JP2009017876A - New pyruvate orthophosphate dikinase and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pyruvate orthophosphate dikinase having excellent storage stability in a low-temperature region and freeze storage stability and high heat stability. <P>SOLUTION: The pyruvate orthophosphate dikinase is derived from Thermotoga maritima. The method for producing the same is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to pyruvate orthophosphate dikinase having excellent storage stability and cryopreservation stability in a low temperature range and high heat stability, and production thereof.

  Pyruvate orthophosphate dikinase ((Pyruvate orthophosphate dikinase (hereinafter sometimes referred to as PPDK), pyruvate phosphate dikinase, EC 2.7.9.1) is adenosine 5 ′ monophosphate in the presence of magnesium ion or the like. Acts on acid (hereinafter sometimes referred to as AMP), phosphoenolpyruvate (hereinafter sometimes referred to as PEP) and pyrophosphate (hereinafter also referred to as PPi), and adenosine 5 ′ triphosphate (Hereinafter also referred to as ATP), an enzyme that catalyzes a reaction that produces pyruvic acid and phosphoric acid (hereinafter sometimes referred to as Pi) and the reverse reaction thereof.

  PPDK is considered to be one of the enzymes constituting the C4 circuit in plants. For example, it is derived from corn leaves (Non-patent Document 1), sugar cane leaves (Non-patent Document 2), and chrysanthemum plants (non-patented). The PPDK of literature 3) has been reported. In addition, as PPDK derived from microorganisms, those derived from Propionibacterium shermanii (Non-patent document 4), Bacteroides symbiosus (Non-patent documents 5 and 6), Acetobacterylinum derived (Non-patent document 7) and the like have been reported.

  PPDK is a tetramer, but dissociates into a dimer or a monomer under a low temperature condition (12 ° C. or lower) and rapidly loses its activity. For example, corn leaf-derived PPDK loses about 80% of its activity after treatment at 0 ° C. for 20 minutes (Non-patent Document 1). In addition, inactivation of PPDK derived from chrysanthemums plants, which are relatively resistant to low temperatures, is observed at 0 ° C. (Non-patent Document 3). Therefore, as a PPDK excellent in storage stability and cryopreservation stability in a low temperature range, PPDK derived from Microvispora was developed (Patent Document 1).

  Since an enzyme is a protein, in order to prevent spoilage and denaturation, the production (purification) process is often performed at a low temperature, and when the production (purification) process is temporarily stopped, it is often stored frozen. Therefore, an enzyme having higher stability in a low temperature range and higher stability in cryopreservation is advantageous in production. From such a viewpoint, among the PPDKs disclosed in the above-mentioned documents, PPDKs derived from plants and normal temperature microorganisms have poor storage stability and cryopreservation stability in a low temperature range, which is disadvantageous in enzyme production. Microbispora-derived PPDK (Patent Document 1) is superior in storage stability and cryopreservation stability in a low temperature range, and is advantageous in production.

  By the way, as a method for producing a target enzyme, a recombinant plasmid containing a gene encoding the target enzyme is prepared, a transformant containing the recombinant plasmid is cultured in a medium, and the target enzyme is introduced into the culture. There is a production method for producing and accumulating a recombinant enzyme and collecting the target enzyme from the culture. In this production method, host microorganisms to which the recombinant plasmid is transferred include microorganisms belonging to Escherichia coli (hereinafter sometimes referred to as E. coli), microorganisms belonging to the genus Bacillus, microorganisms belonging to the genus Streptomyces, Saccharomyces・ Servicier is usually used. Since these host microorganisms usually grow at about 10 to 45 ° C., the proteins and enzymes produced and accumulated by the host microorganism are usually inactivated and easily aggregated by heat treatment at about 80 ° C. Therefore, if the target enzyme is a stable enzyme that does not inactivate or aggregate even at 80 ° C., for example, it can be easily separated from other enzymes by incorporating the heat treatment step into the production step, which is very advantageous in the production of the enzyme. .

  However, PDDK derived from the above-mentioned plants and normal temperature microorganisms is not excellent in thermal stability. Moreover, since the microvispora-derived PPDK developed as having excellent storage stability and cryopreservation stability is a thermophilic actinomycete (Non-patent Document 8), the relatively thermal stability is relatively low. Although it is high, it is still completely deactivated at 70 ° C. (Patent Document 1), so that the manufacturing superiority is insufficient. Therefore, it can be said that PPDK, which is excellent in storage stability in a low temperature range and frozen storage stability and has sufficiently high thermal stability, is very advantageous in production.

JP 08-168375 A Biochemistry 12, 2862-2867 (1973) The Biochemical Journal 114, 117-125 (1969) Plant and Cell Physiology 31, 29 5-297 (1990) Biochemistry 10,721-729 (1971) Methods in Enzymology 42, 199-212 (1975) The Biochemical Journal 125,531-539 (1971) Journal of Bacteriology 104, 211-218 (1970) Biochimica et Biophysica Acta 1431, 363-373 (1999)

  An object of the present invention is to provide PPDK which is excellent in storage stability and cryopreservation stability in a low temperature region and has high thermal stability.

  As a result of intensive research in order to obtain PPDK having excellent storage stability and cryopreservation stability in a low temperature range, which is extremely superior in the production of the enzyme, and the thermostability, the Thermotoga maritima DSM 3109 strain was obtained. Was found to be a gene encoding PPDK, and the physicochemical properties of PPDK derived from Thermotoga maritima DSM 3109 were examined. As a result, Thermotoga maritima DSM 3109 strain PPDK was preserved and frozen in a low temperature range. It has been found that this is a new PPDK having excellent storage stability and high thermal stability. Furthermore, the manufacturing method of this enzyme was established and it came to complete this invention.

  Here, the gene TM0272 of the Thermotoga maritima DSM 3109 strain has 59% identity with the PPDK gene derived from Clostridium symbiosum (Non-patent Document 8 and J. Biol. Chem. 276, 37630-37639 (2001), etc.) The nature of the protein encoded by the gene TM0272 of maritima DSM 3109 strain has not been elucidated so far. That is, Clostridium symbiosum-derived PPDK is unstable to temperature changes such as loss of activity and precipitation upon repeated freezing and thawing (Non-patent Document 5 and Adv. Enzymol. Rel. Area Mol. Biol. 45, 85-155). (1977), etc.), however, PPDK derived from Thermotoga maritima has excellent storage stability and cryopreservation stability in a low temperature range and high thermal stability, and is a very useful novel PPDK discovered for the first time by the present invention. It is.

That is, the present invention relates to the following.
1) Pyruvate orthophosphate dikinase having the following physicochemical properties:
(1) Action In the presence of magnesium ions, etc., a reaction that acts on adenosine 5 ′ monophosphate, phosphoenolpyruvate and pyrophosphate to produce adenosine 5 ′ triphosphate, pyruvate, and phosphate, and vice versa. Catalyze the reaction;
(2) Optimum pH
pH 7-7.5;
(3) pH stability Retains 80% or more of the activity in the range of pH 4.5 to 11 at 50 ° C. for 20 minutes;
(4) Thermal stability Maintains 90% or more activity after heat treatment at 80 ° C for 1 hour, and retains 70% or more activity after storage at 4 ° C for at least 27 days;
(5) When phosphoenolpyruvate and pyrophosphate are used as substrates in the presence of coenzyme-specific magnesium ions, it acts specifically as a coenzyme for adenosine 5 ′ monophosphate, and adenosine 5 ′ diphosphate (Hereinafter also referred to as ADP), inosine 5 ′ monophosphate (hereinafter sometimes referred to as IMP), cytidine 5 ′ monophosphate (hereinafter sometimes referred to as CMP), guanosine 5 ′ monophosphate Does not act on acid (hereinafter sometimes referred to as GMP), thymidine 5 ′ monophosphate (hereinafter sometimes referred to as TMP) or uridine 5 ′ monophosphate (hereinafter sometimes referred to as UMP);
(6) The relative activity is 74% when cobalt ions are used, and 29% when nickel ions are used, when the metal ions magnesium ions are used as 100%;
(7) Molecular weight The molecular weight by SDS polyacrylamide gel electrophoresis is about 83 kDa;
(8) Km value 0.32 mM for phosphoenolpyruvate, 1.12 mM for pyrophosphate, 0.065 mM for adenosine 5 ′ monophosphate;
2) The pyruvate orthophosphate dikinase according to 1), which further has the following physicochemical properties (9):
(9) The pyruvate orthophosphate dikinase according to 1) or 2) above, wherein the pyruvate orthophosphate dikinase is derived from Thermotoga maritima, which retains an activity of 90% or more after storage at −20 ° C. for 2 weeks.
4) The pyruvate orthophosphate dikinase according to any one of 1) to 3) having the following amino acid sequence (a) or (b):
(A) amino acid sequence of SEQ ID NO: 2 in the sequence listing (b) amino acid sequence in which one or several amino acids are deleted, substituted or added from the amino acid sequence of (a) 5) described in SEQ ID NO: 2 of the sequence listing A pyruvate orthophosphate dikinase represented by an amino acid sequence.
6) DNA comprising any one of the following base sequences (a) to (e):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing
(B) DNA encoding a protein having pyruvate orthophosphate dikinase activity, wherein one or more bases are deleted, substituted or added in the base sequence of (a)
(C) DNA in which 1 to 120 bases are substituted in the base sequence of (a) and which encodes a protein having pyruvate orthophosphate dikinase activity
(D) DNA in which 120 bases are substituted in the base sequence of (a) and which encodes a protein having pyruvate orthophosphate dikinase activity
(E) DNA represented by the base sequence set forth in SEQ ID NO: 6 in the sequence listing
7) A recombinant plasmid comprising the DNA of 6) above.
8) A transformant comprising the recombinant plasmid described in 7) above.
9) The transformant according to 8) above is cultured in a medium, and the pyruvate orthophosphate dikinase according to any one of 1) to 5) above is produced and accumulated in the culture, and pyruvate is produced from the culture. A method for producing pyruvate orthophosphate dikinase, which comprises collecting orthophosphate dikinase.
10) A method for producing pyruvate orthophosphate dikinase, comprising a step of heat-treating pyruvate orthophosphate dikinase collected by the method for producing pyruvate orthophosphate dikinase according to 9) above at 80 ° C. for 15 minutes or more.
11) The method for producing pyruvate orthophosphate dikinase according to 10) above, further comprising a column chromatography step using an ion exchanger.
12) The method for producing pyruvate orthophosphate dikinase according to 11) above, further comprising a hydrophobic chromatography step.
13) The method for producing pyruvate orthophosphate dikinase according to 12) above, further comprising a desalting step.
14) A pyruvate orthophosphate dikinase obtained by subjecting the transformant according to 8) above to liquid culture and the following step from the culture solution.
(A) The culture solution is collected by centrifugation, and the cells are washed with physiological saline, suspended in 20 mM HEPES / NaOH (pH 7.5) 4 times the wet weight of the cells, and ultrasonically disrupted. After that, the step of obtaining a crude enzyme solution by centrifugation (b) The step of heat-treating the crude enzyme solution at 80 ° C. for 15 minutes and obtaining the crude purified solution by centrifugation 15) Any one of 1) to 5) A reagent comprising at least one of the pyruvate orthophosphate dikinase described in the above, the DNA described in 6), the recombinant plasmid described in 7), and the transformant described in 8).

  According to the present invention, it is possible to provide pyruvate orthophosphate dikinase which is excellent in storage stability and cryopreservation stability in a low temperature region and has high heat stability. Compared to this, it can be carried out very advantageously.

  Unless otherwise specified, the reagents used in the experiments in the present invention are commercially available, such as Wako Pure Chemical Industries, Ltd., Kokusan Chemical Co., Ltd., Sigma Aldrich Co., Kanto Chemical Co., Ltd., Takara Bio Co., Ltd. We used what was available in

Pyruvate orthophosphate dikinase is EC 2.7.9.1 and is also referred to as pyruvate phosphate dikinase.
<Enzyme action>
The enzymatic action of pyruvate orthophosphate dikinase is as shown in Formula 1 in the presence of magnesium ions and the like (see Enzyme Handbook, Asakura Shoten, 1984). The reaction formula when ATP, pyruvic acid, and Pi are used as substrates is the right direction of Formula 1. The reaction equation when AMP, PEP, and PPi are used as substrates is the left direction of Equation 1. The enzyme activity of PPDK can be measured by the following PPDK activity measurement method 1 or PPDK activity measurement method 2. The following PPDK activity measurement method 1 or PPDK activity measurement method 2 was used depending on the respective characteristics.

  In the PPDK activity measurement method 1, since the first reaction is a temperature (50 ° C.) close to the optimum temperature (80 ° C.) of Thermotoga maritima, the properties close to the original properties of PPDK of the present invention can be investigated. Since trichloroacetic acid is used, it is dangerous in handling, and since the activity is measured in two steps, the operation is complicated and the activity of many samples cannot be measured at once.

  Since the PPDK activity measurement method 2 measures the activity in a single step, the activity of many samples can be measured at one time. On the other hand, in consideration of the thermal stability of lactate dehydrogenase, the measurement is performed at 37 ° C. It is not possible to investigate the nature of.

  Since the activity measurement method 1 is closer to the optimum temperature of PPDK of the present invention, the activity value is higher than that of the activity measurement method 2.

[Formula 1]
ATP + pyruvic acid + Pi <=> AMP + PEP + PPi
The PPDK activity of the present invention refers to the catalytic action of PPDK.

<PPDK activity measurement method 1>
(Reagent composition)
Reaction reagent 1-1 for measuring PPDK activity (concentration is final concentration)
100 mM HEPES / NaOH pH 7.5
5 mM PEP
2 mM AMP
5 mM MgCl2
5 mM PPi
Add enzyme solution and distilled water to make 400 μl.

Reaction reagent 1-2 for measuring PPDK activity (concentration is final concentration)
1.15M Tris / HCl pH 8.0
0.135 mM NADH
Add 400 μl of 5 U / ml lactate dehydrogenase supernatant to 1 ml.
(Measuring method)
The PPDK activity measurement method 1 is a method for measuring activity by dividing an enzyme reaction into two stages. Using the PPDK of the present invention diluted to an appropriate concentration, a reaction reagent 1-1 for measuring PPDK activity was prepared. At this time, the one prepared without adding the enzyme was designated as “blank”, and the one added with the enzyme was designated as “test”. A PPDK solution and distilled water were added to the reaction reagent 1-1 for measuring PPDK activity prepared in advance at 50 ° C. to start the reaction to 400 μl, and incubated for 10 minutes to carry out the first-stage reaction. Subsequently, this reaction solution was ice-cooled, and 50 μl of 50% trichloroacetic acid was added to stop the reaction. 400 μl of the supernatant obtained by centrifuging the reaction solution at 4 ° C. and 15,000 rpm for 10 minutes was transferred to a reaction reagent 1-2 for PPDK activity measurement prepared in advance in a new Eppendorf tube. This was incubated at 37 ° C. for 10 minutes to carry out the second stage reaction, and then the “blank” and “test” absorbances at 340 nm were measured to be Ab and At, respectively. One unit of enzyme activity (one unit) is expressed in millimolar absorbance of NADH in reaction reagent 1-2 for measuring PPDK activity as the amount of enzyme that changes 1 micromole of phosphoenolpyruvate to pyruvate per minute at 37 ° C. The coefficient was 6.22, and the enzyme activity was determined from Ab-At.
<Method 2 for measuring PPDK activity>
(Reagent composition)
Reaction reagent 2 for measuring PPDK activity
100 mM HEPES / NaOH pH 7.5
5 mM PEP
2 mM AMP
10 mM MgCl2
10 mM PPi
0.135 mM NADH
(Measuring method)
PPDK activity measurement method 2 is a method for measuring the activity of an enzyme reaction in one step. 1 ml of reaction reagent 2 for measuring PPDK activity was preheated in a quartz cell having a layer length of 1 cm at 37 ° C. for 1 minute, 1 μl of 5,000 U / ml lactate dehydrogenase was added, and the mixture was further heated for 1 minute. The enzyme reaction was started by mixing 10 μl of the PPDK of the present invention diluted to an appropriate concentration, and the difference in absorbance of NADH at 340 nm from 1 minute to 5 minutes after the start of the reaction was measured (As). One unit of enzyme activity (one unit) was determined as the amount of enzyme that changes 1 micromole of NADH per minute at 37 ° C., and the NADH millimolar extinction coefficient in the above reaction solution was 6.22. .
<Protein concentration measurement method>
The protein concentration was measured using a BioRad protein assay kit (Bradford method) according to the method described in the instruction manual, and calculated using BSA as a standard.
<Optimum pH>
The buffer solution in PPDK activity measurement method 1 (final concentration 100 mM) is a Bis-Tris / HCl buffer solution (marked with a circle in FIG. 1) for pH 6 to pH 7 measurement, and a HEPES / NaOH buffer for pH 7 to pH 8 measurement. The activity of the PPDK of the present invention was measured by changing the solution (Δ in FIG. 1) to glycylglycine / NaOH (□ in FIG. 1) for pH 8 to pH 9 measurement. Appropriate pH was measured. FIG. 1 shows the results as relative activity (%) with the maximum activity as 100%. The optimum pH of PPDK of the present invention was pH 7 to pH 7.5.
<PH stability>
The citric acid / sodium citrate buffer (circle mark in FIG. 2), pH 6.5 for 100 mM pH 4 to pH 6.5 range measurement so that the concentration of the PPDK of the present invention is 0.064 mg / ml. To pH7 range measurement, imidazole / HCl buffer (Δ in FIG. 2), pH7 to pH8 range measurement, HEPES / NaOH buffer (□ in FIG. 2), pH8 to pH9 range measurement, glycylglycine / NaOH buffer (marked with ● in Fig. 2), dissolved in glycine / NaOH buffer for pH 9 to pH 11 measurement (marked with ▲ in Fig. 2), stored at 50 ° C for 20 minutes, and the remaining activity was measured for PPDK activity The residual activity (%) is shown in FIG. 2 with the maximum activity measured by method 1 as 100%. As a result, the PPDK of the present invention retained an activity of 80% or more in the range of pH 4.5 to 11 at 50 ° C. for 20 minutes.
<Coenzyme specificity>
Activity was measured by changing AMP in PPDK activity measurement method 1 to ADP, IMP, CMP, GMP, TMP, and UMP, respectively. As a result, PPDK of the present invention acts as a coenzyme specifically for AMP when phosphoenolpyruvate and pyrophosphate are used as substrates in the presence of magnesium ions, and ADP, IMP, CMP, GMP, TMP, And did not affect UMP.
<Metal ion>
MgCl ₂ in PPDK activity measurement method 1 was changed to CoCl ₂ , NiCl ₂ , CaCl ₂ , and ZnCl ₂ , respectively, and 100 times 20 mM HEPES / NaOH (pH 7) containing 1 mM EDTA and 0.2 M sodium sulfate in advance. The activity of PPDK of the present invention dialyzed twice in 5) was measured. As a result, the PPDK of the present invention showed no activity in the absence of metal ions, in the case of CaCl ₂ and ZnCl ₂ , but in the case of CoCl ₂ , in the case of NiCl ₂ , the case of MgCl ₂ was taken as 100%. The relative activities were 74% and 29%, respectively.
<Molecular weight>
98,102 (calculated value from primary sequence of amino acids). 83,000 (measured value by SDS-PAGE (arrow in FIG. 3)).
<Km value>
The apparent Km for PEP, PPi and AMP was measured. The apparent Km is such that the concentrations of PEP, PPi and AMP in the reaction reagent 1-1 for PPDK activity measurement in PPDK activity measurement method 1 are the concentrations shown on the horizontal axes of FIGS. 4, 5, and 6, respectively. As shown in FIGS. 4, 5, and 6, it was calculated by a line Weber-Burk reciprocal plot. Apparent Km for PEP, PPi and AMP were 0.32 mM, 1.12 mM and 0.065 mM, respectively.

  As one embodiment of the present invention, the amino acid sequence constituting the PPDK of the present invention is represented by 1 to 881 of the amino acid sequence of SEQ ID NO: 2 in the sequence listing.

  In addition, the amino acid sequence constituting the PPDK of the present invention may be a sequence that expresses the same activity as the expression of enzyme activity by a polypeptide consisting of the amino acid sequence represented by 1 to 881 of the amino acid sequence of SEQ ID NO: 2 in the sequence listing. An amino acid sequence substantially equivalent to the amino acid sequence from 1 to 881 of the amino acid sequence of SEQ ID NO: 2, or a partial amino acid sequence not involved in enzyme activity expression, such as one or several Equivalents such as amino acid sequences in which amino acids are deleted, substituted or added are also included. An example of such an equivalent is a protein encoded by the DNA represented by SEQ ID NO: 6 in the Sequence Listing.

  The DNA encoding the amino acid sequence represented by amino acid sequence 1 to 881 of SEQ ID NO: 2 in the sequence listing of the present invention is an amino acid sequence including amino acid residues or polypeptide residues on the N-terminal side and C-terminal side thereof. What is necessary is just DNA which consists of any one codon among the series of codons corresponding to each amino acid.

  In addition, the DNA encoding PPDK of the present invention may be any DNA as long as it expresses the same activity as the enzyme activity expressed by the polypeptide consisting of the amino acid sequence represented by amino acid sequence 1 to 881 of SEQ ID NO: 2 in the sequence listing. DNA that encodes an amino acid sequence substantially equivalent to the amino acid sequence in amino acid sequences 1 to 881 may be used, or a sequence of a part of amino acids not involved in enzyme activity expression may be mutated, for example, 1 Codes an equivalent of an amino acid sequence in which a plurality of (for example, 40, preferably 1 to 40, more preferably 1 to 10, most preferably 1 to several) amino acids are deleted, substituted or added It may also be DNA. That is, from DNA encoding the amino acid sequence represented by amino acid sequence 1 to 881 of SEQ ID NO: 2 of the sequence listing (DNA consisting of the base sequence of SEQ ID NO: 1 of the sequence listing), 1 to plural (for example, 120, Preferably 1 to 120, more preferably 1 to 30, most preferably 1 to several) bases are deleted, substituted or added, and encodes a protein having pyruvate orthophosphate dikinase activity It may be the DNA that is being processed. Furthermore, it is represented by a base sequence having homology (for example, 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the base sequence set forth in SEQ ID NO: 1 in the sequence listing. DNA encoding a protein having pyruvate orthophosphate dikinase activity may be used. An example of such a DNA is the DNA of SEQ ID NO: 6 in the sequence listing. Furthermore, in the DNA encoding the amino acid sequence of PPDK represented by amino acid sequence 1 to 881 of SEQ ID NO: 2 in the sequence listing of the present invention, N of the amino acid sequence represented by 1 to 881 of the amino acid sequence of SEQ ID NO: 2 The terminal side and the C-terminal side may contain an amino acid residue or a polypeptide residue, and the amino acid residue includes a signal peptide or T7 tag, His tag, S tag, Trx tag, CBD tag, DsbA Tags, GST tags, Nus tags, TEE sequences and the like.

  The microorganism that is a donor of the DNA encoding PPDK is not particularly limited as long as it is a bacterium that produces PPDK, but preferably Thermotoga maritima, among which Thermotoga maritima DSM 3109 is mentioned.

  As a vector into which DNA encoding the PPDK of the present invention is incorporated, a phage or plasmid constructed for gene recombination among phages or plasmids that can autonomously grow in the host microorganism is suitable. Examples of the phage vector include Escherichia When using a microorganism belonging to E. coli as a host, λgt · λC, λgt · λB, etc. can be used. Moreover, as a plasmid vector, for example, when Escherichia coli is used as a host, pET vectors (Novagen) such as plasmids pET-3a, pET-21a, and pET-32a, or pBR322, pBR325, pACYC184, pUC12, pUC13, When pUC18, pUC19, pUC118, pINI, BluescriptKS +, Bacillus subtilis are used as hosts, pWH1520, pUB110, pKH300PLK, when actinomycetes are used as hosts, pIJ680, pIJ702, and yeasts, particularly Saccharomyces cerevisiae as hosts. YRp7, pYC1, YEp13, etc. can be used. A vector fragment is prepared by cleaving such a vector with the restriction endonuclease that produces the same end as the DNA end produced by the restriction enzyme used to cleave the DNA encoding PPDK, and the DNA fragment encoding the PPDK and the vector The fragment can be ligated by DNA ligase according to a conventional method, and DNA encoding PPDK can be incorporated into the target vector.

  The host microorganism into which the recombinant plasmid is transferred may be any microorganism as long as the recombinant DNA can be stably and autonomously propagated. For example, when the host microorganism belongs to Escherichia coli, Escherichia coli BL21, Escherichia coli BL21 ( DE3), Escherichia coli BL21trxB, Escherichia coli Rosetta (DE3), Escherichia coli Rosetta, Escherichia coli Rosetta (DE3) pLysS, Escherichia coli Rosetta (DE3) pLaclet, Escherichia coli , Escherichia coli Origami, Escherichia coli Origami, Escherichia coli Tuner, Escherichia coli DH1, Essi Rihia coli JM109, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli C600, or the like can be used. Further, when the host microorganism is a microorganism belonging to the genus Bacillus, Bacillus subtilis, Bacillus megaterium, etc., a microorganism belonging to actinomycetes, Streptomyces lividans TK24, etc., a microorganism belonging to Saccharomyces cerevisiae, Saccharomyces cerevisiae INVSC1 etc. can be used.

  In addition, the PPDK of the present invention may be mutated by a known gene manipulation means to such an extent that the nature of catalyzing the original reaction is not impaired. Such a mutant gene is a gene derived from the PPDK gene of the present invention. It means an artificially mutated gene produced by an engineering technique. This artificially mutated gene uses various genetic engineering methods such as site-specific mutagenesis and substitution of a specific DNA fragment of the target gene with an artificially mutated DNA. Obtained. It is possible to express the mutant PPDK by inserting the PPDK gene thus obtained into a vector and transferring it to a host microorganism, and it is also possible to produce a mutant PPDK having excellent properties.

  The PPDK of the present invention can be produced by culturing a microorganism that produces the PPDK of the present invention, and natural PPDK-producing microorganisms such as Thermotoga maritima DSM 3109 strain may be used. Using this method, PPDK can also be produced using a transformant into which the gene of the PPDK-producing microorganism has been introduced. When PPDK is produced using a transformant, the transformant is cultured in a nutrient medium known per se, the PPDK is produced and accumulated in the cells or in the culture solution, and the PPDK is collected from the culture. Good. When the PPDK is produced in the microbial cells, the microbial cells are collected by means of filtration or centrifugation after completion of the culture, and then the microbial cells are mechanically processed or enzymatic methods such as lysozyme. In addition, if necessary, EDTA and / or an appropriate surfactant are added to concentrate the PPDK, or adsorption concentrate such as ammonium sulfate fractionation, gel filtration, affinity chromatography without concentration. It is possible to obtain PPDK having a high purity by processing by appropriately combining chromatography, ion exchange chromatography, hydrophobic chromatography or desalting. Moreover, it is preferable to heat-process PPDK extract | collected according to these methods at 80 degreeC for 15 minutes. The culture conditions of the transformant may be selected in consideration of the nutritional physiological properties. Usually, the culture is performed by liquid culture in many cases, but industrially, it is advantageous to perform deep aeration and agitation culture. is there. As a nutrient source for the medium, those commonly used for culturing microorganisms can be widely used. The culture temperature can be appropriately changed within the range in which microorganisms serving as a host grow and produce PPDK, but in the case of Escherichia coli, it is preferably about 10 to 45 ° C, more preferably about 15 to 42 ° C. The culture conditions vary slightly depending on the conditions, but the culture may be terminated at an appropriate time in anticipation of the time when PPDK reaches the maximum yield. In the case of Escherichia coli, it is usually about 12 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce PPDK. In the case of Escherichia coli, the pH is preferably about 6 to 8.

  The enzyme of the present invention is mainly contained and accumulated in the fungus body and may be extracted from the fungus body. To illustrate the extraction method, the culture is firstly solid-liquid separated, and the obtained wet cells are dispersed in a solution such as phosphate buffer or Tris / HCl buffer, lysozyme treatment, ultrasonic treatment, French press treatment, The crude enzyme solution of the present invention is obtained by appropriately selecting and combining cell disruption means such as dynomill treatment.

  A purified enzyme is obtained from the crude enzyme solution of the present invention by means of isolation and purification of known proteins and enzymes. For example, the target enzyme is precipitated and recovered by applying a fractional precipitation method using an organic solvent such as acetone, methanol, ethanol or the like, a salting out method using ammonium sulfate, sodium chloride, or the like to the crude enzyme solution of the present invention. Furthermore, after performing dialysis and isoelectric precipitation of this precipitate as necessary, until a single band is shown by electrophoresis or the like, ion exchanger, hydrophobic chromatographic adsorbent, gel filter agent, adsorbent Purify by column chromatography etc. Moreover, when the purification degree of a target enzyme increases by combining these methods appropriately, it can carry out combining suitably. The enzyme of the present invention is a production (purification) process in which heat treatment at 80 ° C. particularly increases the degree of purification.

  Enzymes obtained by these methods can be used as stabilizers in the form of a liquid or a mixture of various salts, saccharides, proteins, lipids, sea surface activators, etc., with or without addition, by methods such as ultrafiltration concentration and freeze-drying. Solid PPDK can be obtained, and may be freeze-dried as appropriate. In this case, saccharose, mannitol, sodium chloride, albumin or the like may be added as a stabilizer in an amount of about 0.5 to 10%.

  The PPDK of the present invention is a novel enzyme having properties different from those of known enzymes that catalyze the same reaction, and exhibits higher stability especially at lower and higher temperatures than known enzymes.

The reagent of the present invention is a reagent comprising at least one of pyruvate orthophosphate dikinase of the present invention, DNA of the present invention, recombinant plasmid of the present invention, and transformant of the present invention. In addition, the reagent of the present invention includes a buffer solution, a reaction stop solution, a reaction product detection agent (eg, lactate dehydrogenase, NADH, etc.), a reaction diluent, adenosine 5′-phosphate, and a metal ion (eg, magnesium). (Ion, cobalt ion, nickel ion) and other substances necessary for carrying out the measurement using a reagent. This reagent can be used, for example, as a reagent for measuring AMP or PEP in a sample.
EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, the scope of the present invention is not limited to a following example. In the present specification, a technique described in accordance with a conventional method includes, for example, the method of Maniatis et al. (See, for example, Maniatis, T., et al. Molecular Cloning. Cold Spring Harbor Laboratory 1982, 1989), and proteins and enzymes. Can be carried out according to the procedures attached to the basic experimental methods (see, for example, revised second edition, Takeichi Horio, Nankodo, 1994) or various commercially available enzymes and kits.

[Example 1]
<Extraction of DNA>
Cell of Thermotoga maritima DSM 3109 purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH with 50 mM Tris / HCl (pH 8.0), 10 mM EDTA, 15 mg sucrose solution After that, SDS was added to a final concentration of 0.25% to dissolve the cells. Further, an equal volume of a 1: 1 mixture of phenol / chloroform was added, stirred for 30 minutes, and then centrifuged (12,000 rpm, 15 minutes) to recover the aqueous layer. A 1/10 volume of 3M sodium acetate (pH 5.5) was mixed with the recovered aqueous layer, and then twice the amount of ethanol was gently overlaid, and the genomic DNA was wrapped around a glass rod and separated. Dissolve the separated genomic DNA in 10 mM Tris / HCl (pH 8.0), 1 mM EDTA aqueous solution (hereinafter sometimes referred to as TE), add an appropriate amount of RNase A, incubate at 37 ° C. for 1 hour, and mix RNA was degraded. Next, an equal amount of a 1: 1 mixture of phenol / chloroform was added and treated in the same manner as above to separate the aqueous layer. One-tenth volume of 3M sodium acetate (pH 5.5) and twice the volume of ethanol were added to the collected aqueous layer, and genomic DNA was separated once again by the method described above. This chromosome was dissolved in TE, a 1: 1 mixture of TE-saturated phenol and chloroform was added to suspend the whole, and the same centrifugation was repeated, and the upper layer was again transferred to another container. To the separated upper layer, 3M sodium acetate buffer (pH 5.5) and ethanol were added, stirred, cooled at -70 ° C for 5 minutes, centrifuged (2,000 G, 4 ° C, 15 minutes), and precipitated. The chromosomes were washed with 75% ethanol and dried under reduced pressure. The DNA preparation of Thermotoga maritima DSM 3109 strain was obtained by the above operation.

[Example 2]
<Amplification of gene shown in SEQ ID NO: 1 by PCR method>
The primers of SEQ ID NO: 3 and SEQ ID NO: 4 were designed so that the gene described in SEQ ID NO: 1 in the sequence listing was inserted into the multicloning site NdeI and HindIII sites of pET21a (+) or pET28a (+) (Novagen). When pUC118 was used, the primers of SEQ ID NO: 4 and SEQ ID NO: 5 were designed so that the gene described in SEQ ID NO: 1 was inserted into the XbaI and HindIII sites at the multiple cloning site. PCR was performed according to a conventional method using KOD DNA polymerase. The obtained approximately 2.7 kbp PCR product was purified according to a conventional method such as using QIAGEN QIAquick PCR Purification Kit.

[Example 3]
<Ligation with expression vector>
The PCR product using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 purified in Example 2 was subjected to restriction enzyme treatment with NdeI and HindIII according to a conventional method (insert A). The PCR product using the primers of SEQ ID NO: 4 and SEQ ID NO: 5 purified in Example 2 was subjected to restriction enzyme treatment with XbaI and HindIII according to a conventional method (insert B). These inserts A and B were purified by a conventional method such as using QIAGEN QIAquick PCR Purification Kit. Insert A was ligated with pET21a (+) or pET28a (+) purified by restriction enzyme treatment with NdeI and HindIII according to a conventional method to prepare pET21a (+) / TM0272 or pET28a (+) / TM0272. Insert B was ligated with pUC118 purified by restriction enzyme treatment with XbaI and HindIII according to a conventional method to prepare pUC118 / TM0272. The recombinant plasmid purified from the positive clone by the colony direct PCR method was DNA sequenced to confirm that the insert sequence was correct.

[Example 4]
<Expression check of pET21a (+) / TM0272, pET28a (+) / TM0272 or pUC118 / TM0272>
pET21a (+) / TM0272, pET28a (+) / TM0272 or Escherichia coli BL21 (DE3), pUC118 / TM0272 was transformed into Escherichia coli JM109 according to a conventional method, and SB medium containing 50 μg / ml ampicillin ( Dissolve 12 g of tryptone, 24 g of yeast extract and 5 ml of glycerol in 0.9 L of distilled water and sterilize for 20 minutes at 121 ° C. Dissolve 12.5 g of potassium monohydrogen phosphate and 3.8 g of potassium dihydrogen phosphate in 0.1 L of distilled water. Sterilized for 20 minutes at 121 ° C. Each was cooled to room temperature and mixed to obtain 1 L of SB medium. After culturing at 37 ° C. for 3 hours, IPTG was added to a final concentration of 1 mM, and further cultured for 3 hours. The culture is centrifuged to collect the cells, and the cells are washed with physiological saline, suspended in 20 mM HEPES / NaOH (pH 7.5) 4 times the wet weight of the cells, sonicated and centrifuged. The supernatant obtained after separation was used as a crude enzyme solution.

[Example 5]
<Heat treatment of crude enzyme solution>
The crude enzyme solution obtained in Example 4 was heat-treated at 80 ° C. for 15 minutes and centrifuged, and the supernatant was used as a crude purified solution. SDS-PAGE of this crude purified solution is shown in FIG.

[Example 6]
<PDK purification 1>
The crude purified solution of Example 5 was applied to a BioRad UnoQ® anion exchange chromatography column equilibrated with 20 mM HEPES / NaOH (pH 7.5). After washing the column with 20 mM HEPES / NaOH (pH 7.5) at 5 times the bed volume, 20 mM HEPES / NaOH (pH 7.5) containing 20 mM HEPES / NaOH (pH 7.5) and 0.5 M NaCl is used. Linear gradient elution was carried out 12 times the bed volume. The active fraction was collected and desalted by dialysis against 100 volumes of 20 mM HEPES / NaOH (pH 7.5).

[Example 7]
<PDK purification 2>
The desalted PPDK of Example 6 was applied to a column using Hydrorad hydroxyapatite equilibrated with 10 mM potassium phosphate (pH 7.5). The column was washed with 10 mM potassium phosphate (pH 7.5) having a bed volume of 5 times or more, and then linear gradient elution was carried out with 12 times the bed volume using 300 mM potassium phosphate (pH 7.5). The active fraction was collected and desalted by dialyzing against 100 volumes of 20 mM HEPES / NaOH (pH 7.5) containing 1 mM DTT to obtain a purified enzyme.

[Example 8]
<PDK purification 3>
The desalted PPDK of Example 6 was applied to a column using Hydrorad hydroxyapatite equilibrated with 10 mM potassium phosphate (pH 7.5). The column was washed with 10 mM potassium phosphate (pH 7.5) having a bed volume of 5 times or more, and then linear gradient elution was carried out with 12 times the bed volume using 300 mM potassium phosphate (pH 7.5). The active fraction was collected and desalted using G-25 (manufactured by GE Healthcare Bioscience), a gel filtration agent equilibrated with 20 mM HEPES / NaOH containing 1 mM DTT, to obtain a purified enzyme.

In Example 4, 500 ml of Escherichia coli JM109 transformed with pUC118 / TM0272 was cultured, and the resulting crude enzyme solution was purified according to Examples 5 to 7, and the PDDK activity obtained in each purification step was measured. The results are shown in Table 1. PPDK activity was measured by PPDK activity measurement method 1. From this, it can be seen that the specific activity is improved 11 times or more by heat-treating the crude enzyme solution at 80 ° C. for 15 minutes and centrifuging, and that the heat-treatment process is a very effective production (purification) process.

[Example 9]
<PDK purification 4>
In Example 4, 500 ml of Escherichia coli BL21 (DE3) transformed with pET28a (+) / TM0272 was cultured, and a crude purified solution was obtained by the method described in Example 5 using the obtained crude enzyme solution. In some cases, the following purification was also performed.

  The above crude purified solution was applied to a Sigma-Aldrich nickel affinity gel chromatography column equilibrated with 20 mM HEPES / NaOH (pH 7.5) containing 0.2 M NaCl. The column was washed with 20 mM HEPES / NaOH (pH 7.5) containing 0.2 M NaCl at least 5 times the bed volume, and then 20 mM HEPES / NaOH (pH 7.5) containing 0.2 M NaCl and 0.2 M imidazole. ) To elute the adsorbed fraction.

  20% ammonium sulfate was added to the eluted active fraction and applied to a phenyl Toyopearl chromatography column (manufactured by Tosoh Corporation) equilibrated with 20 mM HEPES / NaOH (pH 7.5) containing 20% ammonium sulfate. After washing the column with 20 mM HEPES / NaOH (pH 7.5) containing 20% ammonium sulfate at least 5 times the bed volume, linear gradient elution of 12 times the bed volume with 20 mM HEPES / NaOH (pH 7.5) Went. A portion of the eluted active fraction was desalted by dialysis against 20 mM HEPES / NaOH (pH 7.5) containing 100 volumes of 1 mM DTT. Alternatively, desalting was performed using G-25, a gel filtration agent equilibrated with 20 mM HEPES / NaOH containing 1 mM DTT. The specific activity of PPDK obtained in this example when desalted by dialysis and when desalted using G-25 was 7 U / mg.

[Example 10]
<Confirmation of PPDK activity>
The activity of the PPDK of the present invention was confirmed by detecting ATP produced by the reaction when AMP was used as a substrate by high performance liquid chromatography (hereinafter sometimes referred to as HPLC). 10 μl of PPDK obtained in Example 7 was added to a reaction solution composed of 100 mM HEPES / NaOH (pH 7.5), 1 mM AMP, 1 mM MgCl 2, 1 mM PEP, 1 mM PPi, and reacted at 50 ° C. for 20 minutes. The reaction solution was analyzed by HPLC using Asahipak GS-320 (manufactured by Showa Denko KK). The mobile phase was 200 mM sodium phosphate (pH 3), the flow rate was 0.5 ml / min, the column temperature was room temperature, and the absorbance at 260 nm was measured. FIG. 7 shows the result of separating ATP, ADP, AMP and adenosine under these conditions. FIG. 8 shows the result of separating the reaction solution before the reaction. FIG. 9 shows the result of separating the reaction solution after the reaction. As apparent from these analysis results, when AMP was used as a substrate, ATP was generated by the reaction, and PPDK activity was confirmed.

[Example 11]
<PPDK frozen storage stability>
The non-desalted PPDK obtained in Example 9 was stored frozen at −20 ° C. as it was and thawed after 2 weeks. As a result of comparing the activities before and after cryopreservation, there was 94% residual activity even after cryopreservation. It was shown to be excellent in frozen storage stability. PPDK activity was measured by PPDK activity measurement method 2.

[Example 12]
<PPDK refrigerated storage stability>
FIG. 10 shows the results of measuring the activity of the non-desalted PPDK obtained in Example 9 as it was at 4 ° C. and after 0, 5, 8, 12, 19, and 27 days. As a result, it was shown that the PPDK of the present invention has a residual activity of 80% or more even after storage for 27 days under refrigerated storage and is excellent in storage stability in a low temperature range. PPDK activity was measured by PPDK activity measurement method 1.
[Example 13]
<Thermal stability 1>
213 μg / ml of desalted PPDK obtained in Example 7 was heat-treated in the range of 50 ° C. to 100 ° C. for 20 minutes. The residual activity was measured by PPDK activity measuring method 1, and the residual activity (%) is shown in FIG. 11 with the untreated enzyme activity as 100%. As a result, the PPDK of the present invention retained 90% or more of the activity after heat treatment at 90 ° C. for 20 minutes.

[Example 14]
<Thermal stability 2>
213 μg / ml of the desalted PPDK obtained in Example 7 was heat-treated at 80 ° C. or 90 ° C. for 0 to 1 hour. Residual activity was measured by PPDK activity measurement method 1, and the residual activity (%) is shown in FIG. As a result, the PPDK of the present invention retained an activity of 70% or more by heat treatment at 90 ° C. for 1 hour, and retained 90% or more activity by heat treatment at 80 ° C. for 1 hour.
[Example 15]
<Improvement of PPDK expression>
Among the genes shown in SEQ ID NO: 1 in the Sequence Listing, all codons (AGA, AGG, ATA, CGG, CCC, CUA) that are considered to be less frequently used in E. coli are converted into codons that are frequently used in E. coli. The gene of SEQ ID NO: 6 in the column table was created. Codon conversion was performed by a conventional method using PCR using a synthetic primer, and a crude enzyme solution was obtained in the same manner as in Examples 2, 3, and 4. This crude enzyme solution was further heat-treated in the same manner as in Example 5 to obtain a heat-treated crude purified solution. By subjecting this crude purified solution to SDS-PAGE, the expression level of PPDK by the gene of SEQ ID NO: 1 in the sequence listing (FIG. 13A) and the expression level of PPDK by the gene of SEQ ID NO: 6 in the sequence listing (FIG. 13). (B)) was compared. The expression level of PPDK by the gene of SEQ ID NO: 6 in the sequence listing was improved compared to the expression level of PPDK by the gene of SEQ ID NO: 1 in the sequence listing. Moreover, the culture titer of PPDK by the gene of SEQ ID NO: 6 in the sequence listing was about 3 times higher than the culture titer of PPDK by the gene of SEQ ID NO: 1 in the sequence listing.

  Since this enzyme is a pyruvate orthophosphate dikinase which is excellent in storage stability and cryopreservation stability in a low temperature region and has high heat stability, it can be efficiently produced.

The optimum pH of the pyruvate orthophosphate dikinase of the present invention is shown. Figure 2 shows the pH stability of pyruvate orthophosphate dikinase of the present invention. 1 shows SDS-PAGE of pyruvate orthophosphate dikinase of the present invention. The position of the pyruvate orthophosphate dikinase of the present invention is indicated by an arrow. FIG. 2 shows a Reinweber-Burk reciprocal plot for PEP of pyruvate orthophosphate dikinase of the present invention. FIG. 2 shows a Reinweber-Burk reciprocal plot for PPi of pyruvate orthophosphate dikinase of the present invention. FIG. 2 shows a Reinweber-Burk reciprocal plot for AMP of pyruvate orthophosphate dikinase of the present invention. The result of having isolate | separated ATP, ADP, AMP, and adenosine on the HPLC conditions of Example 10 is shown. The result of having separated before the reaction of the reaction liquid described in Example 10 under the HPLC conditions described in Example 10 is shown. The result of separating the reaction solution obtained by adding 10 μl of PPDK obtained in Example 7 to the reaction solution described in Example 10 and reacting at 50 ° C. for 20 minutes under the HPLC conditions described in Example 10 is shown. The stability of the pyruvate orthophosphate dikinase of the present invention stored at refrigeration (4 ° C.) is shown. Figure 2 shows the thermal stability of the pyruvate orthophosphate dikinase of the present invention. Figure 2 shows the thermal stability of the pyruvate orthophosphate dikinase of the present invention. 7 shows SDS-PAGE in which the expression level (A) of PPDK by the gene of SEQ ID NO: 1 in the sequence listing is compared with the expression level (B) of PPDK by the gene of SEQ ID NO: 6 in the sequence listing.

Claims (15)

A pyruvate orthophosphate dikinase having the following physicochemical properties:
(1) Action In the presence of magnesium ions, etc., a reaction that acts on adenosine 5 ′ monophosphate, phosphoenolpyruvate and pyrophosphate to produce adenosine 5 ′ triphosphate, pyruvate, and phosphate, and vice versa. Catalyze the reaction;
(2) Optimum pH
pH 7-7.5;
(3) pH stability Retains 80% or more of the activity in the range of pH 4.5 to 11 at 50 ° C. for 20 minutes;
(4) Thermal stability Maintains 90% or more activity after heat treatment at 80 ° C for 1 hour, and retains 70% or more activity after storage at 4 ° C for at least 27 days;
(5) When phosphoenolpyruvate and pyrophosphate are used as substrates in the presence of coenzyme-specific magnesium ions, it acts specifically as a coenzyme for adenosine 5 ′ monophosphate, and adenosine 5 ′ diphosphate Does not act on inosine 5 ′ monophosphate, cytidine 5 ′ monophosphate, guanosine 5 ′ monophosphate, thymidine 5 ′ monophosphate or uridine 5 ′ monophosphate;
(6) The relative activity is 74% when cobalt ions are used, and 29% when nickel ions are used, when the metal ions magnesium ions are used as 100%;
(7) Molecular weight The molecular weight by SDS polyacrylamide gel electrophoresis is about 83 kDa;
(8) Km value 0.32 mM for phosphoenolpyruvate, 1.12 mM for pyrophosphate, 0.065 mM for adenosine 5 ′ monophosphate; The pyruvate orthophosphate dikinase according to claim 1, which further has the following physicochemical properties (9):
(9) Maintain 90% or more activity after 2 weeks storage at -20 ° C The pyruvate orthophosphate dikinase according to claim 1 or 2, which is derived from Thermotoga maritima. The pyruvate orthophosphate dikinase according to any one of claims 1 to 3, which has the following amino acid sequence (a) or (b):
(A) Amino acid sequence of SEQ ID NO: 2 in the sequence listing (b) Amino acid sequence in which one or several amino acids are deleted, substituted or added from the amino acid sequence of (a) A pyruvate orthophosphate dikinase represented by the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. DNA comprising any one of the following base sequences (a) to (d):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing
(B) DNA encoding a protein having pyruvate orthophosphate dikinase activity, wherein one or more bases are deleted, substituted or added in the base sequence of (a)
(C) DNA in which 1 to 120 bases are substituted in the base sequence of (a) and which encodes a protein having pyruvate orthophosphate dikinase activity
(D) DNA represented by the base sequence set forth in SEQ ID NO: 6 in the sequence listing A recombinant plasmid comprising the DNA according to claim 6. A transformant comprising the recombinant plasmid according to claim 7. The transformant according to claim 8 is cultured in a medium, the pyruvate orthophosphate dikinase according to any one of claims 1 to 5 is produced and accumulated in the culture, and the pyruvate orthophosphate dikinase is accumulated from the culture. A method for producing pyruvate orthophosphate dikinase, which comprises collecting a kinase. The manufacturing method of pyruvate orthophosphate dikinase which has the process of heat-treating pyruvate orthophosphate dikinase extract | collected with the manufacturing method of pyruvate orthophosphate dikinase of Claim 9 at 80 degreeC or more for 15 minutes or more. The method for producing pyruvate orthophosphate dikinase according to claim 10, further comprising a column chromatography step using an ion exchanger. The method for producing pyruvate orthophosphate dikinase according to claim 11, further comprising a hydrophobic chromatography step. The method for producing pyruvate orthophosphate dikinase according to claim 12, further comprising a desalting step. Pyruvate orthophosphate dikinase obtained by subjecting the transformant according to claim 8 to liquid culture and the following step from the culture solution.
(A) The culture solution is collected by centrifugation, and the cells are washed with physiological saline, suspended in 20 mM HEPES / NaOH (pH 7.5) 4 times the wet weight of the cells, and ultrasonically disrupted. (B) A step of heat-treating the crude enzyme solution at 80 ° C. for 15 minutes and centrifuging to obtain a crude purified solution At least among the pyruvate orthophosphate dikinase according to any one of claims 1 to 5, the DNA according to claim 6, the recombinant plasmid according to claim 7, and the transformant according to claim 8. Reagents containing either.