JP2004298184A - Novel thermostable protein with phosphoglycerate kinase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel thermostable protein having phosphoglycerate kinase activity.
SOLUTION: A protein having a specific amino acid sequence has phosphoglycerate kinase activity. This protein is a hyperthermophilic archaeon and is presumed to exhibit thermostable phosphoglycerate kinase activity from the gene sequence of Sulfolobus tokodaii sp. 7 (JCM10545), which is one of aerobic thermoacidophilic crenarchaeons. This gene was obtained by cloning the gene and expressing it using Escherichia coli.
[Selection diagram] Fig. 1

Description

  The present invention relates to a novel thermostable protein having phosphoglycerate kinase activity. This application is an application for a result commissioned by the government.

Phosphoglycerate kinase (EC 2.7.2.3.) Acts on 3-phospho-D-glycerate in the presence of ATP in glycolysis to convert D-1,3-bisphosphoglycerate and ADP. It is one of the phosphotransferases produced. Such enzymes are produced from many sources and are reported in large numbers (see Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, etc.). As phosphoglycerate kinase derived from thermophilic archae, those derived from Methanobacterium bryantii, those derived from Methanothermus fervidus (see Non-Patent Document 6), and Pyrococcus wosei ( Pyrococcus woesei). ) (Non-Patent Documents 7 and 8) and the like.

On the other hand, there is a study on hyperthermophilic archaea (see Non-Patent Document 9), and Sulfolobus tokodaii (JCM10545) (see Non-Patent Document 10), which is a kind of bacteria belonging to the genus Sulfolobus , has the gene thereof. It has already been analyzed (see Non-Patent Document 11). Therefore, if this hyperthermophilic archaea produces phosphoglycerate kinase, it is expected to have excellent thermostability and may have different functions and activities than those from the aforementioned thermophilic archaea. There is.
J. Biol. Chem. 204 (1953) 939-948 Biochim. Biophys. Acta (1947) 292-314 Biochim. Biophys. Acta 65 (1962) 355-357 Biochem. J. 81 (1961) 405-411 Biocheim. Biophys. Acta, 1998 Jul 28; 1386 (1): 179-88 Gene, 91, 19-25 (1990) Gene, 172, 121, (1996) Eur. J. Biochem., 1995 Ocr 1; 233 (1): 227-37 Advances in Protetin Chemistry, Volume 48, Enzymes and Proteins from Hyperthermophilic Microorganisms (M. Adams ed.), Academic Press (1996) Suzuki, T. et al., Extremophiles, 2002 Feb; 6 (1): 39-44 Kawarabayashi, Y. et al., “Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7”, DNA Res. 8 (4), 123-140 (2001)

  The present invention has been made in view of such circumstances, and an object thereof is to provide a novel heat-resistant protein having phosphoglycerate kinase activity.

In order to achieve the above object, the present inventors examined the genome information of Sulfolobus tokodaii (JCM10545), a hyperthermophilic archaeon, and found that this bacterium may produce phosphoglycerate kinase. I found it. As a result of further studies based on this finding, the inventors succeeded in expressing a novel protein having phosphoglycerate kinase activity from this bacterial gene, and reached the present invention. Sulfolobus tokodaii (JCM10545) is stored in the Microbial Strain Preservation Facility of the RIKEN Bio-Institute for Biological Research, and can be purchased at the request of a third party. Since the growth temperature of Sulfolobus tokodaii (JCM10545) is 80 ° C and the growth limit temperature is 87 ° C, the protein of the present invention is active even at a high temperature of 80 to 87 ° C. In addition, while the aforementioned thermophilic archae is anaerobic, Sulfolobus tokodaii (JCM10545) is aerobic, so that phosphoglycerate kinase derived from this strain is different from the conventional one. May have function and activity.

That is, the protein of the present invention is the following protein (a) or (b).
(A) A thermostable protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) a heat-resistant protein having phosphoglycerate kinase activity, wherein the amino acid sequence of SEQ ID NO: 1 has one or more amino acid residues deleted, substituted, added or inserted;

  According to the present invention, a novel thermostable protein having phosphoglycerate kinase activity can be provided.

  The phosphoglycerate kinase activity is a function of acting on 3-phospho-D-glycerate in the presence of ATP to produce D-1,3-bisphosphoglycerate and ADP. The protein of the present invention may catalyze a reverse reaction of the above reaction.

As described above, the novel thermostable protein of the present invention is derived from hyperthermophilic archaebacteria, specifically, from Sulfolobus tokodaii (JCM10545). However, the protein of the present invention is not limited to the protein produced by this fungus, and may be produced by other organisms by genetic engineering techniques.

  Next, the expression vector of the present invention is a vector containing the DNA encoding the protein of the present invention or the DNA of SEQ ID NO: 2.

  Next, the transformant of the present invention is a transformant transformed with the vector of the present invention. The host is not particularly limited, and examples include Escherichia coli.

  Next, the method for producing a protein of the present invention is a production method comprising a step of culturing the transformant of the present invention and a step of collecting the protein expressed in the culturing step.

  Next, the production method of the present invention is a method for producing D-1,3-bisphosphoglycerate and ADP from 3-phospho-D-glycerate in the presence of ATP by an enzymatic reaction, This is a production method in which the above-mentioned protein of the present invention is used as an enzyme, and the above-mentioned enzyme reaction is carried out at a temperature of 80 to 90 ° C.

  Another production method of the present invention is a method for producing 3-phospho-D-glyceric acid and ATP from D-1,3-bisphosphoglycerate in the presence of ADP by an enzymatic reaction, This is a production method in which the above-mentioned protein of the present invention is used as an enzyme, and the above-mentioned enzyme reaction is carried out at a temperature of 80 to 90 ° C.

  As described above, by using the protein of the present invention, the enzyme reaction can be carried out in a high temperature range of 80 to 90 ° C., and as a result, the industrial application of phosphoglycerate kinase is expanded. In addition, in this production method, the pH of the enzyme reaction is preferably in a range of pH 3 to 9.

  In the two production methods of the present invention, the enzyme reaction may be stopped for a certain period of time by a chelating agent. As the chelating agent, ethylenediaminetetraacetic acid (EDTA) is preferable.

  Hereinafter, the present invention will be described in more detail.

The present inventors have determined from the gene sequence of Sulfolobus tokodaii sp. 7 (JCM10545), a hyperthermophilic archaea collected from the ocean floor, which is one of aerobic thermoacidophilic crenarchaeons. The novel heat-resistant protein of the present invention was obtained by cloning a gene (SEQ ID NO: 2) presumed to exhibit synthase activity and expressing it using Escherichia coli. The method for cloning the gene was carried out as described in Example 1 below. The nucleotide sequence of the cloned gene is as shown in SEQ ID NO: 2, and its deduced amino acid sequence is as shown in SEQ ID NO: 1. In addition, if the heat-resistant protein of the present invention has phosphoglycerate kinase activity, one or more or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 may be missing, substituted, added or inserted. It may be. The “deletion, substitution, addition or insertion of an amino acid” in the amino acid sequence can be performed according to a method known to those skilled in the art (for example, a mutagenesis method).

  The protein of the present invention can be produced by the above-described method for producing a protein of the present invention, but is not limited thereto, and may be produced by another production method. For example, as shown in SEQ ID NO: 1, for a protein whose amino acid sequence has been determined, a method known to those skilled in the art based on the sequence, for example, a method of chemically polymerizing individual amino acids to synthesize a protein Can be prepared.

An example of a gene encoding the protein of the present invention is the gene shown in SEQ ID NO: 2. The gene uses, for example, a part of the base sequence represented by SEQ ID NO: 2 from the genome of the hyperthermophilic archaeon Sulfolobus tokodaii (JCM10545) as shown in Example 2 described below as a primer. It can be prepared by a PCR method or a hybridization method using the DNA fragment as a probe. In addition, based on the nucleotide sequence, the gene can be obtained according to a nucleic acid chemical synthesis method known to those skilled in the art, but is not limited thereto.

  The expression vector of the present invention can be obtained by inserting the gene or the DNA of SEQ ID NO: 2 into an appropriate vector. The vector into which the gene of the present invention is inserted is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA, phage DNA, and baculovirus such as AcMNPV. Plasmid DNA can be prepared from Escherichia coli or Agrobacterium by an alkali extraction method or a modification thereof. As a commercially available plasmid, for example, pET-11a (manufactured by Novagen) or a secretory plasmid using a Bacillus host may be used. These plasmids may contain an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and the like.

  Insertion of a gene or the like into a vector includes, for example, a method in which the base sequence of a purified gene is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA at a restriction enzyme site or a multiple cloning site, and ligated to a vector. It can be used, but is not limited thereto. Further, in order to exert the function of the gene of the present invention, the expression vector of the present invention may incorporate a promoter, a terminator, a ribosome binding sequence, and the like in addition to the gene of the present invention. Furthermore, the gene of the present invention may be inserted with a fusion of a sequence encoded by another protein.

  By transforming a host organism with the expression vector, the transformant of the present invention can be obtained. The host organism is not particularly limited as long as it can express the gene of the present invention, and includes, for example, prokaryotic cells such as Escherichia coli, but is not limited thereto. As a transformation method, a known calcium chloride method or the like can be used, but is not limited to these methods.

  The method for producing a protein of the present invention includes a step of culturing the transformant and a step of collecting the protein expressed in the culturing step. The culturing method is performed according to a usual method used for culturing host cells. As a medium for culturing a transformant using a microorganism such as Escherichia coli as a host, a medium containing a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganism can be used as long as the transformant can be efficiently cultured. Any of natural media, synthetic media and the like may be used. The recovery of the protein of the present invention is not particularly limited. When the protein is produced in cells or cells, the cells are recovered by crushing the cells or cells. When the protein of the present invention is produced extracellularly or extracellularly, the culture solution may be used as it is, or after the bacterial cells or cells are removed by centrifugation or the like, used for protein isolation and purification. Isolation and purification of the protein of the present invention from the culture by using common biochemical methods, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. can do. When the culture solution is used as it is, heat treatment deactivates proteins other than the protein of the present invention, so that it can be used substantially as an enzyme solution containing only the protein of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Preparation of chromosomal DNA 10 mL of SSC solution (0.15 M NaCl, 0.015 M sodium citrate) was collected by culturing Sulfolobus tokodaii (JCM10545) overnight at 37 ° C. in L medium and collecting the cells. , 0.5 M EDTA, 100 mg / ml Chicken egg white lysozyme 0.1 mL and 10% nonionic surfactant Brij-58 were added in an amount of 0.5 mL, allowed to stand at 0 ° C. for 30 minutes, and then treated with proteinase K (Merck) 5 mg. Was dissolved in 0.2 mL of 10% SDS and left at 37 ° C. for a few days. A mixed solution of water-saturated phenol, chloroform and isoamyl alcohol was added to this solution, and the mixture was allowed to stand at 37 ° C. for 1 hour. The aqueous layer was separated, and ethanol was added thereto to precipitate and concentrate DNA. This DNA precipitate was dissolved in 10 mL of a TE solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0)), and 0.25 mL of ribonuclease (final concentration 0.25 mg / mL) was added thereto. , And precipitated with ethanol. Next, after dissolving the DNA in 5 mL of the TE solution, the DNA concentration was determined from the absorbance at 260 nm (Clarke, L. & Carbon, J. (1979) Methods Enzymol. 68, 396-408).

Construction of expression plasmid and gene expression Construction of Expression Plasmid The following DNA primers were synthesized for the purpose of constructing a DNA containing the restriction enzymes BamHI and NotI sites behind the translation region of the heat-resistant phosphoglycerate kinase gene, and the heat-resistant phosphoglycerin was subjected to PCR using this primer. A restriction enzyme site was introduced after the translation region of the acid kinase gene. The DNA polymerase used was KOD-plus- (Toyobo).
Forwadr primer (SEQ ID NO: 3): 5'-TGATCAAAGTAGCTGACCTTAATATTCCTGTT-3 '
Reverse primer (SEQ ID NO: 4): 5'-ATATGGATCCGCGGCCGCTTATTAACTTTTTCCACTTTGCAT-3 '

  After the PCR reaction, the amplified fragment was digested with the restriction enzyme BamHI, and the gene fragment was purified. pET-11a (manufactured by Novagen) was digested with the restriction enzyme NdeI, the ends were blunt-ended with Klenow fragment, and further digested with the restriction enzyme BamHI was purified, and purified with the above structural gene and T4 ligase at 15 ° C., 30 ° C. The reaction was allowed to take place for minutes and ligated. A part of the ligated DNA was introduced into competent cells of Escherichia coli DH5α, spread on an LB agar plate containing ampicillin in an appropriate amount, and cultured at 37 ° C. overnight to obtain a transformant colony. The obtained transformant was cultured for 24 hours in an LB medium (18 mL) containing ampicillin, and the expression plasmid was purified from the culture by a modified alkaline SDS method. The nucleotide sequence of the insert of the purified plasmid was determined using a BigDye Terminator kit (registered trademark: manufactured by Applied Biosystems) and ABI PRISM 3700 DNA Analyzer (registered trademark: manufactured by Applied Biosystems). The correct sequence of the phosphoglycerate kinase gene was confirmed.

2. Expression of recombinant gene Competent cells of Escherichia coli Rosetta-gami (DE3) (Novagen) were thawed and transferred to a Falcon tube at 0.1 mL. Among them, 1. After adding 0.002 mL of the purified expression plasmid solution and leaving it on ice for 20 minutes, heat shock at 42 ° C. for 90 seconds, leaving it on ice for 1 minute, and then LB agar plate containing chloramphenicol and ampicillin , And cultured overnight at 37 ° C to obtain a transformant. The obtained transformant was cultured in an LB medium (5 mL) containing ampicillin for 18 hours to express a thermostable phosphoglycerate kinase gene. After the culture, the cells were collected by centrifugation (13,000 G, 10 minutes).

  0.2 mL of a disrupted solution (20 mM Tris-HCl, 100 mM KCl, pH 7.5) was added to the collected cells, the cells were disrupted with an ultrasonic generator, and the suspension was added in two 0.1 mL portions. Divided into sample tubes. One sample tube was centrifuged (13,000 G, 10 minutes) to separate into a supernatant and a precipitate, and the precipitate was suspended in 0.1 mL of a crushed liquid. The other sample tube was subjected to heat treatment (70 ° C., 10 minutes) and then centrifuged (13,000 G, 10 minutes) to separate into a supernatant and a precipitate, and the precipitate was suspended in 0.1 mL of a crushed liquid. . Some of these samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) to confirm the expression. The results are shown in the SDS-PAGE photograph of FIG.

  After performing SDS polyacrylamide gel electrophoresis on a sample in which the expression of thermostable phosphoglycerate kinase was observed, the sample was transferred to a PVDF membrane by electroblotting and visualized by staining. The band of the kinase was cut out, and the amino-terminal sequence was analyzed using a protein sequencer Model492Procise (manufactured by Applied Biosystems). As a result, the amino-terminal sequence of 6 residues was determined as shown in SEQ ID NO: 5. From this sequence, it was confirmed that the expressed protein was a thermostable phosphoglycerate kinase. This expressed protein was composed of 415 amino acid residues, and had an estimated molecular weight of 45.4 kD, which almost coincided with the result of FIG.

Mass cultivation of recombinant Escherichia coli Using the plasmid DNA (pET-11a vector) prepared in the same manner as in Example 2, Escherichia coli DH5α strain was transformed according to a conventional method. Plasmid DNA was extracted from the transformed Escherichia coli DH5α using the alkaline SDS method. This plasmid DNA was used to transform Escherichia coli Rosetta-Gami (DE3) strain. Three loops of the colony growing on the plate were weighed and 5 mL of LBL medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% lactose, 50 μg / mL ampicillin, 40 μg / mL) (ml chloramphenicol) and precultured at 37 ° C. for about 6 hours until immediately before the start of culture. The total volume of the preculture was added to 3 L of 4 × LBL medium (4% peptone, 2% yeast extract, 2% NaCl, 50 μg / mL ampicillin), and the mixture was placed at 37 ° C. The cells were cultured under the control of a computer program at a pH of 7.2 and a pressure of 0.02 Pa. The pH was adjusted with autoclaved 2M HCl (manufactured by Wako Pure Chemical Industries, Ltd.) and 2M NaOH (manufactured by Wako Pure Chemical Industries, Ltd.). Approximately 23 hours before collection, an autoclaved 300 mL expression induction solution (10% lactose, 20% glycerol) was added. When the growth of Escherichia coli reached the stationary phase (50 hours after the start of culture), the cells were collected using a large centrifuge (AvantiHP-30I manufactured by Beckman). The collected cells were stored at −30 ° C. At this time, a small amount of the cells is separately taken, dissolved and suspended in 150 mM NaCl, 20 mM Tris-HCl (pH 8), 5 mM β-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.). 201). This solution was divided into two equal parts, one was centrifuged at 9100 G at 4 ° C. for 10 minutes, separated into a supernatant and a precipitate, and the other was placed in a thermostat (TAITEC DryThermounit DTU-1C) set at 75 ° C. for 10 minutes. After heating, the mixture was centrifuged at 9100 G and 4 ° C. for 10 minutes to separate a supernatant and a precipitate. A denaturant (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 2.4%) was added to these four kinds of supernatants and precipitates (the precipitates were resuspended in a cell lysate). β-mercaptoethanol, 0.005% bromophenol blue (manufactured by Wako Pure Chemical Industries, Ltd.) were added, and the mixture was denatured by heating at 95 ° C. for 5 minutes. These denatured protein solutions were added to 12.5% or 15% (depending on the molecular weight of the protein to be expressed) polyacrylamide gel, and subjected to electrophoresis by SDS-PAGE. Using a staining solution (Quick-CBB, manufactured by Wako Pure Chemical Industries), the gel after electrophoresis was stained and decolorized to confirm the expression of the target protein.

Purification of Recombinant Protein In Example 3, the cells stored at −30 ° C. were dissolved and suspended in 150 mM NaCl, 20 mM Tris-HCl (pH 8), and 5 mM β-mercaptoethanol. It was crushed in a TOMY UD-201), heated in a thermostat (TAITEC, DryThermoUnit DTU-1C) set at 75 ° C. for 10 minutes, and then quickly cooled. Next, the disrupted cell fluid was centrifuged at 100,000 G for 1 hour using a large centrifuge (Avanti HP-30I manufactured by Beckman), and the supernatant was collected.

Next, this supernatant was replaced with a buffer solution of 1.05 M ammonium sulfate, 20 mM Tris-HCl (pH 8.0), and 5 mM β-mercaptoethanol using a protein purification device (AKTA explorer, manufactured by Amersham Biosciences). Then, the solution was passed through a hydrophobic exchange column (Amersham Biosciences, RESOURCE ^™ Phe 6 ml). Elution was performed with a concentration gradient of 1.05 M → 0 ammonium sulfate. Each fraction was confirmed by SDS-PAGE, and a fraction of the target protein was collected.

Next, the collected fraction was replaced with a buffer solution of 20 mM Tris-HCl (pH 8.0) and 5 mM β-mercaptoethanol, and then passed through an anion exchange column (Amersham Biosciences, RESOURCE ^™ Q 6 ml). Elution was performed with sodium chloride, and each eluted fraction was confirmed by SDS-PAGE, and a fraction of the target protein was collected.

  Next, the collected fraction was replaced with 10 mM phosphate buffer solution (pH 7.0) and 0.5 mM β-mercaptoethanol, and then concentrated by centrifugation using a centrifugal concentration tube (VIVASPIN10000 manufactured by Millipore). It was used for activity measurement.

Activity measurement The protein purified in the measurement method Example 4 is phosphoglycerate kinase (PGK), and PGK produces ATP and 3-phosphoglycerate from the substrate ADP and 1,3-bisphosphoglycerate in vivo. The reaction is catalyzed. In this example, 3-phosphoglycerate (PGA) is used, and under the following various conditions, the activity of the reverse reaction (3-phosphoglycerate + ATP → 1,3-bisphosphoglycerate + ADP) is measured. Was done. That is, a reaction solution containing 30 mM of NaCl, 50 mM of MgCl ₂ , 20 mM of ATP (manufactured by Oriental Yeast), and 20 mM of PGA (manufactured by Sigma-Aldrich) was held for 1 minute at each measurement temperature described below. Thereafter, the protein purified in Example 4 was added to a final concentration of 10 nM and held for 5 minutes (ie, a reaction time of 5 minutes). Thereafter, an equivalent amount of 0.1 M H ₃ PO ₄ to the reaction solution was added to stop the reaction. The measurement was performed with a cation exchange column (TSKgel DEAE 2SW manufactured by Tosoh Corporation) using HPLC (AKTA explorer manufactured by Amersham Biosciences), 15 μL of the reaction solution that had been deproteinized by filtration was collected, and a phosphoric acid concentration gradient (50 mM to 500 mM) was collected. ). The eluted ATP and ADP were detected at a wavelength of 256 nm, and peaks were obtained. The concentration of the product (ADP) was determined from the ratio of the sum of the peak areas of the obtained ATP and ADP chromatography and the peak area of ADP.

2. The measurement was performed at each of the optimum temperatures of 30, 40, 50, 60, 70, 80, 90, and 95 ° C. to determine the specific activity. The specific activity at each temperature was calculated based on the value measured at 80 ° C. (100%). The measurement results are shown in Table 1 and the graph of FIG. As shown in the figure, the optimum temperature was found to be 90 ° C.

3. Effects of metal ions and chelating agents on activity
1) Based on the activity when the above measurement was performed by further adding 5 mM MgCl ₂ as a reference (100%), 2) When the above measurement was performed by further adding 0.2 M EDTA, 3) Further 20 mM ZnCl ₂ was added The effects of the metal ions and the chelating agents on the activities were examined by comparing the activities when the above measurements were performed. The reaction temperature was 80 ° C. The measurement results are shown in Table 2 and the graph of FIG. As shown in the figure, when 0.2 M EDTA was added, no activity was observed, indicating that the chelating agent was effective in inhibiting the reaction. In addition, when 20 mM ZnCl ₂ was added, the activity was retained, but was clearly reduced, indicating that Zn ²⁺ was effective in inhibiting the reaction.

4. Optimum pH
The buffer solution was changed to one having a target pH at a measurement temperature of 70 ° C., and the above measurement was performed to determine the specific activity. Table 3 shows the relationship between the buffer solution used for the measurement and the target pH.

The specific activity at each pH was calculated based on the measured value at the highest activity at pH 6.0 (100%). The measurement results are shown in Table 4 and the graph of FIG. As shown, at all pHs, it had an activity of 65% or more of the highest activity, pH 6.0. Since the decrease in activity at pH 7.0 was near the isoelectric point, it is considered that the decrease was caused by the isoelectric point precipitation. Therefore, it can be said that this enzyme has a uniform activity in the pH range of 3 to 9 except for around the isoelectric point.

5. Thermal stability Prior to the measurement, a 1 mM protein solution (20 mM Tris-HCl, 5 mM β-Me) purified in Example 4 was pretreated at 80 ° C. for 30, 60, and 120 minutes. The above-mentioned measurement was performed using the above-mentioned pretreated product, and the specific activity was obtained. The final concentration of the protein purified in Example 4 was 100 nM instead of 10 nM, and the reaction temperature was 80 ° C. In addition, the pretreatment time of 0 hour was a time when no pretreatment was performed, and the relative activity at each pretreatment time was determined using this as a reference (100%). The measurement results are shown in Table 5 and the graph of FIG. As shown in the figure, no significant change was observed in the activity, indicating that there was no complete inactivation of the protein purified in Example 4 in this range.

  According to the present invention, a novel thermostable protein having phosphoglycerate kinase activity can be provided. The protein of the present invention can be used at a high temperature, and can be used for many purposes such as increasing the substrate concentration, improving the reaction efficiency, removing contaminating microorganisms, extending the storage period and the service life, as well as expanding industrial applications. Benefits are brought.

FIG. 1 is an SDS-PAGE photograph of the recombinant protein in one example of the present invention. FIG. 2 is a graph showing the relationship between temperature and specific activity in one example of the present invention. FIG. 3 is a graph showing the effect of a metal ion and a chelating agent on the activity of a purified protein in one example of the present invention. FIG. 4 is a graph showing the relationship between pH and specific activity in one example of the present invention. FIG. 5 is a graph showing the relationship between the heat treatment time at 80 ° C. and the specific activity in one example of the present invention.

SEQ ID NO: 1: Amino acid sequence of thermostable phosphoglycerate kinase SEQ ID NO: 2: Base sequence of thermostable phosphoglycerate kinase SEQ ID NO: 3: Introduction of restriction enzyme sites BamHI and NotI at the end of the structural gene of thermostable phosphoglycerate kinase 1 shows a forward primer for performing the following.
SEQ ID NO: 4: shows a reverse primer for introducing restriction enzyme sites BamHI and NotI into the end of the structural gene of thermostable phosphoglycerate kinase.
SEQ ID NO: 5: N-terminal amino acid sequence

Claims (12)

The following heat-resistant protein of (a) or (b).
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 1;
(B) a heat-resistant protein having phosphoglycerate kinase activity, wherein the amino acid sequence of SEQ ID NO: 1 has one or more amino acid residues deleted, substituted, added or inserted; The protein according to claim 1, wherein the phosphoglycerate kinase activity is a function of generating D-1,3-bisphosphoglycerate and ADP by acting on 3-phospho-D-glycerate in the presence of ATP. . 3. The protein according to claim 1, which is derived from a hyperthermophilic archaeon. The protein according to claim 3, wherein the hyperthermophilic archae is Sulfolobus tokodaii (JCM10545). A vector comprising the DNA encoding the protein according to any one of claims 1 to 4 or the DNA according to SEQ ID NO: 2. A transformant transformed by the vector according to claim 5. A method for producing the protein according to any one of claims 1 to 4, comprising a step of culturing the transformant according to claim 6, and a step of collecting the protein expressed in the culturing step. . 5. A method for producing D-1,3-bisphosphoglycerate and ADP from 3-phospho-D-glycerate in the presence of ATP by an enzyme reaction, wherein the enzyme is any one of claims 1 to 4. A production method wherein the enzyme reaction is carried out using the protein described in 1 above at a temperature of 80 to 90 ° C. A method for producing 3-phospho-D-glyceric acid and ATP from D-1,3-bisphosphoglycerate in the presence of ADP by an enzymatic reaction, wherein the enzyme is any one of claims 1 to 4. A production method wherein the enzyme reaction is performed using the protein described in the above at a temperature of 80 to 90 ° C. The method according to claim 8, wherein the pH of the enzyme reaction is in a range of pH 3 to 9. 10. The method according to any one of claims 8 to 10, wherein after performing the enzyme reaction for a certain period of time, the enzyme reaction is stopped with a chelating agent. The method according to claim 11, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

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