WO1998048031A1

WO1998048031A1 - Process for producing adenosine 5'-triphosphate and use thereof

Info

Publication number: WO1998048031A1
Application number: PCT/JP1998/001711
Authority: WO
Inventors: Kazuya Ishige; Toshitada Noguchi
Original assignee: Yamasa Corporation
Priority date: 1997-04-18
Filing date: 1998-04-15
Publication date: 1998-10-29
Also published as: JP3764755B2

Abstract

A process for producing adenosine 5'-triphosphate (ATP) by reacting adenosine 5'-monophosphate (AMP) with a polyphosphate kinase, an adenylate kinase and a polyphosphoric acid. ATP can be easily produced at a low cost by this process.

Description

Description Method for producing adenosine 5'-triphosphate and its application

The present invention provides a method for producing adenosine 5′-triphosphate (ATP) by reacting adenosine 5′-monophosphate (AMP) with polyphosphate kinase, adenylate kinase and polyphosphate. It is about its application. Background art

Recent advances in genetic engineering technology have made it possible to prepare inexpensive large quantities of various enzymes.Useful bioactive substances that have been conventionally synthesized by microbial conversion or fermentation using microbial cells can be produced at low cost by direct enzymatic reactions. It is becoming possible to manufacture.

By the way, for enzymatic reactions requiring high energy such as phosphorylation and amination, ATP is required as an energy donor or a phosphate donor. In conventional microbial conversion or fermentation production, ATP is supplied from the organism of the microorganism used, but in enzymatic methods, ATP must be added to the reaction system or an efficient ATP regeneration system must be developed. Is essential. At present, ATP is synthesized from AMP or adenine using a chemical synthesis method or a microorganism or yeast. However, an inexpensive method for synthesizing ATP has not been established at present, and commercially available ATP is extremely expensive. In addition, as a system for regenerating ATP, a combination of phosphocreatine and phosphocreatine kinase is not practical because the power, substrates, and enzymes used at the laboratory level are extremely expensive. Also, a combination of polyphosphate kinase and polyphosphate is being studied, but the use of expensive ATP or ADP is still indispensable, and has not yet been put to practical use. Thus, while ATP is extremely expensive, AMP can be produced relatively cheaply. Therefore, in an enzyme reaction system using ATP, it is desired to develop a method for producing ATP from inexpensive AMP or a method for efficiently regenerating consumed ATP, instead of adding expensive ATP. Was.

Therefore, an object of the present invention is to provide a method for producing or regenerating expensive ATP more efficiently than AMP. Disclosure of the invention

The present inventors have conducted studies to achieve the above object, and found that the activity of synthesizing ATP from AMP by coupling to adenylate kinase in the presence of polyphosphate kinase And completed the present invention.

That is, the present invention provides a method for producing adenosine 5'-triphosphate, which comprises reacting adenosine 5'-monophosphate with polyphosphate kinase, adenylate kinase and polyphosphate. Is what you do.

Further, the present invention provides a method for producing a compound using an enzymatic reaction consuming adenosine 5'-triphosphate, wherein adenosine 5'-monophosphate is replaced by polyphosphate kinase, adenylate kinase and polylysine. A process for producing the compound, characterized in that adenosine 5′-triphosphate is produced by the action of an acid and supplied to the enzyme reaction.

Further, the present invention provides a method for producing a compound using an enzymatic reaction consuming adenosine 5'-triphosphate, wherein the produced adenosine 5'-phosphate and Z or adenosine 5'-diphosphate are produced. A method for producing the compound, characterized in that the enzyme reaction is carried out while adenosine 5'-triphosphate is regenerated by reacting polyphosphate kinase, adenylate kinase and polyphosphate with the enzyme. Is what you do.

Furthermore, the present invention relates to an adenosine 5'-to adenosine 5'-triamine comprising a combination of polyphosphate kinase, adenylate kinase and polyphosphate. The present invention provides a system for synthesizing an acid. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows changes in AMP, ADP, and ATP in the ATP synthesis system of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The polyphosphate kinase (EC 2.7.4.1) and adenylate kinase (EC 2.7.4.3) used in the present invention are both known enzymes, and are derived from animals, plants and microorganisms. Those of origin and the like can be used. Among them, microorganisms, particularly Escherichia coli-derived polyphosphate kinase and adenylate kinase, are advantageous in terms of ease of enzyme preparation and the like. In addition, the polyphosphate kinase gene or adenylate kinase gene is cloned using recent gene recombination technology, and polyphosphate kinase or adenylate kinase is mass-produced using E. coli or the like as a host. It is also possible to prepare the above two types of enzymes respectively (J. Biol. Chem., 267, 22556-22561 (1992), Nucleic Acids Res., 13, 7139-7151 (1985)).

The polyphosphate kinase and adenylate kinase added to the reaction system may be in any form as long as they have the activity. Specifically, the preparation of the cells of the _c microorganism, which can be exemplified by the cells of the microorganism, the processed product of the cell, or the enzyme preparation obtained from the processed product, is performed by using a medium in which the microorganism can grow. It can be carried out by a conventional method, followed by culturing by a conventional method, and collecting cells by centrifugation or the like. To be more specific, bacteria belonging to the genus Bacillus or E. coli are described as examples. The culture medium is bouillon medium, LB medium (1% tryptone, 0.5% yeast extract, 1% salt) or 2XYT A medium (1.6% tryptone, 1% yeast extract, 0.5% salt) can be used.After inoculating the medium with the inoculum, 30 to 50 ° C, 10 to 50 hours About need It is possible to prepare microbial cells having polyphosphoric acid kinase activity or adenylate kinase activity by culturing with further stirring and centrifuging the obtained culture solution to collect microbial cells. it can.

The treated cells of the microorganisms can be obtained by mechanically crushing the above microorganisms (using a ring blender, French press, homogenizer, mortar, etc.), freeze-thawing, self-digesting, drying (freeze-drying, air-drying, etc.) ), Enzyme treatment (eg, with lysozyme), ultrasonic treatment, chemical treatment (eg, with acid or alkali treatment), etc. A modified product of the above can be exemplified.

As the enzyme preparation, a fraction having polyphosphoric acid kinase activity or adenylate kinase activity from the above treated cells is purified by a conventional enzyme purification method (salt-out treatment, isoelectric point precipitation treatment, organic solvent precipitation treatment). , Dialysis, various types of chromatography, etc.).

Commercially available AMP can be used for the present invention. The concentration used, eg if 1 to 2 0 O mM, preferably. 1 to 5 0 mM range _c and may be appropriately set from, polyphosphate can be used a commercially available added. The concentration used can be appropriately set, for example, in the range of 1 to 100 mM, preferably 10 to 100 mM in terms of inorganic phosphoric acid.

ATP can be produced by, for example, adding AMP and polyphosphoric acid to an appropriate buffer having a pH in the range of 4 to 9, and further adding 0.001 units or more, preferably 0.01 to 1 unit. 0.1 units of polyphosphate kinase, and 0.01 units or more, preferably 0.01 to 100 units // of adenylate kinase, and added at 20 ° C or more. The reaction can be carried out preferably at 30 to 40 ° C. for about 1 to 50 hours with stirring as necessary.

The ATP thus prepared can be isolated and purified by known methods.c Also, in a method for producing a compound using an enzyme reaction consuming ATP, The compound can be produced by reacting AMP with the above-mentioned polyphosphate kinase, adenylate kinase and ATP produced by reacting polyphosphoric acid to the enzyme reaction to supply the compound to the enzyme reaction. . In particular, in a method for producing a compound using an enzymatic reaction that consumes ATP, it is possible to carry out the reaction while regenerating ATP using AMP and / or ADP generated by the enzymatic reaction as a raw material. The target compound can be specifically produced, for example, galactose-1-phosphate synthesis system using galactokinase, UDP synthesis system using UMP kinase, phosphocollage using corin kinase. It can be applied to any enzymatic reaction that consumes ATP, such as a synthesis system.

The reaction conditions for such an ATP synthesis system and the enzymatic reaction may be appropriately determined by a small-scale test, and the target compound can be isolated and purified by a known method. Example

Hereinafter, the present invention will be described in detail with reference to Examples, but it is apparent that the present invention is not limited thereto. In the Examples, DNA preparation, restriction enzyme digestion, DNA ligation using T4 DNA ligase, and transformation of E. coli were all described in `` Moiecular cloningj (Maniatis et al., Edited by Cold spring Harbor Laboratory, Cold Spring Harbor, New York ( 1982)) Restriction enzymes, Amp1i TaqDNA polymerase and T4 DNA ligase were obtained from Takara Shuzo Co., Ltd. Quantification of nucleotides in the reaction solution was performed by the HPLC method. the _c specifically went, the separation using 0DS- AQ 3 1 2 column manufactured by YMC was used 0. 5M monopotassium phosphate solution as an eluent.

Example 1 Synthesis of ATP

(1) Cloning of Escherichia coli polyphosphate kinase gene

Chromosomal DNA of E. coli K12 strain JM109 (obtained from Takara Shuzo Co., Ltd.) was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). this Using DNA as a template, the following two primers, DNA, were synthesized in a conventional manner, and the E. coli polyphosphate kinase (ppk) gene was amplified by PCR.

Primer (A) 5'-TAC

A-3,

Primer (B) 5 'ATGG ATC

GAGTGA- 3 '

Amplification of the ppk gene by PCR was performed in a reaction mixture of 10 (5 OmM chloride chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin , Temperate DNA 0.1 lg, Primer DNA (A) (B) 0.2 、 M each, Ampli Taq DNA polymerase 2.5 units) were replaced with Perkin-ElmerCetus Instrume. Thermal denaturation (94 ° C, 1 minute), annealing (55 ° C, 1.5 minutes), polymerase reaction (72, 1.5 Min) was repeated 25 times.

After gene amplification, the reaction solution was treated with a mixture of phenol Z-cloth form (1: 1), and the DNA was precipitated by adding twice the volume of ethanol to the water-soluble fraction. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning. Supra), and a DNA fragment equivalent to Okb was purified. The DNA was cleaved with restriction enzymes NcoI and BamHI, and plasmid pTrc99A (obtained from Pharmacia Biotech), also digested with restriction enzymes NcoI and BamHI, and T4 DNA ligase Was connected using Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pTrc-PPK was isolated from the obtained ampicillin-resistant transformant. pTr c-PPK is obtained by inserting an NcoI-BamHI DNA fragment containing the Escherichia coli ppk gene into the NcoI-BamHI cleavage site downstream of the trc promoter of pTrc99A. (2) Preparation of Escherichia coli polyphosphate kinase

Escherichia coli JM109 carrying plasmid pTrc-Ppk was inoculated into 2XYT medium 30 containing 10OigZ ^ ampicillin, and cultured with shaking. Once at the 4 X 1 0 ³ bacteria to a final concentration of 1 mM to the culture solution

I PTG was added, and shaking culture was further continued at 30 for 5 hours. After completion of the culture, the cells were collected by centrifugation (9,000 X g, 10 minutes), and the cells were collected in 60 buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0. 1% Triton X—100,

0.2 mg Z7 ^ rib team). After incubating at 37 ° C for 1 hour, the cells were sonicated to disrupt the cells, and the cells were removed by centrifugation (20,000 xg, 10 minutes). The supernatant fraction thus obtained was treated with 5 mM magnesium chloride and

It was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 1 mM 2-mercaptoethanol to obtain a crude enzyme solution.

The specific activity of the polyphosphate kinase in the crude enzyme solution was 0.19 unit / _mg protein, which was the specific activity of the control bacterium (E. coli JM109 bacterium harboring pTrc99A). 0.18 units (Zmg protein). Next, the crude enzyme solution was fractionated using DEAE Toyopearl 6.5 M (Toy Corporation) with a concentration gradient of 0 to 0.5 M Na to obtain a polyphosphate kinase fraction. This fraction was used as a polyphosphate kinase enzyme preparation. The specific activity of polyphosphate kinase in this enzyme preparation was 0.6 unit Zmg protein.

The unit (unit) of polyphosphate kinase activity in the present invention is measured and calculated by the following method. That is, the enzyme preparation was added to a 25 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 10 OmM ammonium sulfate, 5 mM ADP, and polyphosphoric acid (100 as inorganic phosphoric acid). Te, 3 7 ° to carry out the reaction by incubating at C, and ΑΤΡ in the reaction solution with 1 0 0 ° C, 1 minute annealing _c high-performance liquid α Matogurafi one to stop the reaction by (HP LC) Quantify the activity to produce 1 mo ^ e of ATP per minute at 37 ° C. O

(3) Cloning of E. coli adenylate kinase

Chromosomal DNA of Escherichia coli 12 strain JM109 (obtained from Takara Shuzo Co., Ltd.) was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). Using this DNA as a template, the following two primers, DNA, were synthesized according to a conventional method, and the Escherichia coli adduct kinase (adk) gene was amplified by PCR.

Primer (A): 5'-ATGGATCCCGTTTCAGCCCCAGG

TGCC- 3 '

Primer (B): 5 '— ATAAGCTTGGC CTGAGATTGCTG

ATAAG- 3 '

Amplification of the adk gene by PCR was performed in a reaction mixture of 100 mM (50 mM chloride, 10 mM Tris-HCl (pH 8.3), 1.5 raM magnesium chloride, 0.001% 0.1 g of gelatin and temperate DNA, 0.1 M of each of primer DNA (A) and (B), 2.5 M of Amp1i Taq DNA polymerase (2.5 units) were added to Perkin-Elmer C Thermal denaturation (94 min., 1 min.), annealing (56 ° C, 1.0 min.), polymerization (72 min.) were performed using DNA Thermal Cyc 1 er manufactured by etus International. And 3.0 minutes) were repeated 25 times.

After gene amplification, the reaction solution was treated with a mixture of funinol Z-cloth form (1: 1), and DNA was precipitated by adding twice the volume of ethanol to the water-soluble fraction. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning. Supra), and a DNA fragment equivalent to Okb was purified. The DNA was digested with the restriction enzymes BamHI and HindIII, and the plasmid pUC18 (obtained from Takara Shuzo) and T4 DNA digested with the restriction enzymes BamHI and HindIII. Ligation was performed using ligase. Escherichia coli JM109 using ligation reaction solution And the plasmid pUC-ADK was isolated from the resulting ampicillin-resistant transformant. pUC-ADK is obtained by inserting a BamHI-Hind UI DNA fragment containing the E. coli adk gene into the BamHI-HindII cleavage site downstream of the lac promoter overnight in pUC18.

(4) Preparation of E. coli adenylate kinase

Escherichia coli JM109 carrying plasmid pUC—ADK was inoculated into 300 XYT medium containing 100 Ozg / ampicillin and cultured at 37 ° C with shaking. Once at the 4 X 1 0 ⁸ bacteria Bruno, to a final concentration of 1 mM to the culture solution

IPTG was added, and shaking culture was continued at 30 ° C. for 5 hours. After completion of the culture, the cells were collected by centrifugation (9,000 X g., 10 minutes), and the buffer of 60 ^^ (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0 mM 1% Triton X—100, 0.2 mg Z ^ rhizodium). After incubating at 37 ° C for 1 hour, the cells were sonicated to disrupt the cells, and the cells were removed by centrifugation (20,000 X g., 10 minutes).

The supernatant fraction thus obtained was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 5 mM magnesium chloride and 1 mill 2-mercaptoethanol to obtain a crude enzyme solution. The specific activity of adenylate kinase in the crude enzyme solution was 134 units of Zmg protein, and the specific activity of the control bacterium (E. coli JM109 carrying pUC18) (1.9 units / unit). mg protein). Then the crude enzyme solution DEAE preparative Yono, ⁰ - fractionated by gradient 0~ 0. 5 M Na C _β with Le 6 5 0 M (DOO one source i (Inc.)), Ade two rate Fractions with kinase activity were collected. This fraction was used as an adenylate kinase enzyme preparation. The specific activity of polyphosphate kinase in this enzyme preparation was 344 unit Zmg protein. The unit (unit) of the adenylate kinase activity in the present invention was also measured and calculated by the following method. That is, an enzyme preparation was added to 50 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 5 mM ATP, and 5 mM AMP. The reaction is carried out by keeping the temperature at 37 ° C, and the reaction is stopped by heat treatment at 100 ° C for 1 minute. Quantify ADP in the reaction solution using HP LC, and define the activity to produce 2 / mo ^ e of ADP per minute at 37 ° C as 1 unit (unit).

(5) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 1) Contains 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphate (75 mM as inorganic phosphate), and 4 mM AMP The enzyme preparation or the cell extract was added to 5 OmM Tris-HCl buffer (pH 7.8), and the mixture was kept at 37 ° C for 150 minutes. After completion of the reaction, nucleotides in the reaction solution were quantified using HPLC. The result is displayed

O shown in 1

Table 1 Added enzyme preparations ATP (mM) ADP (raM)

PPK fraction (0.02 units Ζτηβ) <0.01 <0.01 ADK fraction (0.2 units / m <0.01 <0.01

PPK fraction (0.02 unit Z) + ADK fraction (0.2 unit τηβ) 2.58 1.82

ΡΡΚ fraction (0.02 unit Z) + ADK fraction (0.02 unit Ζτηβ) 0.78 1.11

PPK fraction (0.02 unit Z) + JM109 [pUC18] crude extract (2 // g / m6) <0.01 <0.01

PPK fraction (0.02 unit Z) + JM109 [pliC-AM] crude extract (2 g / τηβ) 0.20 1.37

PPK: Polyphosphate kinase, ADK: Adenylate kinase

As shown in Table 1, polyphosphate kinase and adenylate kinase alone do not phosphorylate AMP and have no activity to produce ADP and ATP, and only when both are present at the same time do AMP phosphorylation. Oxidation occurred. AMP phosphorylation does not occur when a crude enzyme solution prepared from normal E. coli (JM109 CpUC18)) is mixed with polyphosphoric acid kinase, but adenylate kinase-producing strain ( When the crude enzyme solution prepared from JM109CpUC-ADK)) was mixed with polyphosphate kinase, a marked AMP phosphorylation reaction occurred. From the above, it is clear that co-presence of polyphosphate kinase and adenylate kinase causes AMP phosphorylation.

(6) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 2) 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphate (OmM and 3 OmM as inorganic phosphate, respectively) , 5τ ^ polyphosphate kinase and 0.5 unit in 5 OmM Tris-HCl buffer (pH 7.8) containing 4 mM AMP / τηβ adenylate kinase was added, and the mixture was kept at 37 for 120 minutes. After completion of the reaction, nucleotides in the reaction solution were quantified using HPLC. The results are shown in Table 2.

Table 2 Polyphosphate concentration (as phosphoric acid) ATP (mM) ADP (mM)

OmM <0.01 over 0.01

30 0.37 0.76

75 0.67 1.52

150 <0.01 0.09 As shown in Table 2, the phosphorylation activity of polyphosphate kinase and adenylate kinase depends on the concentration of polyphosphate, but in the presence of high concentrations of polyphosphate, Its activity was also impaired. (7) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 3) 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphate (75 mM as inorganic phosphate), 4 mM Add 0.1 unit of polyphosphate kinase and various concentrations of adenylate kinase enzyme to 5 OmM Tris-HCl buffer (pH 7.8) containing AMP. Keep warm for a minute. After completion of the reaction, nucleotides in the reaction solution were quantified using HPLC. The results are shown in Table 3.

Table 3 Polyphosphate kinase adenylate kinase ATP (mM) ADP (mM)

0.10 unit mk 0.05 unit m. 0.18 0.14

0.10 0.25 0.85 1.16

0.10 0.50 0.89 1.26

0.10 2.50 1.04 1.20

(8) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 4) 100 mM magnesium chloride, 100 mM ammonium sulfate, polyphosphate (75 as inorganic phosphate), 4 mM AMP Add 0.1 units of polyphosphate kinase and 2.5 units of adenylate kinase enzyme to 5 OmM Tris-HCl buffer (pH 7.8) containing Keep warm for a minute. After the reaction was completed, nucleotides in the reaction solution were quantified using HPLC. Figure 1 shows the results. At the end of the reaction, the nucleotide concentrations in the reaction solution were ATP 2.3 mM, ADP 1.3 mM, and AMP 0.4 mM.

Example 2 Production of Galactose-1 Monophosphate Using ATP Regeneration System

(1) Preparation of E. coli galactokinase

Escherichia coli JM109 containing plasmid PDR540 (Gene, 20, 231 (1982), obtained from Pharmacia) containing the Escherichia coli galactokinase gene was isolated from 100 g / r of manpicillin. Inoculate 2X YT medium 300 containing The cells were cultured at 37 ° C with shaking. Once at the 4 x 1 0 ⁸ bacteria, was added I PTG to a final concentration of 1 mM to the culture solution was continued for an additional 3 0 ° C for 5 hours with shaking culture. After completion of the culture, the cells were collected by centrifugation (9,000 X g, 10 minutes), and a buffer of 6ητηβ (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0 mM 1% Triton X—100, 0.2 mg Z7 ^ ribzyme). After incubation at 37 ° C for 1 hour, sonication was performed to disrupt the cells, and centrifugation (20,000 X g,

(10 minutes) to remove bacterial cell residues. The supernatant fraction obtained in this manner is treated with 5 OmM Tris-HCl containing 5 mM magnesium chloride and 1 mM 2-mercaptoethanol.

(pH 7.8) was dialyzed to obtain a galactokinase enzyme solution. The specific activity of galactokinase in the enzyme solution was 1.39 units / mg protein.

The unit (unit) of galactokinase activity was measured and calculated by the following method. Add enzyme preparation to 10 OmM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 1 OmM ATP, and 10 mM galactose.

The reaction is carried out by keeping the temperature at 37 ° C, and the reaction is stopped by heat treatment at 100 T: 1 minute. Galactose in the reaction solution using a sugar analyzer (Dionex)

The 1-phosphoric acid is quantified, and the activity to produce 1 mo ^ e of galactose- 1-phosphoric acid per minute at 37 ° C is defined as 1 unit (unit).

(2) ATP regeneration by polyphosphate kinase and adenylate kinase and synthesis of galactose-1-phosphate by galactokinase

1 Omm magnesium chloride, 1 0 Omm ammonium sulfate, 0.1 poly-phosphate 1 0 0 mM Tris-HCl buffer containing and 4 mM AMP (7 5 m as inorganic phosphate) (ρΗ7 · 8) 1 unit / ^/ poly Phosphate kinase and 2.5 units / adenylate kinase enzyme preparation were added, and the mixture was incubated at 37 ° C for 120 minutes. The nucleotide concentrations in the reaction solution at the end of the AMP phosphorylation reaction were ATP 1.4 mM, ADP 1.7 mM, and AMP 0.9 mM. D (10) galactose was added to this reaction solution to a final concentration of 40 mM, and galactokinase was further added to 1.0 unit /. Was added and kept at 37 ° C. for 4.5 hours. Analysis of the reaction solution using a sugar analyzer (Dionex) confirmed the production of 28.4 mM galactose-1-phosphate. Industrial applicability

According to the present invention, it has become possible to easily and inexpensively produce ATP from AMP. By combining this ATP synthesis system with an ATP-consuming enzyme reaction system, it has become possible to efficiently produce the target compound.

Claims

The scope of the claims

1. Adenosine 5'-a method for producing adenosine 5'-triphosphate, which comprises reacting monophosphate with polyphosphate kinase, adenylate kinase and polyphosphate.

2. In a method for producing a compound using an enzymatic reaction that consumes adenosine 5'-triphosphate, adenosine 5'-monophosphate is reacted with polyphosphate kinase, adenylate kinase and polyphosphate. Adenosine 5′-triphosphate is produced by the reaction and supplied to the enzymatic reaction.

3. Adenosine 5 '-In the production of compounds using an enzymatic reaction that consumes triphosphate, adenosine 5'--phosphate and Z or adenosine 5 '-diphosphate are converted to polyphosphate kinase, adenylate. A process for producing the compound, characterized in that the enzyme reaction is carried out while adenosine 5'-triphosphate is regenerated by reacting monokinase and polyphosphate.

4. Synthetic system of adenosine 5'-adenosine 5'-triphosphate by combining polyphosphate kinase, adenylate kinase and polyphosphate.