Adenosine kinase mutant with high thermal stability and application thereof

Abstract

The invention discloses an adenosine kinase mutant with high thermal stability and application thereof, and relates to the technical field of enzyme engineering, and the adenosine kinase mutant with high thermal stability is obtained by performing point mutation on the 253rd site or the 82nd site of adenosine kinase as shown in SEQ ID NO: 1. According to the invention, an adenosine kinase mutant is used as a biocatalyst in an enzyme catalysis manner; the method is used for enzymatic synthesis of 5 '-adenylic acid, adenosine and adenosine triphosphate react to prepare adenylic acid, generation of by-products and impurities in the reaction is reduced to a certain extent, production efficiency and purity of adenylic acid are improved, reaction conditions are mild in preparation in an enzyme catalysis mode, pollution to the environment is avoided, and the method is suitable for industrial production. And the high-thermal-stability adenosine kinase mutant is applied to adenylic acid preparation, so that the preparation efficiency and yield of adenylic acid can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to an adenosine kinase mutant with high thermal stability and application thereof. Background Art

[0002] Adenylic acid is a compound of adenine, ribose and phosphate. It is one of the four main mononucleotides that make up the RNA of animal cells. Adenylic acid is widely used in medicine and industry. There are four common production methods. The current process for synthesizing adenylic acid mainly uses chemical methods. There are three by-products in the preparation process of existing adenylic acid, and their content accounts for a relatively large proportion, which seriously affects the purity and efficiency of the preparation of adenylic acid.

[0003] The existing enzymatic method for preparing adenylic acid has the following defects:

[0004] 1. Patent document CN107602648B discloses a method for preparing 5'-adenylic acid, which mainly considers the use of a solvent-free synthesis method to reduce reaction impurities and be environmentally friendly, but does not consider how to improve the yield and conversion efficiency of adenylic acid;

[0005] 2. Patent document CN105603028B discloses a method for simultaneously preparing glutathione and adenylic acid by enzymatic method. It mainly considers how to prepare GSH and AMP simultaneously, but does not consider how to reduce the interference of other proteins or impurities, so as to more effectively convert adenosine into adenylic acid.

[0006] 3. Patent document CN103833813B discloses a method for preparing adenylic acid crystals based on isoelectric dissolution coupling, which mainly considers how to prepare adenylic acid crystals and obtain high-purity products in a short period of time, but does not consider how to improve the efficiency of the preparation process and improve the purity of the product;

[0007] 4. The existing adenylate preparation process does not consider how to improve the efficiency of screening, thereby helping to improve the efficiency of adenylate preparation. Summary of the invention

[0008] The object of the present invention is to provide a highly thermally stable adenosine kinase mutant and its application to solve the problems raised in the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solution: a highly thermally stable adenosine kinase mutant and its application.

[0010] In one aspect, the present invention provides a highly thermostable adenosine kinase mutant.

[0011] Specifically, the adenosine mutant is obtained by mutating the 253rd or 82nd position of the adenosine kinase shown in SEQ ID NO: 1 (nucleotide sequence is the 623581-624603 sequence of Saccharomyces cerevisiae S288C Chromosome: X; NC_001142.9).

[0012] More specifically, the mutation at position 253 is K253R. The mutation at position 82 is V82C.

[0013] In another aspect, the present invention provides a gene encoding the above-mentioned adenosine kinase mutant.

[0014] In yet another aspect, the present invention provides an expression vector comprising the above encoding gene.

[0015] Specifically, the vector includes but is not limited to: pET28a(+), pet30a(+), pETduet, pREST or pTriEX.

[0016] In certain specific embodiments of the present invention, the vector is pET28a(+).

[0017] In yet another aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned adenosine kinase mutant and / or the above-mentioned encoding gene and / or the above-mentioned expression vector.

[0018] Specifically, the genetically engineered bacteria include but are not limited to Escherichia coli, Salmonella, and Pichia pastoris.

[0019] In certain specific embodiments of the present invention, the engineered bacteria is Escherichia coli BL21 (DE3).

[0020] In another aspect, the present invention provides the use of the adenosine kinase mutant and / or the genetically engineered bacteria of the expression vector in the preparation of adenylic acid.

[0021] In certain specific embodiments of the present invention, the genetically engineered bacteria are cultured and then centrifuged to obtain wet bacteria.

[0022] In certain specific embodiments of the present invention, the wet bacteria are ultrasonically disrupted to obtain adenosine kinase crude enzyme solution.

[0023] In certain specific embodiments of the present invention, the adenosine kinase is purified by suspending the wet bacteria in a buffer, disrupting them by ultrasonication, centrifuging them, taking the supernatant, and purifying the protein using a Ni affinity column.

[0024] Specifically, the genetically engineered bacteria, wet bacteria, crude enzyme solution, and adenosine kinase can all be used for the preparation of adenylic acid.

[0025] In yet another aspect, the present invention provides a method for producing adenylate.

[0026] Specifically, the method uses the adenosine kinase mutant as claimed in claim 1 as a catalyst.

[0027] More specifically, the method uses adenosine, disodium adenosine triphosphate, and magnesium chloride hexahydrate as substrates; uses adenylate kinase and mutant adenosine kinase as catalysts to construct a reaction system to synthesize adenylic acid in a one-step reaction.

[0028] In certain specific embodiments of the present invention, the reaction system contains 20-374 mM adenosine, 10-226 mM adenosine disodium triphosphate, and 10-100 mM magnesium chloride hexahydrate; and the reaction system contains 60.7-121.3 U of adenylate kinase and 16.8-33.5 U of mutant adenosine kinase per 100 mL.

[0029] Specifically, the one-step reaction is carried out at 30-50°C.

[0030] In certain specific embodiments of the present invention, the reaction is carried out at 40°C and 50°C.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention uses an enzyme catalytic method and utilizes an adenosine kinase mutant as a biocatalyst for the enzymatic synthesis of 5'-adenylic acid, reacts adenosine with adenosine triphosphate to obtain adenosine acid, which reduces the generation of by-products and impurities in the reaction to a certain extent, thereby improving the production efficiency and purity of adenosine acid. In addition, the reaction conditions in the preparation by the enzyme catalysis are mild and will not cause pollution to the environment. In addition, the high thermal stability adenosine kinase mutant is used in the preparation of adenosine acid, which is beneficial to improving the efficiency and yield of the preparation of adenosine acid.

[0033] 2. The present invention reduces the interference of other proteins or impurities by purifying adenosine kinase, can more effectively convert adenosine into adenylic acid, reduce the occurrence of side reactions, and make the final adenylic acid purer. Under the catalysis of highly active purified enzyme, the cost of enzyme use can be reduced, and at the same time, an efficient catalytic reaction can be achieved, thereby improving the reaction efficiency and reducing the cost to a certain extent.

[0034] 3. The present invention can effectively screen out adenosine kinases with poor activity and poor thermal stability by measuring the activity and thermal stability of adenosine kinase, which helps to improve the quality of adenosine kinases that participate in the subsequent adenosine and adenosine triphosphate reaction process, so as to further improve the efficiency of the preparation process and improve the purity of the product.

[0035] 4. The present invention helps to improve the thermal stability of ArK through ArK molecular dynamics simulation and homologous protein multiple sequence alignment, thereby improving the efficiency of screening, and further helping to improve the efficiency of preparing adenylic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The figure is a schematic diagram of the measurement results of the thermal stability of adenosine kinase at different temperatures of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The present invention provides an adenosine kinase mutant with high thermal stability. The preparation method of the adenosine kinase mutant is as follows:

[0039] Step S1: Add an NcoⅠ restriction site before the start codon of the nucleotide sequence of adenosine kinase, then delete the stop codon, and add an XhoⅠ restriction site at the end of the sequence to obtain a new sequence, wherein the nucleotide sequence is the 623581-624603 sequence of Saccharomyces cerevisiae S288CChromosome:X; NC_001142.9, and then perform full gene synthesis on the obtained new sequence, and then use the NcoⅠ / XhoⅠ double restriction site to connect the new sequence to the pET28a(+) vector to construct a pET28a(+)-ArK expression vector, followed by centrifugation at a centrifugal speed of 10 rpm for 10 minutes, and then add 50 μL of ddH2O thereto, and place it on a vortex for dissolution, and then transform the dissolved substance into Escherichia coli E.coliB L21 (DE3), and then activate the transformed Escherichia coli E. coli BL21 (DE3), wherein the activation temperature is 37°C, the stirring speed is 220 rpm, and the time is 1 hour. After activation, it is coated on a 50 μg / mL kanamycin-resistant LB plate, and cultured overnight at a culture temperature of 37°C. On the second day, a single colony is picked and inoculated into a TB liquid culture medium containing 50 μg / mL kanamycin, and glucose and 20 g / L lactose with a final concentration of 5 g / L are added to the culture medium, and stirred at a speed of 220 rpm at 37°C until OD600 reaches 0.8-1.2, and then the TB liquid culture medium is cooled to 28°C, and stirred at a speed of 220 rpm, and cultured for 14 hours, and then centrifuged at a speed of 8000 rpm for 10 minutes at 4°C to obtain wet bacteria containing adenosine kinase;

[0040] Step S2: Use 5 column volumes of buffer A to balance the Ni column until the baseline is stable, then pass the supernatant containing the target protein through the Ni affinity column at a flow rate of 1 mL / min, wherein the concentration of the target protein in the solution is 25-40 mg / mL, then rinse with 6 column volumes of buffer A until the baseline is stable, wherein the flushing flow rate is 1 mL / min, elute with buffer B, and collect the target protein, wherein the elution flow rate is 1 mL / min, and dialyze the obtained target protein in a buffer of pH 8.0, 20 mM Tris overnight to obtain purified adenosine kinase, and then rinse the Ni column with 5 column volumes of buffer C until the baseline is stable, and rinse with 5 column volumes of buffer C until the baseline is stable. The Ni column is preserved in ultrapure water containing 20% ​​ethanol by volume, and adenosine kinase is finally obtained, wherein buffer A is a mixture of NaCl, imidazole and Tris buffer, wherein the concentration of NaCl is 0.5M, the concentration of imidazole is 20mM, the pH is 8.0, and the concentration of Tris buffer is 20mM; buffer B is a mixture of NaCl, imidazole and Tris buffer, wherein the concentration of NaCl is 0.5M, the concentration of imidazole is 400mM, the pH is 8.0, and the concentration of Tris buffer is 20mM; buffer C is a mixture of NaCl and Tris buffer, wherein the concentration of NaCl is 0.5M, the concentration of Tris buffer is 20mM, and the pH is 8.0;

[0041] Step S3: reacting 13.35 g / L adenosine, 81.5 g / L ATP-Na2, 10 g / L MgCl2·6H2O and enzyme solution at 35°C and pH 8.0 for 20 min, then performing HPLC detection and analysis, diluting adenosine kinase, and then placing in an environment of 30°C, 35°C, 40°C, 45°C and 50°C, respectively, and taking samples every hour to measure the enzyme activity, the duration is 24 hours;

[0042] Step S4: Use SWISS-MODEL to predict the three-dimensional structure of ArK, then align the amino acid sequence of ArK with the amino acid sequence of the template protein, and then use GMQE therein to determine the three-dimensional model and perform modeling, then use GROMACS to perform molecular dynamics (MD) simulation, generate trajectory files, and generate RMSD and RMSF by inputting commands, use the amino acid sequence of ArK as a probe in the NCBI database, query enzyme proteins that are highly similar to the ArK sequence, and select all amino acid sequences whose identity with the ArK sequence is greater than 35%. Multiple sequence alignment was performed, and then the homologous proteins were aligned using the BioXM tool. Combined with molecular dynamics simulation and multiple sequence alignment of homologous proteins, the amino acid residue sites were selected to construct a mutation library, and the following 21 single-point mutants were constructed: V6L, V7C, L8F, L8C, V37A, K45H, T69S, A70M, A74Q, V82C, M83G, Y84F, S87C, G117P, G178A, F192L, V200F, T213D, A217G, C226A, K253R;

[0043] Step S5: Amplify the target DNA fragment by PCR amplification technology, catalyze with KOD high-fidelity enzyme polymerase at 95°C, keep for 2 minutes, and then continue to complete 30 cycles, each cycle includes keeping at 55°C for 20 seconds, then keeping at 71°C for 100 seconds, and keeping at 72°C for 10 minutes after the cycle is completed, and then use electrophoresis to separate agarose gel electrophoresis to separate the target DNA fragment, and then cut out the gel part containing the target DNA fragment by gel recovery, and recover the PCR product, and then add DpnI enzyme to the PCR product recovered by gel, incubate at 37°C for 2 hours, degrade the initial template, and finally leave a pure PCR product, and use restriction endonuclease to eliminate the PCR product obtained in step S52. coli BL21 (DE3) strain, and the transformed cells were plated on LB agar plates containing 50 μg / mL kanamycin, and the LB agar plates were placed in an incubator at 37°C for overnight culture. The positive clones containing the correct target DNA fragments were obtained by antibiotic screening and sequencing verification on the next day, i.e., recombinant bacteria containing ArK enzyme mutants. The obtained recombinant bacteria were treated according to the method in step S1 to obtain a crude enzyme solution of the ArK enzyme mutant, and the mutant was subjected to a thermal stability test according to the method in step S3, the test temperature being 50°C, and the mutant crude enzyme solution was purified according to the purification method in step S2 to finally obtain a pure enzyme solution of the mutant, and the obtained highly thermally stable adenosine kinase mutant was used for adenylate preparation.

[0044] Embodiment 1:

[0045] The highly thermostable adenosine kinase mutant K253R was applied to the preparation of adenylic acid. NaOH solution was first added to dissolve adenosine, and then adenosine triphosphate was added to make the molar ratio of adenosine to adenosine triphosphate 2:1, wherein the concentration of adenosine was 187 mM, the amount added was 50 g, the concentration of adenosine triphosphate was 93.5 mM, the amount added was 54.5 g, and then 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added to obtain a reaction system of 500 ml, and the adenosine kinase mutant K253R was added to the reaction system. 16,800 U of ATP, 60,700 U of adenylate kinase, adenosine and adenosine triphosphate generate adenylic acid and adenosine diphosphate under the action of adenosine kinase, adenosine diphosphate generates adenylic acid and adenosine triphosphate under the action of adenylate kinase, the reaction is allowed to stand at 50°C for 1 hour, then the pH is adjusted back to 7.0 with NaOH solution, and samples are taken during the reaction for HPLC analysis. The final reaction data are AMP: 92.234%, Ar: 6.653%, ADP: 1.054%, ATP: 0.022%;

[0046] After the reaction liquid heat is terminated, the protein and other insolubles are removed by suction filtration, and the A259nm is measured to be 4423. Then the solution is preheated to 60°C, stirring is maintained, and the pH is adjusted to 3.0 with HCl solution, and the heating is turned off. After the crystals are precipitated, HCl solution is gradually added to adjust the pH to 2.0. After stabilizing and dropping to room temperature, the A259nm of the crystallization mother liquor is measured after filtration and is 587. The yield is low, and the purity of the wet powder is 99.256%.

[0047] Embodiment 2:

[0048] The highly thermostable adenosine kinase mutant K253R was applied to the preparation of adenylic acid. NaOH solution was first added to dissolve adenosine, and then adenosine triphosphate was added to make the molar ratio of adenosine to adenosine triphosphate 2:1, wherein the concentration of adenosine was 374mM, the amount added was 100g, the concentration of adenosine triphosphate was 187mM, the amount added was 109g, and then 4.07g of magnesium chloride hexahydrate with a concentration of 20mM and a pH of 7.0 was added to obtain a reaction system of 1000ml, and the adenosine kinase mutant was added to the reaction system. 33,500 U, adenylate kinase 121,300 U, adenosine and adenosine triphosphate generate adenylic acid and adenosine diphosphate under the action of adenosine kinase, and adenosine diphosphate generates adenylic acid and adenosine triphosphate under the action of adenylate kinase. The reaction is allowed to stand at 50°C for 1 hour, and then the pH is adjusted back to 7.0 with NaOH solution. Samples are taken during the reaction for HPLC analysis. The final reaction data are AMP: 91.525%, Ar: 6.967%, ADP: 1.387%, ATP: 0.022%;

[0049] After the reaction liquid heat is terminated, the protein and other insolubles are removed by suction filtration, and then the solution is preheated to 60°C, stirring is maintained, and the pH is adjusted to 3.0 with HCl solution, and the heating is turned off. After crystals are precipitated, HCl solution is gradually added to adjust the pH to 2.0. After stabilization and dropping to room temperature, adenosine crude product is obtained after filtration, with a purity of 99.063% and a wet weight of 282g. After refining, adenosine finished product is obtained, with a net weight of 185.77g, a purity of 99.646%, and a content of 99.5%.

[0050] Embodiment three:

[0051] The highly thermostable adenosine kinase mutant K253R was used to prepare adenosine. Adenosine and adenosine triphosphate were mixed in a molar ratio of 2:1.1. The concentration of adenosine was 374 mM and the amount added was 100 g. The concentration of adenosine triphosphate was 206 mM and the amount added was 120 g. Then, 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added. The resulting reaction system was 1000 ml. 33,500 U of adenosine kinase mutant and 12.5 U of adenylate kinase were added to the reaction system. 130,000 U, adenosine and adenosine triphosphate generate adenylic acid and adenosine diphosphate under the action of adenosine kinase, and adenosine diphosphate generates adenylic acid and adenosine triphosphate under the action of adenylate kinase. The reaction is stirred at 50°C. The pH is automatically controlled at 7.0 for 4 hours before the reaction and at 7.5 after 4 hours by an automatic pH adjustment system. Samples are taken during the reaction for HPLC analysis. The final data after 18 hours of reaction are AMP: 93.967%, Ar: 2.579%, ADP: 3.240%, ATP: 0.098%;

[0052] After the reaction liquid heat is terminated, the protein and other insolubles are removed by suction filtration, and then the solution is preheated to 60°C, stirring is maintained, and the pH is adjusted to 3.0 with HCl solution, and the heating is turned off. After crystals are precipitated, HCl solution is gradually added to adjust the pH to 2.0. After stabilization and cooling to room temperature, the crude adenylate is obtained by filtration, and the finished adenylate is obtained after drying, with a net weight of 165.62 g, a purity of 99.683%, and a content of 99.3%.

[0053] Embodiment 4:

[0054] The highly thermostable adenosine kinase mutant K253R was used to prepare adenosine. Adenosine and adenosine triphosphate were mixed in a molar ratio of 2:1.2. The concentration of adenosine was 374 mM and the amount added was 100 g. The concentration of adenosine triphosphate was 225 mM and the amount added was 131 g. Then, 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added. The resulting reaction system was 1000 ml. 33,500 U of adenosine kinase mutant and 12. 130,000 U, adenosine and adenosine triphosphate generate adenylic acid and adenosine diphosphate under the action of adenosine kinase, and adenosine diphosphate generates adenylic acid and adenosine triphosphate under the action of adenylate kinase. Stir the reaction at 50°C, and use an automatic pH adjustment system to automatically control the pH at 7.0 for 4 hours before the reaction and at 7.5 after 4 hours. Samples are taken during the reaction for HPLC analysis. After 18 hours of reaction, the final data are AMP: 90.266%, Ar: 0.981%, ADP: 8.323%, ATP: 0.325%;

[0055] After the reaction liquid heat is terminated, the protein and other insolubles are removed by suction filtration, and then the solution is preheated to 60°C, stirring is maintained, and the pH is adjusted to 3.0 with HCl solution, and the heating is turned off. After crystals are precipitated, HCl solution is gradually added to adjust the pH to 2.0. After stabilization and cooling to room temperature, adenylic acid wet product is obtained after filtration, and adenylic acid finished product is obtained after drying, with a net weight of 170.53 g, a purity of 99.541%, and a content of 99.2%.

[0056] Embodiment five:

[0057] The highly thermostable adenosine kinase mutant K253R was applied to the preparation of adenylic acid. Adenosine was mixed with adenosine triphosphate, and the molar ratio of adenosine to adenosine triphosphate was 2:1.1. The concentration of adenosine was 374 mM, and the amount added was 100 g. The concentration of adenosine triphosphate was 206 mM, and the amount added was 120 g. Then, 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added. The resulting reaction system was 1000 ml. 16,800 U of adenosine kinase mutant and 6.0 U of adenylate kinase were added to the reaction system. 70,000 U, adenosine and adenosine triphosphate generate adenylic acid and adenosine diphosphate under the action of adenosine kinase, and adenosine diphosphate generates adenylic acid and adenosine triphosphate under the action of adenylate kinase. The reaction is stirred at 40°C. The pH is automatically controlled at 7.0 for 22 hours before the reaction and at 7.5 after 22 hours by an automatic pH adjustment system. Samples are taken during the reaction for HPLC analysis. The final data after 36 hours of reaction are AMP: 93.551%, Ar: 1.755%, ADP: 4.506%, ATP: 0.101%;

[0058] After the reaction liquid heat is terminated, the protein and other insolubles are removed by suction filtration, and then the solution is preheated to 60°C, stirring is maintained, and the pH is adjusted to 3.0 with HCl solution, and the heating is turned off. After crystals are precipitated, HCl solution is gradually added to adjust the pH to 2.0. After stabilization and cooling to room temperature, adenylic acid wet product is obtained after filtration, and adenylic acid finished product is obtained after drying, with a net weight of 167.34 g, a purity of 99.663%, and a content of 99.4%.

[0059] The results of the thermal stability test of adenosine kinase mutants at 50°C are as follows:

[0060]

[0061]

[0062] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A highly thermostable adenosine kinase mutant, characterized in that: The highly thermostable adenosine kinase mutant is obtained by performing a point mutation at position 253 or position 82 of the adenosine kinase shown in SEQ ID NO:

1.

2. The highly thermostable adenosine kinase mutant according to claim 1, characterized in that: The mutation position at site 253 is: K253R.

3. The highly thermostable adenosine kinase mutant according to claim 1, characterized in that: The mutation position at site 82 is: V82C.

4. A gene encoding the adenosine kinase mutant according to any one of claims 1 to 3.

5. An expression vector comprising the coding gene according to claim 4.

6. The expression vector according to claim 5, characterized in that: The expression vector was constructed based on pET28a(+).

7. A genetically engineered bacterium comprising the adenosine kinase mutant according to any one of claims 1 to 3, the coding gene according to claim 4 and / or the expression vector according to any one of claims 5 to 6.

8. The genetically engineered bacterium according to claim 7, characterized in that: The genetically engineered bacteria is Escherichia coli BL21 (DE3).

9. Use of the adenosine kinase mutant according to any one of claims 1 to 3 and / or the encoding gene according to claim 4 and / or the expression vector according to any one of claims 5 to 6 and / or the genetically engineered bacteria according to any one of claims 8 to 9 in the preparation of adenylic acid.

10. A method for preparing adenylic acid, characterized in that: The method uses the adenosine kinase mutant described in any one of claims 1 to 3 and / or the genetically engineered bacteria described in any one of claims 7 to 8 as a catalyst.

11. The method according to claim 10, characterized in that: The method uses adenosine, disodium adenosine triphosphate and magnesium chloride hexahydrate as substrates; uses adenylate kinase and mutant adenosine kinase as catalysts to construct a reaction system and synthesize adenylate in one step.

12. The method according to claim 11, characterized in that: The reaction system contains 20-374 mM adenosine, 10-226 mM adenosine disodium triphosphate, and 10-100 mM magnesium chloride hexahydrate.

13. The method according to claim 11, characterized in that: The reaction system contains 60.7-121.3 U of adenylate kinase and 16.8-33.5 U of mutant adenosine kinase per 100 mL.

14. The method according to claim 11, characterized in that: The one-step reaction is carried out at 30-50°C.

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