JP2006288391A - New heat-resistant protein having enolase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new heat-resistant protein having an enolase activity. <P>SOLUTION: This protein comprising a specific amino acid sequence and having an enolase activity. This protein is obtained by cloning a gene estimated to encode a protein having the enolase activity, from the gene sequence of Aeropyrumpernix K1 (JCM9820) which is a hyperthermophilic archaebacterium and is one of aerobic hyperthermophilic archaeon, and then expressing the cloned gene in Escherichia coli. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel thermostable protein having enolase activity. In addition, this application is an application of the result which concerns on national commission.

  Enolase has a function of acting on 2-phosphoglyceric acid to produce phosphoenolphurbic acid and water. It has been proposed to use this function to measure phosphoglycerate mutase concentration in blood, thereby distinguishing Alzheimer's dementia and vascular dementia in patients with dementia (for example, patents) Reference 1). In addition, as another application, a gene encoding enolase is introduced into a coryneform bacterium, and the production of L-lysine or L-glutamic acid is increased by amplifying enolase activity (see, for example, Patent Document 2). It has also been proposed to use it as a thrombolytic agent (see, for example, Patent Document 3). Furthermore, since enolase plays an important role in cell survival and maintaining ATP levels in cardiomyocytes, it has been studied to improve cardiac function by directly administering enolase to ischemic myocardium. (For example, refer to Patent Document 4).

As enolase, those derived from various organisms are known, and among them, those derived from heat-resistant bacteria are known. Examples of the thermostable bacteria include Pyrococcus furiosus , which is a hyperthermophilic archaeon, and Thermotoga maritima , which is a thermophilic eubacteria (for example, Non-Patent Document 1 and Non-Patent Documents). 2). Such an enolase derived from a thermostable bacterium is expected to expand industrial applications, but those derived from the two types of bacteria are still insufficient.

On the other hand, there has been research on hyperthermophilic archaea other than the above-mentioned bacteria (see Non-Patent Document 3), and Aeropyrum pernix (JCM9820) (JCM9820) (see Non-Patent Document 4), which is a kind of bacteria belonging to the genus Aeropyrum . The gene has already been analyzed (see Non-Patent Document 5). Thus, if this hyperthermophilic archaea produces a protein with enolase activity, it is expected to have excellent heat resistance and stability, which may further expand industrial applications.

JP 11-239500 A JP 2003-180355 A US Pat. No. 6,190,659 Japanese Patent Laid-Open No. 2004-081111 Arch. Biochem. Biophysics, 1994, September, 313 (2), 280-286 Protein Science 4, 228-236 (1995) Advances in Protein Chemistry, Volume 48, Enzymes and Proteins from Hyperthermophilic Microorganisms (M.W.W.Adams ed.), Academic Press (1996) Int. J. Syst. Bacteriol. 1996 October; 46 (4): 1070-1077 DNA Res. 1999 April 30; 6 (2): 83-101, 145-152

  The present invention has been made in view of such circumstances, and an object thereof is to provide a novel thermostable protein having enolase activity.

In order to achieve the above object, the genome information of Aeropyrum pernix (JCM9820), which is a hyperthermophilic archaeon, was examined, and it was found that this bacterium may produce enolase. As a result of further research based on this finding, the inventors succeeded in expressing a novel thermostable protein having enolase activity from the gene of this bacterium, and reached the present invention.

That is, the protein of the present invention is the following heat-resistant protein (a) or (b).
(A) A heat-resistant protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) A heat-resistant protein having an enolase activity, comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 1.

Aeropyrum pernix (JCM9820) is stored in the microbial strain storage facility of RIKEN Bioinformatics and can be sold at the request of a third party.

Since the optimum growth temperature of Aeropyrum pernix (JCM9820) is 90-95 ° C. and the growth limit temperature is 100 ° C., the protein of the present invention is active even at a high temperature of 90-100 ° C. is there. Therefore, the protein of the present invention can be used at a high temperature, has high stability at a high temperature, and can expand industrial applications.

  The enolase has a function of acting on 2-phosphoglyceric acid to produce phosphoenolphurbic acid and water. In addition, the enolase has a function of acting on phosphoenolpyruvate to generate 2-phosphoglycerate.

As described above, the novel thermostable protein of the present invention is derived from a hyperthermophilic archaea, specifically, from Aeropyrum pernix (JCM9820). However, the protein of the present invention is not limited to those produced by this bacterium, and may be produced by other organisms by genetic engineering techniques.

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

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

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

  Next, the method for producing phosphoenolpyruvate according to the present invention is a method for producing phosphoenolpyruvate from 2-phosphoglycerate by enzymatic reaction, wherein the protein of the present invention is used as an enzyme. It is a manufacturing method.

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

The inventors of the present invention are a hyperthermophilic archaeon collected from the sulfur pores on the coast of Kotajima Island, and a gene sequence of Aeropyrum pernix species K1 (JCM9820), which is a kind of aerobic hyperthermophilic archaeon. Was cloned from a gene (SEQ ID NO: 2) presumed to encode a protein exhibiting enolase activity and expressed using Escherichia coli, whereby the novel heat-resistant protein of the present invention was obtained. The gene cloning method was performed as described in Example 1 described later. The base sequence of the cloned gene is as shown in SEQ ID NO: 2, and the deduced amino acid sequence is as shown in SEQ ID NO: 1. In addition, as long as the novel heat-resistant protein of the present invention has enolase activity, one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 are missing, substituted, added or inserted. Also good. The “amino acid deletion, substitution, addition or insertion” in this amino acid sequence can be carried out according to a method known to those skilled in the art (eg, mutagenesis).

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

An example of a gene encoding the protein of the present invention is the gene shown in SEQ ID NO: 2. The gene is, for example, from the genome of the hyperthermophilic archaeon Aeropyrum pernix (JCM9820) as shown in Example 2 described later, for example, using a part of the base sequence represented by SEQ ID NO: 2 as a primer. It can be prepared by the PCR method used or the hybridization method using the DNA fragment as a probe. Moreover, although the said gene can also be obtained according to the nucleic acid chemical synthesis method etc. which are well-known to those skilled in the art based on the base sequence, this invention is not limited to these.

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

  The insertion of a gene or the like into a vector includes, for example, a method in which the base sequence of the purified gene is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Although it can be used, it is not limited to these. In addition, the expression vector of the present invention incorporates a promoter, terminator, ribosome binding sequence and the like in addition to the gene encoding the protein of the present invention so that the function of the gene encoding the protein of the present invention is exhibited. Also good. Furthermore, a gene encoding the protein of the present invention may be inserted by fusing a sequence encoded by another protein.

  If a host organism is transformed 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 encoding the protein of the present invention, and examples thereof include, but are not limited to, prokaryotic cells such as Escherichia coli. As the transformation method, a known calcium chloride method or the like can be used, but it is not limited to these methods.

The protein production method of the present invention is a production method comprising a step of culturing the transformant and a step of recovering the protein expressed in the culture 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, any medium that contains a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the microorganism can be used. 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 protein is recovered by crushing the cells or cells. When the protein of the present invention is produced outside the cells or cells, the culture solution is used as it is or after removing the cells or cells by centrifugation or the like, it is used for protein isolation and purification. The protein of the present invention can be isolated and purified from the culture by using general biochemical methods such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography alone or in appropriate combination. . When the culture solution is used as it is, other proteins are inactivated by heat treatment, so that it can be used substantially as an enzyme solution containing only the protein of the present invention.

The protein of the present invention can produce 2-phosphoglyceric acid from phosphoenolpyruvate, for example, by its enzymatic reaction. Therefore, the present invention includes, as one aspect thereof, a method for producing 2-phosphoglyceric acid from phosphoenolpyruvate by an enzymatic reaction, and the method using the heat-resistant protein of the present invention as an enzyme. The said enzyme reaction can be performed on 70-85 degreeC temperature conditions, for example. The enzyme reaction is preferably performed in the presence of divalent metal ions such as Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Ca ^2+, and Co ²⁺ , and each may be one type or two or more types. May be used in combination. Among them, the presence of Mg ²⁺ is preferable. For the enzyme reaction, for example, a reaction solution containing MgCl ₂ , MnCl ₂ , NiCl ₂ , CaCl ₂ and CoCl ₂ can be used. The reaction solution may further contain, for example, phenylmethanesulfonyl fluoride (PMSF) and mercaptoethanol. The buffer solution for the reaction solution is not particularly limited, and examples thereof include a MES buffer solution and a Tris / HCl buffer solution. The pH of the reaction solution is preferably in the range of pH 4.5 to 6.5, more preferably in the range of pH 4.5 to 6.0. The concentration of the protein of the present invention in the reaction solution is not particularly limited, but is, for example, 3.2 to 14 U / mg, and the concentration of phosphoenolpyruvate is, for example, 0.25 to 2 mM.

  Next, as described above, the method for producing phosphoenolpyruvate according to the present invention is a method for producing phosphoenolpyruvate from 2-phosphoglycerate by enzymatic reaction, and the thermostable protein according to the present invention is used as an enzyme. Is the way to use. If the heat-resistant protein of the present invention is used, an enzyme reaction can be carried out in a high temperature region. Reaction conditions (for example, temperature conditions, divalent metal ions, buffer solution, pH, etc.) in the enzyme reaction are the same as in the method for producing 2-phosphoglycerate. The concentration of the protein and substrate of the present invention in the reaction solution is not particularly limited, and can be appropriately determined from the concentration in the production of 2-phosphoglycerate, for example.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Preparation of chromosomal DNA Aeropyrum pernix (JCM9820) was cultured overnight at 37 ° C. in L medium and collected into an SSC solution (0.15 M NaCl, 0.015).
M sodium citrate) solution (10 mL), 0.5 M EDTA solution (0.2 mL), 100 mg / mL chicken egg white lysozyme (0.1 mL) and 10% nonionic surfactant Brij-58 solution (0.5 mL) were added, and 0 ° C was added. Was added for 30 minutes, and then 5 mg of proteinase K (manufactured by Merck) dissolved in 0.2 mL of 10% SDS solution was added, and the mixture was allowed to stand at 37 ° C. for a few days. A mixed solution of water-saturated phenol, chloroform, and isoamyl alcohol was added to this solution and allowed to stand at 37 ° C. for 1 hour. The aqueous layer was separated, ethanol was added thereto, and DNA was precipitated and concentrated. This DNA precipitate was dissolved in 10 mL of TE solution (10 mM Tris / HCl (pH 7.5), 1 mM EDTA (pH 8.0)), 0.25 mL of ribonuclease (final concentration 0.25 mg / mL) was added, And allowed to stand overnight, and then precipitated with ethanol. The DNA was then dissolved in 5 mL of TE solution, and the DNA concentration was determined from the absorbance at 260 nm (Clarke, L. & Carbon, J. (1979) Methods Enzymol. 68, 396-408).

Construction of expression vector and gene expression Construction of an expression vector 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 enolase gene, and PCR of the thermostable enolase gene was carried out using this primer. Restriction enzyme sites were introduced before and after the translation region. KOD Dash (manufactured by Toyobo Co., Ltd.) was used as the DNA polymerase.
Forward primer (SEQ ID NO: 3): 5'-ATATCATATGGGGGTAAATGACTTTGCTATTGAGCGCGTT -3 '
Reverse primer (SEQ ID NO: 4): 5'-ATATGGATCCGCGGCCGCTTATTAGCGCCTGCCCATTACGCC -3 '

  After the PCR reaction, deoxyadenosine was added to the 3 ′ end of the amplified fragment using Ex Taq (Takara Shuzo), and then reacted with pGEM-T Easy Vector (Promega) and T4 ligase at 15 ° C. for 30 minutes. Connected. The ligated DNA was introduced into competent cells of E. coli DH5α to obtain transformant colonies. The obtained transformant was cultured in an LB medium (18 mL) containing ampicillin for 24 hours, and the expression vector was purified from the culture solution by a modified alkaline SDS method. The presence of the inserted DNA fragment of the expected size in the purified vector was confirmed by agarose electrophoresis. The base sequence of the inserted DNA fragment of the purified vector was determined using BigDye Terminator kit (registered trademark: Applied Biosystems) and ABI PRISM 3700 DNA Analyzer (registered trademark: Applied Biosystems). The sequence was confirmed to be the correct sequence of the thermostable enolase gene. A part of the vector having the correct sequence was completely digested with restriction enzymes NdeI and BamHI (2 hours at 37 ° C.), and then the thermostable enolase gene was purified by agarose electrophoresis. pET-11a (manufactured by Novagen) was cleaved and purified with restriction enzymes NdeI and BamHI, reacted with T4 ligase, and ligated with the above structural gene. A part of the ligated DNA was introduced into competent cells of Escherichia coli DH5α, and an appropriate amount was spread on an LB agar plate containing ampicillin and cultured at 37 ° C. overnight to obtain transformant colonies. The obtained transformant was cultured in an LB medium (18 mL) containing ampicillin for 24 hours, and the expression vector was purified from the culture solution by a modified alkaline SDS method.

2. Expression of recombinant gene 0.1 mL of competent cells of Escherichia coli Rosetta-gami (DE3) (manufactured by Novagen) were transferred to a falcon tube. Among them, the above 1. 0.002 mL of the purified expression vector solution was added and left on ice for 20 minutes. Next, heat shock was performed at 42 ° C. for 90 seconds and left on ice for 1 minute, and then an appropriate amount was spread on an LB agar plate containing chloramphenicol and ampicillin and cultured at 37 ° C. overnight to obtain a transformant. . The obtained transformant was cultured in LB medium (5 mL) containing ampicillin for 18 hours to express the thermostable enolase gene. After incubation, the cells were collected by centrifugation (13,000 G, 10 minutes).

  To the collected cells, 0.2 mL of a cell disruption solution (20 mM Tris / HCl, 100 mM KCl, pH 7.5) is added, the cells are disrupted with an ultrasonic generator, and the suspension is divided into 0.1 mL each 2 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 the cell disruption solution. The other sample tube was heat-treated (80 ° C., 10 minutes) and then centrifuged (13,000 G, 10 minutes) to separate the supernatant and the precipitate. Suspended. A part of these samples was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and the expression could be confirmed. The results are shown in the SDS-PAGE photograph of FIG.

  After performing SDS polyacrylamide gel electrophoresis on the sample in which the expression of thermostable enolase was observed, it was transferred to a PVDF membrane by electroblotting, and the band of thermostable enolase that is the target recombinant protein visualized by staining was cut out. As a result of analyzing the amino terminal sequence using a protein sequencer Model492Procise (Applied Biosystems), an amino terminal sequence of 7 residues was determined as shown in SEQ ID NO: 5. This sequence confirmed that the expressed protein was a thermostable enolase. This expressed protein is composed of 432 amino acid residues, and its estimated molecular weight is 46.8 kDa, which almost coincides with the result of FIG.

Mass Recombination of DNA Recombinant E. coli The expression vector was purified in the same manner as in Example 2, and Escherichia coli Rosetta-Gami (DE3) was transformed with this expression vector. Take 3 platinum loops of the colonies that grew on the plate, 5 mL of LBL medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% lactose, 50 μg / mL ampicillin, 40 μg / (mL chloramphenicol) and inoculated for about 6 hours at 37 ° C. The total amount of this pre-culture solution is added to 3 L of 4 × LBL medium (4% peptone, 2% yeast extract, 2% NaCl, 50 μg / mL ampicillin), and a predetermined density in a high-density culture tank (manufactured by ABLE). The culture was carried out under computer program control under conditions (37 ° C., pH 7.2, pressure 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 18 hours before collection (about 24 hours before the start of culture), 300 mL of an autoclaved expression induction solution (10% lactose, 20% glycerol) was added. When the degree of growth of E. coli entered the stationary phase (42 hours after the start of culture), the cells were collected using a large-scale centrifuge (trade name Avanti HP-30I manufactured by Beckman). A small amount was collected from the collected cells, and the remaining cells were stored at -30 ° C. The sorted cells are suspended in a crushing solution (150 mM NaCl, 20 mM Tris-HCl (pH 8) and 5 mM β-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.)), and an ultrasonic crushing device (manufactured by TOMY, trade name). UD-201). This solution was divided into two equal parts, and one was centrifuged at 9,100 G for 10 minutes at 4 ° C. to separate into a supernatant and a precipitate. The other was heated for 10 minutes in a thermostatic chamber set to 75 ° C. (trade name DTU-1C, manufactured by TAITEC), and then centrifuged at 9,100 G for 10 minutes at 4 ° C. to separate into a supernatant and a precipitate. The resulting precipitate was resuspended in the crushed liquid. A denaturant (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 2.4% β-mercaptoethanol, 0.005% bromophenol was added to these four supernatants and the precipitate suspension. Blue (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was denatured by heating at 95 ° C. for 5 minutes. Each denatured protein solution was added to 12.5% polyacrylamide gel, and electrophoresis was performed by SDS-PAGE. As a result of staining and decoloring the gel after electrophoresis using a staining solution (trade name Quick-CBB, manufactured by Wako Pure Chemical Industries, Ltd.), expression of the target protein (enolase) was confirmed.

1. Purification of DNA recombinant protein The cells stored at -30 ° C. in Example 3 were suspended in a disruption solution (150 mM NaCl, 20 mM Tris-HCl (pH 8) and 5 mM β-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.)). The sample was crushed with an ultrasonic crusher (trade name UD-201, manufactured by TOMY). This cell disruption solution was heated for 10 minutes in a thermostatic bath set to 80 ° C. (trade name DTU-1C, manufactured by TAITEC), and then quickly cooled. Subsequently, the mixture was centrifuged at 100,000 G for 1 hour using a large-scale centrifuge (Beckman, product name Avanti HP-30I), and the supernatant was collected.

Next, this supernatant was subjected to a buffer solution containing 1.35 M ammonium sulfate, 20 mM Tris-HCl (pH 8.0) and 5 mM β-mercaptoethanol using a protein purification apparatus (trade name: AKTA ^™ explorer manufactured by Amersham Biosciences). Then, the solution was passed through a hydrophobic exchange column (manufactured by Amersham Biosciences, trade name RESOURCE ^™ Phe 6 mL). Elution was carried out with an ammonium sulfate concentration gradient of 1.35M → 0M, each fraction was confirmed by SDS-PAGE, and the fraction of the target protein was recovered.

Next, the collected fraction was replaced with a buffer solution containing 50 mM MES (pH 6.0) and 5 mM β-mercaptoethanol, and then applied to an anion exchange column (trade name TSK-GEL BioAssist ^™ Q 6 mL, manufactured by Tosoh Corporation). I passed. Elution was performed with a buffer solution containing 0 to 1 M sodium chloride, and each fraction eluted at 0.48 M sodium chloride concentration was confirmed by SDS-PAGE, and a fraction of the target protein was collected.

Next, the collected fraction is centrifuged and concentrated using a centrifugal concentration tube (trade name VIVASPIN ^™ 10000, manufactured by Millipore), and 20 mM Tris-HCl (produced by GILSON) is used. The solution was passed through a gel filtration column (manufactured by Amersham Biosciences, trade name HiLoad ^™ 16/60, Superdex ^™ 75) equilibrated with a solution containing 5 mM β-mercaptoethanol and 150 mM NaCl. Each eluted fraction was confirmed by SDS-PAGE electrophoresis, and the fraction of the target protein was recovered.

Next, the collected fraction was replaced with a solution containing 150 mM NaCl, 20 mM Tris-HCl (pH 8.5) and 5 mM β-mercaptoethanol, and then a centrifugal concentration tube (manufactured by Sartorius, trade name VIVASPIN ^™ 10000) was used. And then concentrated by centrifugation and used as a sample for the following measurements.

2. Determination of optimum temperature Reaction solution containing 2.0 mM phosphoenolpyruvate (PEP) (manufactured by SIGMA), 1.0 mM MgCl ₂ (manufactured by Wako Pure Chemical Industries), 50 mM Tris / HCl (manufactured by Nacalai Tesque) (pH 7.5) And the reaction solution was set at a predetermined temperature (25, 45, 55, 65, 75, 85 and 90 ° C.). Subsequently, the purified sample was added to the reaction solution to start an enzyme reaction, and the absorbance at a wavelength of 240 nm derived from phosphoenolpyruvate was measured immediately after the start of the reaction and over time from the start of the reaction (trade name U-3000 Spectrophotometer). : Made by HITACHI). Since the molar extinction coefficient of phosphoenolpyruvate changes depending on the Mg concentration, etc., the amount of 2-phosphoglyceric acid generated from the change in absorbance is calculated using the molar extinction coefficient by calculation from the actual measurement value under each condition. The amount of 2-phosphoglyceric acid (μmol) produced by 1 mg of heat-resistant protein per minute was calculated as the specific activity (μmol / min / mg). The result is shown in FIG. As shown in the figure, the highest activity was shown at 95 ° C. The amount of protein in the sample was quantified by an absorbance method.

3. Determination of optimal pH Except that the temperature conditions were set to 85 ° C. and the enzyme activity was measured at various pHs using the buffer shown in Table 1 below instead of the Tris / HCl buffer, the above “2. Measurements were performed in the same manner as in “Determination of optimum temperature”. The result is shown in FIG. As shown in the figure, the thermostable protein showed the highest activity at pH 5.5 (Tris / HCl buffer).

4). Thermal stability (1) CD spectrum
After adding the thermostable protein sample to potassium phosphate buffer (pH 7.0), increase the solution temperature from 15 ° C. to 95 ° C. or decrease the θ ₂₂₂ value from 95 ° C. to 15 ° C. It was measured. In addition, the brand name Spectropolarimeter J-720-W (made by JASCO) was used for the measurement. The result is shown in FIG. As shown in the figure, the thermostable protein was stable in the range of 15 to 95 ° C. Note that. As the temperature increased, the absorbance increased, but no precipitation occurred in the cell when the temperature reached 95 ° C. This increase was due to measurement error or baseline shift. it is conceivable that.

(2) Activity measurement The sample of the thermostable protein was incubated at a predetermined temperature (70, 85 and 95 ° C) (trade name GeneAmp PCR System 2700: manufactured by Applied Biosystems), and the residual activity was measured over time. The buffer used was a 50 mM Tris / HCl (pH 5.5) buffer, and the activity was measured in the same manner as in “2. Determination of optimum temperature”. The result is shown in FIG. FIG. 5 (a) shows the specific activity, and FIG. 5 (b) shows the relative activity (%) when the specific activity at 0 minutes is taken as 100. As shown in these figures, at 95 ° C., the residual activity decreased to 8.8% after 2 hours of incubation, but at 70 and 85 ° C., the activity was high and stable even after 36 hours. It was.

5). pH stability After incubation of the thermostable protein sample with a buffer solution having a predetermined pH shown in Table 2 below at 85 ° C. for 1 hour (trade name: DTU-N: manufactured by TAITEC), the above-mentioned “2. Determination of Optimal Temperature” The activity was measured in the same manner as above. The result is shown in FIG. FIG. 6 (a) shows the specific activity, and FIG. 6 (b) shows the relative activity (%) when the specific activity when the Tris / HCl (pH 5.5) buffer is used is taken as 100. As shown in these figures, the thermostable protein showed enzyme activity in all pH ranges and was stable.

6). It was determined Km and Vmax for calculating PEP and MgCl ₂ in Km value and Vmax value. The results are shown in Table 3 below. The activity was measured in the same manner as in “2. Determination of optimum temperature” except that 50 mM Tris / HCl (pH 5.5) buffer was used.

7). Inhibitory effect of divalent metal, EDTA, mercaptoethanol and PMSF PMSF (manufactured by Wako Pure Chemical Industries), mercaptoethanol (Mer-EtOH) (manufactured by Nacalai Tesque) and EDTA (manufactured by Nacalai Tesque) are added to the reaction solution, respectively. The effect of sex protein on enzyme activity was examined. Except for using 50 mM Tris / HCl (pH 5.5) buffer, the activity was measured in the same manner as in “2. Determination of optimum temperature”, the final EDTA concentration was 5 mM, and the other final concentrations were 1 mM. It was. The result is shown in FIG. FIG. 7 (a) shows the specific activity, and FIG. 7 (b) shows the relative activity (%) when the specific activity is 100 when using a reaction solution containing MgCl ₂ (final concentration 16 mM). . As shown in these figures, the reaction solution containing no Mg ²⁺ does not exhibit enzyme activity, and the activity decreases when EDTA, which is a divalent metal chelator, is added. It was found that Mg ²⁺ is essential for enzyme activity. In addition, when PMSF and mercaptoethanol were added, there was no big influence.

8). In place of the metal-dependent MgCl ₂ , other divalent metals (NiCl ₂ , MnCl ₂ , CaCl ₂ and CoCl ₂ ) were added to the reaction solution, and a 50 mM Tris / HCl (pH 5.5) buffer solution was used. The enzyme activity was measured in the same manner as in “2. Determination of optimum temperature”. The result is shown in FIG. FIG. 8 (a) shows the specific activity, and FIG. 8 (b) shows the relative activity (%) when the specific activity is 100 when using a reaction solution containing MgCl ₂ . As shown in these figures, the enzyme activity in the presence of Mn ²⁺ and Ca ²⁺ is 60% of Mg ²⁺ , and the enzyme activity in the presence of Ni ²⁺ is 50% of Mg ²⁺ , It was 40% of Mg ²⁺ in the Co ²⁺ presence.

  According to the present invention, a novel thermostable protein having enolase activity can be provided. The protein of the present invention can be used at high temperatures, and has a wide range of industrial applications, as well as many substrate concentrations, increased reaction efficiency, removal of contaminating microorganisms, extended shelf life and longevity, etc. Benefits are provided.

FIG. 1 is an SDS-PAGE photograph of a recombinant protein in one example of the present invention. FIG. 2 is a graph showing the enzyme activity of a protein under a predetermined temperature condition in another example of the present invention. FIG. 3 is a graph showing the enzyme activity of a protein at a predetermined pH in another example of the present invention. FIG. 4 is a graph showing the thermal stability of a protein under a predetermined temperature condition in another example of the present invention. FIG. 5 (a) is a graph showing the enzyme activity of a protein under a predetermined temperature condition in another example of the present invention, and FIG. 5 (b) is a graph showing its relative activity. FIG. 6 (a) is a graph showing the enzyme activity of a protein at a predetermined pH in another example of the present invention, and FIG. 6 (b) is a graph showing its relative activity. FIG. 7 (a) is a graph showing the enzyme activity of the protein in the reaction solution to which various metals etc. are added in other examples of the present invention, and FIG. 7 (b) is a graph showing the relative activity. . FIG. 8 (a) is a graph showing the enzyme activity of a protein in the presence of various metals in other examples of the present invention, and FIG. 8 (b) is a graph showing its relative activity.

SEQ ID NO: 1: amino acid sequence of thermostable enolase SEQ ID NO: 2: base sequence encoding amino acid sequence of thermostable enolase SEQ ID NO: 3: restriction enzyme sites NdeI and Bam at the end of the structural gene of thermostable enolase
Forward primer for introducing HI, NotI SEQ ID NO: 4: Reverse primer for introducing restriction enzyme sites NdeI and BamHI, NotI at the end of the thermostable enolase structural gene SEQ ID NO: 5: N-terminal amino acid sequence

Claims (10)

The heat-resistant protein of the following (a) or (b).
(A) A heat-resistant protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) A heat-resistant protein having an enolase activity, comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 1. The protein according to claim 1, wherein the enolase activity has a function of acting on 2-phosphoglyceric acid to produce phosphoenolphurbic acid and water. The protein according to claim 1, wherein the enolase activity has a function of acting on phosphoenolphurbic acid to produce 2-phosphoglycerate. The protein according to any one of claims 1 to 3, which is derived from a hyperthermophilic archaea. The protein according to claim 4, wherein the hyperthermophilic archaea is Aeropyrum pernix (JCM9820). A vector comprising the DNA encoding the protein of any one of claims 1 to 5 or the DNA of SEQ ID NO: 2. A transformant transformed with the vector according to claim 6. A method for producing the protein according to any one of claims 1 to 5, comprising a step of culturing the transformant according to claim 7, and a step of recovering the protein expressed in the culturing step. . A method for producing phosphoenolpyruvate from 2-phosphoglycerate by enzymatic reaction, wherein the protein according to any one of claims 1 to 5 is used as an enzyme. The production method according to claim 9, wherein the enzyme reaction is performed in the presence of a divalent metal ion selected from the group consisting of Mg ²⁺ , Ca ²⁺ , Co ²⁺ , Mn ²⁺ and Ni ²⁺ .