate sodium salt was 0.8 mol / L.

[0081] The reaction solution was reacted at 40°C, and the pH of the reaction solution was adjusted to 7.0 with sodium hydroxide every 4-6 hours.

[0082] The catalytic reaction was continued, and liquid phase detection was performed. The reaction was terminated when the concentration of the product 3,7-dimethylguanosine triphosphate no longer changed significantly. The terminated reaction solution was centrifuged, the supernatant was obtained, and after dilution by a certain multiple, liquid phase detection was performed. The detection results showed that the concentration of the product 3,7-dimethylguanosine triphosphate was 0.16 mol / L (88.16 g / L), and the conversion rate was 80%. The reaction solution was treated with 4 times the volume of ethanol, and the precipitate was collected. The precipitate was re-dissolved with an equal volume of purified water, and centrifuged after full dissolution. The liquid portion was retained as the crude 3,7-dimethylguanosine triphosphate solution.

[0083] Take the crude 3,7-dimethylguanosine triphosphate solution, add MgCl2 with a final concentration of 0.3g / L and 20g / L inorganic pyrophosphatase bacteria (Gene ID: 852296) to the reaction system for treatment, and react in a 25°C water bath. Samples are taken at intervals during the reaction, and the consumption of 3,7-dimethylguanosine triphosphate and the generation of the product 3,7-dimethylguanosine diphosphate are detected by liquid phase. When the concentration of 3,7-dimethylguanosine diphosphate no longer changes, the reaction is terminated, the terminated reaction solution is centrifuged, the supernatant is obtained, and after dilution by a certain multiple, liquid phase detection is performed. The detection results show that the concentration of the product 3,7-dimethylguanosine diphosphate is 0.13mol / L (61.36g / L), and the conversion rate is 82%.

[0084] During the reaction, the pH value of the reaction solution was maintained at about 7.5 with saturated sodium carbonate.

[0085] Through this example, the applicant unexpectedly discovered that inorganic pyrophosphatase can be used to catalyze the formation of 3,7-dimethylguanosine diphosphate from 3,7-dimethylguanosine triphosphate with a high conversion rate.

[0086] Example 4

[0087] To a phosphate buffer system containing a magnesium chloride concentration of 0.2 g / L, the bacterial cells expressing the recombinant bacteria of deoxyguanylate kinase prepared in Example 2 were added at a concentration of 10 g / L, the bacterial cells expressing the recombinant bacteria of acetate kinase were added at a concentration of 10 g / L, and the bacterial cells expressing the recombinant bacteria of inorganic pyrophosphatase (Gene ID: 852296) were added at a concentration of 100 g / L to prepare a reaction system, wherein the concentrations of the buffer salts disodium hydrogen phosphate and sodium dihydrogen phosphate were both 0.05 mol / L, and the catalytic substrates 3,7-dimethylguanosine monophosphate, adenosine triphosphate (ATP) sodium salt and acetyl phosphate sodium salt were added to the reaction system, wherein the concentration of 3,7-dimethylguanosine monophosphate was 0.1 mol / L, the concentration of adenosine triphosphate (ATP) sodium salt was 0.004 mol / L, and the concentration of acetyl phosphate sodium salt was 0.6 mol / L.

[0088] The reaction solution was reacted at 60°C, and the pH of the reaction solution was adjusted to 7.0 with sodium hydroxide every 4-6 hours;

[0089] After the catalytic reaction was continued for 48 hours, the reaction was terminated by boiling for 3 minutes. The terminated reaction liquid was centrifuged to obtain the supernatant, which was diluted a certain number of times and then subjected to liquid phase detection. The detection results showed that the concentration of the product 3,7-dimethylguanosine diphosphate was 0.082 mol / L (38.71 g / L) and the conversion rate was 82%.

[0090] Example 5

[0091] To a phosphate buffer system containing a magnesium chloride concentration of 0.03 g / L, the bacterial cells expressing the recombinant bacteria of deoxyguanylate kinase prepared in Example 2 were added at a concentration of 60 g / L, the bacterial cells expressing the recombinant bacteria of acetate kinase were added at a concentration of 2 g / L, and the bacterial cells expressing the recombinant bacteria of inorganic pyrophosphatase (Gene ID: 852296) were added at a concentration of 10 g / L to prepare a reaction system, wherein the concentrations of the buffer salts disodium hydrogen phosphate and sodium dihydrogen phosphate were both 0.5 mol / L, and the catalytic substrates 3,7-dimethylguanosine monophosphate, adenosine triphosphate (ATP) sodium salt and acetyl phosphate sodium salt were added to the reaction system, wherein the concentration of 3,7-dimethylguanosine monophosphate was 0.05 mol / L, the concentration of adenosine triphosphate (ATP) sodium salt was 0.001 mol / L, and the concentration of acetyl phosphate sodium salt was 0.1 mol / L.

[0092] The reaction solution was reacted at 35°C, and the pH of the reaction solution was adjusted to 7.0 with sodium hydroxide every 4-6 hours.

[0093] After the catalytic reaction was continued for 120 hours, the reaction was terminated by boiling for 3 minutes. The terminated reaction liquid was centrifuged to obtain the supernatant, which was diluted a certain number of times and then subjected to liquid phase detection. The detection results showed that the concentration of the product 3,7-dimethylguanosine diphosphate was 0.043 mol / L (20.29 g / L) and the conversion rate was 86%.

[0094] Example 6

[0095] To a Tris-HCl buffer system containing a magnesium chloride concentration of 0.3 g / L, the bacterial cells expressing the recombinant bacteria of deoxyguanylate kinase prepared in Example 2 were added at a concentration of 100 g / L, the bacterial cells expressing the recombinant bacteria of acetate kinase were added at a concentration of 5 g / L, and the bacterial cells expressing the recombinant bacteria of inorganic pyrophosphatase (Gene ID: 852296) were added at a concentration of 50 g / L to prepare a reaction system, wherein the concentration of the buffer salt Tris-HCl was 0.5 mol / L, and the catalytic substrates 3,7-dimethylguanosine monophosphate, adenosine triphosphate (ATP) sodium salt and acetyl phosphate sodium salt were added to the reaction system, wherein the concentration of 3,7-dimethylguanosine monophosphate was 0.2 mol / L, the concentration of adenosine triphosphate (ATP) sodium salt was 0.005 mol / L, and the concentration of acetyl phosphate sodium salt was 0.8 mol / L.

[0096] The reaction solution was reacted at 40°C, and the pH of the reaction solution was adjusted to 7.0 with sodium hydroxide every 4-6 hours.

[0097] After the catalytic reaction was continued for 96 hours, the reaction was terminated by boiling for 3 minutes. The terminated reaction liquid was centrifuged to obtain the supernatant, which was diluted a certain number of times and then subjected to liquid phase detection. The detection results showed that the concentration of the product 3,7-dimethylguanosine diphosphate was 0.152 mol / L (71.74 g / L) and the conversion rate was 76%.

[0098] Through Examples 4 to 6, the applicant can also obtain 3,7-dimethylguanosine diphosphate with a high yield by a one-pot method, that is, adding all materials at once, indicating that inorganic pyrophosphate is not easily interfered by other reaction materials during catalysis. Compared with the step-by-step addition in Example 1, the step of separating and obtaining the crude 3,7-dimethylguanosine triphosphate solution is omitted, which not only reduces the required equipment but also greatly shortens the overall production time.

[0099] Compared with the chemical method, the enzyme catalysis method provided by the present invention has fewer by-products, and has no other by-products except unreacted raw materials, thereby effectively reducing the purification steps and reducing the production cost.

Claims

1. An application of inorganic pyrophosphatase, characterized in that: Hydrolyzes nucleoside triphosphates to nucleoside diphosphates.

2. An application of inorganic pyrophosphatase, characterized in that: Hydrolyzes 3,7-dimethylguanosine triphosphate to 3,7-dimethylguanosine diphosphate.

3. A catalytic system, characterized in that: The catalytic system comprises: auxiliary metal ions, nucleotide kinase, acetate kinase, acetyl phosphate or its salt, phosphate donor, and inorganic pyrophosphatase.

4. The catalytic system according to claim 3, characterized in that: The auxiliary metal ions are divalent metal ions.

5. A method for enzymatically synthesizing nucleoside diphosphates, characterized in that: At least an inorganic pyrophosphatase and an auxiliary metal ion are added to a solution containing nucleoside triphosphates.

6. A method for enzymatically synthesizing nucleoside diphosphates, characterized in that: At least auxiliary metal ions, nucleotide kinase, acetate kinase, acetyl phosphate or its salt, phosphate donor, and inorganic pyrophosphatase are added to the solution containing nucleoside monophosphate.

7. The method according to claim 5 or 6, characterized in that: The auxiliary metal ions are divalent metal ions.

8. The method according to claim 7, characterized in that: The nucleoside diphosphate is 3,7-dimethylguanosine diphosphate, and the nucleoside triphosphate is 3,7-dimethylguanosine triphosphate.

9. The method according to claim 7, characterized in that: The reaction temperature is not lower than 10°C.

10. The method according to claim 7, characterized in that: The reaction temperature is 30-70°C.

11. The method according to claim 7, characterized in that: The reaction process includes pH control of the reaction solution.

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