JP4961544B2 - Phosphorylation method using E. coli - Google Patents

Description

  The present invention relates to a method for producing a phosphorylation reaction using Escherichia coli in a bioprocess, and more specifically, a method for phosphorylating an arbitrary substance using polyphosphate and polyphosphate kinase in Escherichia coli. It is.

  Many biological reactions have positive changes in free energy and do not occur spontaneously. However, it is known that when the hydrolysis of adenosine triphosphate (ATP), which is a bioenergy, is coupled, the overall free energy change becomes negative and the reaction occurs spontaneously (ATP Conjugation reaction).

  Industrial production of substances using enzymes as catalysts is possible. However, when the change in free energy is positive or a phosphorylation step using ATP is included, a reaction for regenerating ATP (ATP regeneration system) is necessary.

  Polyphosphoric acid, which can be easily chemically synthesized and used as a food additive, is a substance having the same high energy phosphate bond as ATP, and an ATP regeneration system using polyphosphoric acid as a phosphate donor has been constructed. For example, polyphosphate derived from Acinetobacter johnsonii using polyphosphate as a phosphate donor is converted to ADP by AMP phosphotransferase, and the resulting two molecules of ADP are converted into one molecule of ATP and AMP by E. coli-derived adenylate kinase. (See Non-Patent Document 1). Utilizing this regeneration system, galactose 1-phosphate has been successfully synthesized by galactokinase.

  In the reaction method described in Non-Patent Document 1, the enzyme used is a purified enzyme extracted from a microorganism or recombinant Escherichia coli. However, since this purification step requires complicated work, polyphosphate kinase (an enzyme that converts ADP to ATP using polyphosphate as a phosphate donor) present in E. coli is not removed from the cell. The method of using is also known (refer nonpatent literature 2). That is, the cells are treated with a surfactant or the like, and the cell membrane is crushed to allow permeation of polyphosphate into the cells. Thereafter, polyphosphoric acid is added and taken up into the cell to regenerate ATP in the cell.

In the method described in Non-Patent Document 2, a new production method has been developed in which a dipeptide is directly and irreversibly bound in E. coli using an ATP regeneration system using polyphosphoric acid. By this method, a large amount of dipeptide can be supplied at low cost.
Journal of Biotechnology Vol.80, No.12, Page590 (2002) Bioscience and Industry Vol.63, No.8, Page555-556 (2005)

  However, the method described in Non-Patent Document 2 still has some problems. That is, it is necessary to treat cells with a surfactant, an organic solvent or the like in order to incorporate polyphosphate into the cells. This operation is a process necessary for incorporating polyphosphate, which is not originally taken into the cell, into the cell. However, surfactants are substances that cause problems when mixed into products as impurities.

  In addition, there are problems that there are enzymes that hydrolyze polyphosphate in cells, and there are many enzymes that generate by-products when the reaction is performed.

  The present inventor uses thermophosphate polyphosphate kinase as a method for solving all of these problems. That is, the cell membrane is destroyed by heat treatment (heating at 70 ° C. for about 10 minutes) of Escherichia coli into which a polyphosphate kinase gene of a thermophilic bacterium has been introduced, so that polyphosphate can be removed from the outside without using a surfactant. It is used for reproducing ATP.

  Further, by expressing an enzyme that phosphorylates a substance (X) to be phosphorylated (hereinafter referred to as “X kinase”) simultaneously with polyphosphate kinase, X can be phosphorylated in E. coli cells. . Here, since the activities of E. coli-derived enzymes other than X kinase are lost by heat treatment, only the desired phosphorylation reaction can be performed with polyphosphoric acid. This solves the problem that there are many enzymes that produce by-products.

  Escherichia coli into which the genes of thermophosphate polyphosphate kinase and X kinase are introduced can cope with phosphorylation of many substances X by changing only X kinase. Escherichia coli into which a thermophosphate polyphosphate kinase has been introduced becomes a general-purpose host for phosphorylating many substances X. An outline of the present invention is shown in FIG.

  By using the present invention, polyphosphoric acid can permeate into cells without using a surfactant which is an impurity, and it is possible to safely avoid the problem that there are many enzymes that produce by-products in the cells. . Moreover, by changing only X kinase, it becomes a general-purpose host that can cope with phosphorylation of a large number of substances X. This general-purpose host has the effect of allowing inexpensive and safe polyphosphoric acid to be widely used industrially as a phosphorylation substrate.

[Polyphosphate kinase]
Polyphosphate kinase is an enzyme whose EC (enzyme commission) number is EC2.7.4.1, and synthesizes ATP from polyphosphate and ADP. Depending on the type of polyphosphate kinase, a reverse reaction may occur, but it is not limited in the present invention.

  The inventor has already determined the chromosomal DNA sequence in order to find a polyphosphate kinase that maintains its activity even after heat treatment at 70 ° C. for about 10 minutes, at which the normal enzyme of E. coli loses its activity. Based on the registered chromosomal DNA sequence of Thermus thermophilus HB27, a gene sequence similar to E. coli polyphosphate kinase was searched. As a result, a gene (No. TTC0637) having about 30% homology with E. coli polyphosphate kinase was found in Thermus thermophilus.

  The reason for choosing Thermus thermophilus here is that this bacterium is classified as a thermophile. In general, a thermophilic bacterium is a microorganism having an optimum growth temperature of 45 ° C. or more, and it is said that thermophilic bacterium has many thermostable enzymes. In particular, Thermus thermophilus is a microorganism that can grow at 65-85 ° C., and was expected to have the desired thermostable polyphosphate kinase.

  Thermus thermophilus polyphosphate kinase gene was expressed in E. coli and the activity was measured. As a result, it was confirmed that the activity was not lost by heating at 70 ° C. for about 10 minutes. It was confirmed that Thermus thermophilus polyphosphate kinase has an optimum temperature around 70 ° C. and has a specific activity comparable to that of E. coli polyphosphate kinase at 37 ° C. Thereby, it was considered that an ATP regeneration system at a high temperature could be constructed.

  However, the polyphosphate kinase used in the present invention can be derived from any enzyme as long as the normal enzyme of E. coli loses its activity and the activity is maintained under the heating conditions in which the cell permeability of polyphosphate is obtained. It is not particularly limited, and any organism such as bacteria, yeast, animals, plants may be used.

[Escherichia coli expressing thermostable polyphosphate kinase]
After the polyphosphate kinase gene of Thermus thermophilus was expressed in E. coli, it was heat-treated at 70 ° C. for about 10 minutes, and centrifuged to separate the precipitate containing the supernatant and cells. As a result of adding polyphosphoric acid and ADP to the supernatant and the precipitate, respectively, the activity of synthesizing ATP from polyphosphoric acid and ADP was observed in the precipitate. As a result, by exposing Escherichia coli expressing the Thermus thermophilus polyphosphate kinase gene to a heat treatment at 70 ° C. for about 10 minutes, polyphosphate and ADP can permeate into the cells. confirmed.
[Heating conditions]
The heating condition is exemplified by heat treatment at 70 ° C. for about 10 minutes, but may be heat treatment at 80 ° C. for about 5 minutes. Conversely, when heat treatment is performed at a relatively low temperature of 60 ° C., the time may be lengthened. The heating conditions are not particularly limited as long as the normal enzyme of E. coli loses its activity and the cell permeability of polyphosphate can be obtained.
[X kinase]
The present invention is characterized by including a step of phosphorylating the substance X. An enzyme that phosphorylates substance X is referred to as X kinase. X kinase uses ATP to phosphorylate substance X to produce ADP. The produced ADP is regenerated into ATP by polyphosphate and thermostable polyphosphate kinase. When considering the overall reaction, substance X is phosphorylated with polyphosphate by X kinase and polyphosphate kinase.

  In the present invention, in order to show that it is possible to cope with phosphorylation of any substance X by changing X kinase introduced into Escherichia coli expressing thermostable polyphosphate kinase, fructose 6 phosphate is used as substance X, and fructose 6 phosphorus is used. The step of converting acid to fructose 1,6-diphosphate is illustrated. Fructose 1,6-diphosphate is a substance expected as a drug for ischemic heart disease. The X kinase in this case is phosphofructose kinase, but the substance X and the corresponding X kinase are not particularly limited.

[Phosphofructose kinase]
Phosphofructose kinase is an enzyme of EC 2.7.1.56, which synthesizes ADP and fructose 1,6-diphosphate from ATP and fructose 6-phosphate.

  In order to find a phosphofructose kinase whose activity is maintained even after heat treatment at 70 ° C. for about 10 minutes at which the normal enzyme of Escherichia coli loses its activity, the inventor uses phosphofructose of Propionibacterium freudenreichii based on the DNA sequence of Thermus thermophilus. A gene having homology with kinase (P29495) was searched. As a result, a gene (gene number TTC1597) having about 28% homology was found.

  The reason for choosing Thermus thermophilus here is that this bacterium is classified as a thermophile. In particular, Thermus thermophilus is a microorganism that can grow at 65-85 ° C., and was expected to have a thermostable phosphofructose kinase.

  Thermus thermophilus phosphofructose kinase gene was expressed in E. coli and activity was measured. As a result, it was confirmed that ADP and fructose 1,6-diphosphate were synthesized from ATP and fructose 6-phosphate at 70 ° C.

  However, the phosphofructose kinase used in the present invention is derived from the normal enzyme of Escherichia coli as long as the activity is maintained even under heating conditions in which the activity of the normal enzyme of E. coli is lost and the cell permeability of polyphosphate is obtained. It is not particularly limited, and any organism such as bacteria, yeast, animals, plants may be used.

[Escherichia coli expressing thermostable polyphosphate kinase and thermostable phosphofructose kinase genes]
A plasmid expressing the phosphofructose kinase gene was introduced into Escherichia coli carrying the polyphosphate kinase gene of Thermus thermophilus. Escherichia coli expressing both genes was heat-treated at 70 ° C. for about 10 minutes, and as a result of adding polyphosphoric acid and fructose 6-phosphate, it was confirmed that fructose 1,6-diphosphate was produced.

[Example 1: Construction of plasmid expressing thermostable polyphosphate kinase]
First, Escherichia coli expressing the phosphate phosphate of Thermus thermophilus was constructed. From the DNA sequence of Thermus thermophilus HB27 registered in the database (DDBJ), a sequence similar to that of E. coli polyphosphate kinase was searched by DDBJ gene search software. As a result, a gene (TTC0637) having 30% homology with E. coli polyphosphate kinase was found in Thermus thermophilus HB27.
Two oligonucleotide primers GGAATTCCATATGCACCTCCTTCCCGAAGC (NdeI site addition, SEQ ID NO: 3) and GAAGTCGACTAGCTCCAGGCGCTGGGCGT (SalI site addition, SEQ ID NO: 4) were prepared, and PCR was performed using the chromosomal DNA of Thermus thermophilus as a template. The reaction used KOD Plus DNA polylase (Takara Bio). The reaction conditions were maintained at 96 ° C. for 2 minutes, then repeated 10 cycles of 94 ° C. for 30 seconds, 59 ° C. for 30 seconds, 68 ° C. for 2 minutes, 94 ° C. for 30 seconds, and 68 ° C. for 2 minutes. The reaction was repeated for 25 cycles.

  The PCR product and the expression vector pET21-a (Novagen) were treated with restriction enzymes NdeI and SalI at 37 ° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each DNA fragment excised from the gel was ligated with Ligation High (TOYOBO) at 16 ° C. for 2 hours, and transformed into Escherichia coli MV1184 strain. A plasmid with the desired DNA fragment inserted was extracted from the obtained colony to construct pETPPK. FIG. 2 shows the structure of pETPPK.

[Example 2: Expression and purification of thermostable polyphosphate kinase]
BL21 (Rosetta) (Novagen) transformed with pETPPK was cultured at 37 ° C. in an LB medium until the OD600 reached 0.5, and 0.2 mM IPTG was added and further cultured at 22 ° C. for 8 hours. After collection, the cells were crushed with ultrasonic waves and heat-treated at 70 ° C. for 10 minutes. Thereafter, the mixture was centrifuged, and the supernatant was purified with a HisTrap HP 1 ml (Amersham Bioscience) column. After washing with 10 ml of buffer A (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 15% glycerol) containing 50 mM imidazole, elution was performed with buffer A containing 0.5 M imidazole. Finally, 1 ml fraction containing the fusion protein was obtained.

[Example 3: Activity of thermostable polyphosphate kinase]
A solution containing 5 mM polyphosphoric acid (Sigma phosphate glass P65), 80 μM ADP, 50 mM HEPES · KOH (pH 7.2), 40 mM (NH ₄ ) ₂ SO ₄ , 4 mM MgCl ₂ pH 7.2 was kept at 75 ° C. and purified. Polyphosphate kinase (0.56 mg) was added. The solution was fractionated over time, and the synthesized ATP was measured with a bioluminescence kit (Roche). FIG. 3 shows the ATP synthesis reaction by Thermus thermophilus polyphosphate kinase. At 75 ° C., ATP is produced from polyphosphoric acid and ADP.

  Then, the optimum temperatures of Thermus thermophilus polyphosphate kinase and Escherichia coli polyphosphate kinase were compared. Only the temperature was changed under the conditions described above, and the ATP synthesis rate at each temperature was measured. E. coli polyphosphate kinase had the highest activity at 30 ° C. to 40 ° C., and the activity decreased with increasing temperature. On the other hand, it can be seen that Thermus polyphosphate kinase shows the highest activity at 60 to 80 ° C., particularly at 70 ° C. The optimum temperature is shown in FIG.

  Next, the thermostability of Thermus thermophilus polyphosphate kinase and Escherichia coli polyphosphate kinase were compared. Under the above-mentioned conditions not containing ADP, after keeping the temperature at 30 to 80 ° C. for 10 minutes, ADP was added and the ATP synthesis rate was measured. The ATP synthesis rate before heating was expressed as 100%. No significant difference was observed at 30 to 40 ° C., and the activity of E. coli polyphosphate kinase was greatly reduced from 50 ° C., and the activity was completely lost after 60 ° C. In contrast, Thermus thermophilus polyphosphate kinase remained active even at temperatures above 50 ° C. A graph of residual activity after heat treatment at each temperature is shown in FIG.

[Example 4: Thermostable phosphofructokinase]
E. coli expressing the phosphofructokinase of Thermus thermophilus was constructed. A sequence similar to the phosphofructose kinase (P29495) of Propionibacterium freudenreichii was searched for from the DNA sequence of Thermus thermophilus HB27 registered in the database (DDBJ) using the gene search software of DDBJ. As a result, a gene (TTC1597) having about 28% homology with E. coli kinase was found in Thermus thermophilus.
Two oligonucleotide primers GGCCATATGAAACGCATCGGGGTGTT (NdeI site added, SEQ ID NO: 5) and TATAAGCTTGAGGGCCAGCACCTGCGATA (HindIII site added, SEQ ID NO: 6) were prepared, and PCR was performed using Thermus thermophilus chromosomal DNA as a template. The reaction used KOD Plus DNA polylase (Takara Bio). The reaction conditions were maintained at 94 ° C. for 2 minutes, followed by 35 cycles of 94 ° C. for 15 seconds, 56 ° C. for 30 seconds, and 68 ° C. for 90 seconds, and held at 72 ° C. for 10 minutes.

  The PCR product and the expression vector pET21-b (Novagen) were treated with restriction enzymes NdeI and HindIII at 37 ° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each DNA fragment excised from the gel was ligated with Ligation High (TOYOBO) at 16 ° C. for 2 hours, and transformed into E. coli MV1184 strain. From the obtained colony, a plasmid in which the target DNA fragment was inserted was extracted to construct pETPFK. FIG. 6 shows the structure of pETPFK.

[Example 5: Purification and activity of thermostable phosphofructokinase]
BL21 (Rosetta) (Novagen) transformed with pETPFK was cultured at 28 ° C. in 2YT medium until the OD600 reached 0.5, and 0.5 mM IPTG was added and further cultured for 7 hours. After collection, the cells were disrupted with lysozyme and ultrasonic waves, and the centrifuged supernatant was purified with a HisTrap HP 1 ml (Amersham Bioscience) column. After washing with 10 ml of buffer A (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 15% glycerol) containing 10 mM imidazole, elution was performed with buffer A containing 0.5 M imidazole. Finally, 1 ml fraction containing the fusion protein was obtained.

A solution containing 10 mM polyphosphoric acid (Sigma phosphate glass P65), 20 mM fructose 6 phosphoric acid, 5 mM ADP, 50 mM MOPS · NaOH (pH 6.5), 25 mM KCl, 5 mM MgCl ₂ was kept at 70 ° C. and purified polyphosphorus. 5 mg of acid kinase and 5 mg of phosphofructokinase were added. The solution was fractionated over time, and the synthesized fructose 1,6-diphosphate was measured by the method described in Extremophiles, 3, 121-129 (1999).

    In this reaction, a reaction not containing polyphosphate kinase was used. As a result, fructose 1,6 diphosphate is synthesized in reactions involving polyphosphate kinase and phosphofructokinase, and fructose 1,6 diphosphate is synthesized in reactions involving only phosphofructokinase without polyphosphate kinase. It turns out not to be. In addition, fructose 1,6-diphosphate is not synthesized even in a reaction system that does not contain polyphosphoric acid. From this, it was found that ATP was regenerated by polyphosphoric acid, and thereby fructose 1,6-diphosphate was synthesized. FIG. 7 shows a synthetic reaction of fructose 1,6-diphosphate using purified polyphosphate kinase and phosphofructokinase.

[Example 6: Construction of Escherichia coli expressing thermostable polyphosphate kinase and phosphofructokinase]
pETPPK and pETPFK are plasmids expressing polyphosphate kinase and phosphofructokinase, respectively, but cannot coexist in the same E. coli. Therefore, the phosphofructokinase gene was transferred to a pACYC plasmid that can coexist with the pET vector. Two oligonucleotide primers GAAGAATTCAATGAAACGCATCGGGGTGTT (EcoRI site addition, SEQ ID NO: 7) and TATAAGCTTGAGGGCCAGCACCTGCGATA (HindIII site addition, SEQ ID NO: 8) were prepared, and PCR was performed using pETPFK as a template. The reaction used KOD Plus DNA polylase (Takara Bio). The reaction conditions were maintained at 96 ° C. for 2 minutes, then repeated 10 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute, 94 ° C. for 30 seconds, 61 ° C. for 30 seconds, The reaction at 68 ° C. for 1 minute was repeated 15 cycles.

  The PCR product and expression vector pACYC (Novagen) were treated with restriction enzymes EcoRI and HindIII at 37 ° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each DNA fragment excised from the gel was ligated with Ligation High (TOYOBO) at 16 ° C. for 2 hours, and transformed into Escherichia coli MV1184 strain. A plasmid with the desired DNA fragment inserted was extracted from the obtained colonies to construct pACYCPFK. FIG. 8 shows the structure of pACYCPFK.

  pACYCPFK was introduced into E. coli BL21 (Novagen) carrying pETPPK. BL21 (Novagen) having pETPPK and pACYCPFK in the same microbial cell was cultured at 37 ° C. until the OD600 reached 0.5 in LB medium, and then 0.2 mM IPTG was added and cultured at 22 ° C. for 8 hours.

[Example 7: Synthesis of fructose 1,6-diphosphate from fructose 6-phosphate using E. coli expressing thermostable polyphosphate kinase and phosphofructokinase]
A solution containing 10 mM polyphosphoric acid (Sigma phosphate glass P65), 10 mM fructose 6 phosphoric acid, 5 mM ADP, 50 mM MOPS · NaOH (pH 6.5), 25 mM KCl, 5 mM MgCl ₂ is kept at 70 ° C., and pETPPK and pACYCPFK BL21 (DE3) cells (2 × 10 ⁷ ) having the same microbial cell were mixed.

    The solution was collected over time, and the synthesized fructose 1,6-diphosphate was measured by the method described in Extremophiles, 3, 121-129 (1999). FIG. 9 shows that fructose 1,6-diphosphate is synthesized using E. coli expressing thermostable polyphosphate kinase and phosphofructokinase.

  The present invention is applicable to a bioprocess that uses inexpensive and safe polyphosphoric acid as a substrate for phosphorylation. There are many enzymes that can permeate polyphosphoric acid into cells without the need for complicated enzyme purification, and without the use of impurities as surfactants, and produce by-products in the cells. This is advantageous in that the problem can be avoided.

Outline reaction process diagram of the present invention Conceptual diagram showing the configuration of pETPPK Graph showing the thermostable activity of Thermus thermophilus polyphosphate kinase Graph showing comparison of optimum temperature of Escherichia coli polyphosphate kinase and Thermus thermophilus polyphosphate kinase Graph showing a comparison of heat resistance between Escherichia coli polyphosphate kinase and Thermus thermophilus polyphosphate kinase Conceptual diagram showing the structure of pETPFK Graph showing the synthesis reaction of fructose 1,6-diphosphate with purified polyphosphate kinase and phosphofructokinase Conceptual diagram showing the structure of pACYCPFK Synthesis of fructose 1,6-diphosphate using Escherichia coli expressing thermostable polyphosphate kinase and phosphofructokinase

Claims (10)

Escherichia coli containing a polyphosphate kinase gene of a thermophilic bacterium, in a state where the normal enzyme of E. coli loses its activity and the cell permeability of polyphosphate is obtained, in a state where the cell membrane of the E. coli is destroyed by heating, by transmitting polyphosphoric acid into the cell to regenerate the ATP, the polyphosphate kinase, 70 ° C., all SANYO activity does not disappear for 10 minutes,
The E. coli, phosphorylation method using E. coli, characterized in Rukoto heated at 60 ° C. to 80 ° C..   The polyphosphate kinase is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. The phosphorylation reaction method according to claim 1. A method for phosphorylating substance X comprising the following steps (a) to (d), wherein the following polyphosphate kinase and X kinase do not lose their activity upon treatment at 70 ° C. for 10 minutes:
(A) E. coli containing a gene for polyphosphate kinase and a gene for an enzyme (X kinase) that phosphorylates substance X to be phosphorylated can be obtained by losing the activity of normal enzymes of E. coli and cell permeability of polyphosphate. Heating to obtain a heat-treated product having a disrupted cell membrane of E. coli under conditions , wherein the E. coli is heated at 60 ° C. to 80 ° C. (b) polyphosphoric acid and substance X (C) a step of phosphorylating the substance X by the X kinase to generate a phosphorylated substance X and ADP; and (d) a step of regenerating ADP to ATP by the polyphosphoric acid. The polyphosphate kinase is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. The phosphorylation method according to claim 3 . The phosphorylation method according to claim 3 , wherein the X kinase is phosphofructose kinase. The phosphorylation method according to claim 3 , wherein there are a plurality of enzymes that phosphorylate the substance X to be phosphorylated. A method of phosphorylation using E. coli comprising the following steps (e) to (g), wherein the activity of the following polyphosphate kinase is not lost by treatment at 70 ° C. for 10 minutes:
(E) a step of culturing Escherichia coli containing a polyphosphate kinase gene of a thermophilic bacterium and accumulating polyphosphate (f) the Escherichia coli accumulating the polyphosphate, wherein an ordinary enzyme of Escherichia coli loses its activity and polyphosphate The step of destroying the cell membrane by heating under the condition that the cell permeability is obtained , wherein the E. coli is heated at 60 ° C. to 80 ° C. (g) A solution containing ADP is added to regenerate to ATP Process. The polyphosphate kinase is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. The phosphorylation reaction method according to claim 7 . The phosphorylation reaction method according to claim 7 , which is an ATP regeneration system in which polyphosphate and polyphosphate kinase react, and includes an ATP regeneration reaction at 50 ° C to 80 ° C. The polyphosphate kinase is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. The phosphorylation reaction method according to claim 9 .