JP2004298185A - New thermostable protein having phosphoglyceric acid dehydrogenase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new thermostable protein having phosphoglyceric acid dehydrogenase activity. <P>SOLUTION: This protein comprising a specific amino acid sequence has the phosphoglyceric acid dehydrogenase activity. The protein is obtained by cloning a gene estimated to exhibit the thermostable phosphoglyceric acid dehydrogenase activity from a gene sequence of a Sulfolobustokodaii species 7 (JCM10545) which is one species of aerophilic thermoacidophilic crenarchaeon of hyperthermophilic archaebacteria, and then expressing the protein from the cloned product by using Escherichia coil. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

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

  Phosphoglycerate dehydrogenase (EC1.1.1.95), also called D-3-phosphoglycerate dehydrogenase, acts on 3-phosphoglycerate in the presence of NAD (+) to produce 3-phosphohydroxypyruvate. Is a dehydrogenase that produces Serine, on the other hand, is a major intermediate in the biosynthesis of a wide range of cellular metabolites including economically important compounds such as choline, glycine, cysteine and tryptophan. The serine biosynthetic pathway is commonly found in a wide range of tissues and microorganisms, from 3-phospho-D-glyceric acid (PGA) to 3-phosphohydroxypyruvic acid (PHA), and from 3-phospho-L-serine. L-serine is produced. Here, conversion of 3-phospho-D-glyceric acid (PGA) to 3-phosphohydroxypyruvic acid (PHA) is performed by phosphoglycerate dehydrogenase (PGD). The conversion of 3-phosphohydroxypyruvic acid (PHA) to 3-phospho-L-serine is performed by phosphoserine aminotransferase, and the conversion of 3-phospho-L-serine to L-serine is It is performed by phosphoserine phosphatase. The gene encoding PGD has already been cloned from E. coli and sequenced, and the amino acid sequence of the PGD subunit has been deduced (see Non-Patent Document 1). It is known that phosphoglycerate dehydrogenase is produced not only in microorganisms such as prokaryotes (particularly bacteria) and yeasts, but also in eukaryotes. Such phosphoglycerate dehydrogenase is useful in the biosynthesis of serine and serine-related products and is industrially important. Therefore, it can be said that thermostable phosphoglycerate dehydrogenase has a wide range of use.

On the other hand, there is research on hyperthermophilic archaea (see Non-Patent Document 2), and the gene of Sulfolobus tokodaii (JCM10545) (see Non-Patent Document 3), which is a kind of bacteria belonging to the genus Sulfolobus , is disclosed. It has already been analyzed (see Non-Patent Document 4). Therefore, if this hyperthermophilic archaea produces phosphoglycerate dehydrogenase, it is expected to have excellent thermostability.
Tobey and Grant, J. Biol. Chem., 261: 12179-12183 (1986) 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 of the present invention is to provide a novel heat-resistant protein having phosphoglycerate dehydrogenase activity.

To achieve the above object, the present inventors examined the genome information of the hyperthermophilic archaeon Sulfolobus tokodaii (JCM10545), and found that this bacterium may produce phosphoglycerate dehydrogenase. I found it. Based on this finding, further studies have led to the successful expression of a novel protein having phosphoglycerate dehydrogenase activity from this bacterial gene, and have 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.

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 thermostable protein having an amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues are deleted, substituted, added, or inserted, and having phosphoglycerate dehydrogenase activity.

  As described above, the present invention can provide a novel thermostable protein having phosphoglycerate dehydrogenase activity.

  The phosphoglycerate dehydrogenase activity of the protein is a function of acting on 3-phosphoglycerate in the presence of NAD (+) to generate 3-phosphohydroxypyruvate. The NAD (+) becomes NADH. 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 3-phosphohydroxypyruvic acid from 3-phosphoglyceric acid in the presence of NAD (+) by an enzymatic reaction, wherein the enzyme of the present invention is used as the enzyme. This is a production method in which the enzyme reaction is carried out at a temperature of 75 to 95 ° C. using a protein. The NAD (+) becomes NADH. As described above, by using the protein of the present invention, an enzymatic reaction can be performed in a high temperature range of 75 to 95 ° C., and as a result, the industrial application of phosphoglycerate dehydrogenase is expanded. Further, the production method of the present invention is useful for industrial production of economically important compounds such as serine, such as choline, glycine, cysteine and tryptophan.

  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 dehydrogenase activity, one or more or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 are 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 Sulfolobus tokodaii (JCM10545) was cultured overnight in an L medium at 37 ° C. and collected, and 10 mL of an SSC solution (0.15 M NaCl, 0.015 M sodium citrate) was added. , 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 (manufactured by 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, the DNA was dissolved in 5 mL of a TE solution, and the DNA concentration was determined from the absorbance at 260 nm (see 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 DNA containing restriction enzymes NdeI, BamHI and NotI sites before and after the translation region of the thermostable phosphoglycerate dehydrogenase gene, and this primer was used. Restriction enzyme sites were introduced before and after the translation region of the thermostable phosphoglycerate dehydrogenase gene by PCR. The DNA polymerase used was KOD Dash (Toyobo).
Forward primer (SEQ ID NO: 3): 5'-ATATCATATGGCTATCTATACCGTAAAGGCCTTAATAACA-3 '
Reverse primer (SEQ ID NO: 4): 5'-ATATGGATCCGCGGCCGCTTATTATATCATCCCTAACTCTTT-3 '

  After the PCR reaction, deoxyadenosine was added to the 3 'end of the amplified fragment using Ex Taq (manufactured by Takara Shuzo), followed by reaction with pGEM-T Easy Vector (manufactured by Promega) and T4 ligase at 15 ° C. for 30 minutes. And connected. The ligated DNA was introduced into competent cells of Escherichia coli DH5α to obtain a transformant colony. The obtained transformant was cultured in an LB medium (18 mL) containing ampicillin for 24 hours, and the plasmid was purified from the culture by a modified alkaline SDS method. The presence of an insert of the expected size in the plasmid was confirmed by agarose electrophoresis, as shown in FIG. 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 sex phosphoglycerate dehydrogenase gene was confirmed. After a part of the plasmid having the correct sequence was completely digested with the restriction enzymes NdeI and BamHI (at 37 ° C. for 2 hours), the structural gene of thermostable phosphoglycerate dehydrogenase was purified by agarose electrophoresis. After pET-11a (manufactured by Novagen) was cut and purified with restriction enzymes NdeI and BamHI, it was ligated by reacting with the above structural gene using T4 ligase. 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 in an LB medium (18 mL) containing ampicillin for 24 hours, and the expression plasmid was purified from the culture by a modified alkaline SDS method.

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 dehydrogenase 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 a 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 result is shown in the SDS-PAGE photograph of FIG.

  After performing SDS polyacrylamide gel electrophoresis on a sample in which the expression of thermostable phosphoglycerate dehydrogenase was observed, the sample was transferred to a PVDF membrane by electroblotting and visualized by staining. As a result of analyzing the amino-terminal sequence using a protein sequencer Model492Procise (manufactured by Applied Biosystems), the amino-terminal sequence of 6 residues was found as shown in SEQ ID NO: 5. I was able to decide. Based on SEQ ID NO: 5, it was confirmed that the expressed protein was subjected to post-translational processing and the first Met was dropped, and that the second and subsequent amino terminal sequences of SEQ ID NO: 5 matched the amino terminal sequences expected from the genomic sequence. As a result, it was confirmed that the expressed protein was thermostable phosphoglycerate dehydrogenase. This expressed protein was composed of 313 amino acid residues, and its estimated molecular weight was 34.7 kD, which was almost consistent 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.). About 21 hours before the harvest, 300 mL of an autoclaved expression induction solution (10% lactose, 20% glycerol) was added. When the growth of Escherichia coli reached the stationary phase (44 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, the supernatant was replaced with a buffer solution of 0.6 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). The fraction was eluted with a 0.6M → 0 ammonium sulfate concentration gradient, each fraction was confirmed by SDS-PAGE, and the fraction of the target protein was collected.

  Next, the collected fraction was replaced with a buffer solution of 20 mM MES (2-Morpholinoethanesulfonic acid) pH 6.0 and 5 mM β-mercaptoethanol using a protein purification device (AKTA explorer, manufactured by Amersham Biosciences). The solution was passed through a cation exchange column (TSK-GEL Bioassist S manufactured by Tosoh Corporation). Elution was performed with sodium chloride, and each eluted fraction was confirmed by SDS-PAGE electrophoresis, and a fraction of the target protein was collected.

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

Activity measurement Measurement Method A 10 mM phosphate buffer solution (pH 7.0 at 25 ° C.) containing 10% of the protein purified in Example 4 and 15% glycerol was prepared, and the CD (circularly polarized light) of the protein solution was prepared under the following various conditions. (Dichroism) was measured. J-720W manufactured by JASCO Corporation was used for the measurement. The measurement results are corrected to the intensity per residue.

2. Spectrum Measurement The spectrum of the reaction solution at 200C to 250nm at 25C and the spectrum of 200 to 250nm after the reaction solution was held at 95C for 10 minutes were compared. The measurement results are shown in the graph of FIG. As shown in the figure, no difference was observed in the spectrum, and it was found that the secondary structure of the purified protein of Example 4 after holding at 25 ° C. and 95 ° C. for 10 minutes was almost the same.

3. Temperature change measurement The change in the spectrum at 222 nm when the temperature of the reaction solution was changed from 25 ° C. to 95 ° C. at 1 ° C./min was measured. Note that 222 nm used here is an absorption extreme wavelength derived from α-helix. The measurement results are shown in the graph of FIG. As shown in the figure, the continuous temperature change from 25 ° C. to 95 ° C. showed that the secondary structure of the purified protein of Example 4 was almost maintained.

4. Time change at 95 ° C. The time change of the 222 nm spectrum when the reaction solution was kept at 95 ° C. for 10 minutes was measured. The measurement results are shown in the graph of FIG. As shown in the figure, it was found that the secondary structure of the purified protein of Example 4 was kept at 95 ° C. for 10 minutes.

  From the above results, it was found that the purified protein of Example 4 was stable at 95 ° C.

  As described above, the present invention can provide a novel thermostable protein having phosphoglycerate dehydrogenase activity. 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 a PAGE photograph of an insert DNA of a purified plasmid in one example of the present invention. FIG. 2 is an SDS-PAGE photograph of the recombinant protein in one example of the present invention. FIG. 3 is a graph showing a spectrum comparison of the purified protein in one example of the present invention after holding at 25 ° C. and 95 ° C. for 10 minutes. FIG. 4 is a graph showing a change in spectrum when the temperature of the purified protein is changed from 25 ° C. to 95 ° C. in one example of the present invention. FIG. 5 is a graph showing a time change of a spectrum when the purified protein in one example of the present invention is kept at 95 ° C. for 10 minutes.

SEQ ID NO: 1: Amino acid sequence of thermostable phosphoglycerate dehydrogenase SEQ ID NO: 2: Nucleotide sequence of thermostable phosphoglycerate dehydrogenase SEQ ID NO: 3: Structure of thermostable phosphoglycerate dehydrogenase The forward primer for introducing restriction enzyme sites NdeI, BamHI, and NotI into the end of the gene is shown.
SEQ ID NO: 4: shows a reverse primer for introducing restriction enzyme sites NdeI, BamHI, and NotI into the end of the structural gene of thermostable phosphoglycerate dehydrogenase.
SEQ ID NO: 5: N-terminal amino acid sequence

Claims (8)

The following heat-resistant protein of (a) or (b).
(A) A thermostable protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) A thermostable protein having an amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues are deleted, substituted, added, or inserted, and having phosphoglycerate dehydrogenase activity. The protein according to claim 1, wherein the phosphoglycerate dehydrogenase activity is a function of acting on 3-phosphoglycerate in the presence of NAD (+) to generate 3-phosphohydroxypyruvate. 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. . A method for producing 3-phosphohydroxypyruvic acid from 3-phosphoglycerate in the presence of NAD (+) by an enzyme reaction, wherein the protein according to any one of claims 1 to 4 is used as the enzyme. A production method in which the enzyme reaction is carried out at a temperature of 75 to 95 ° C.

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