JP4250762B2 - Novel thermostable protein having metabolic activity of 6-N-hydroxyaminopurine - Google Patents

Description

  The present invention relates to a novel thermostable protein having 6-N-hydroxyaminopurine metabolic activity. This application is a result of a national commission.

  Nucleic acid metabolism-related enzymes are known to be involved in, for example, the synthesis or degradation of nucleotides, DNA, and RNA. Furthermore, enzymes having the activity of metabolizing mutagens that induce mutations are known to exist widely in archaea, eubacteria and eukaryotes. As an enzyme having an activity of metabolizing a mutagen that induces a mutation, for example, an enzyme having a 6-N-hydroxyaminopurine metabolism function together with an NTPase activity is known. Hydroxyaminopurine sensitivity is known (see Non-Patent Document 1). However, from thermostable bacteria, the presence of the gene is predicted, but it has not yet been collected as a protein. The range of utilization of thermostable nucleic acid metabolism-related enzymes is thought to expand. No heat-resistant 6-N-hydroxyaminopurine metabolizing enzyme is known. The thermostable 6-N-hydroxyaminopurine metabolizing enzyme is expected to be usable in a wide range of applications such as industrial applications.

On the other hand, there has been research on hyperthermophilic archaea (see Non-Patent Document 2). Sulfolobus tokodaii (JCM10545) (see Non-Patent Document 3), which is one of the genus Sulfolobus , has its gene It has already been analyzed (see Non-Patent Document 4). Therefore, if this hyperthermophilic archaea produces 6-N-hydroxyaminopurine metabolizing enzyme, it is expected to have excellent heat resistance.
Genetica 1997 May; 33 (5): 591-8 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 6-N-hydroxyaminopurine metabolic activity.

In order to achieve the above-mentioned object, the genome information of Sulfolobus tokodaii (JCM10545), a hyperthermophilic archaeon, was examined. This bacterium could produce 6-N-hydroxyaminopurine metabolizing enzyme. I found out that there is sex. As a result of further research based on this finding, the inventors succeeded in expressing a novel thermostable protein having 6-N-hydroxyaminopurine metabolic activity from the bacterial gene, and reached the present invention. In addition, Sulfolobus tokodaii (JCM10545) is preserve | saved in the microorganisms preservation | save facility of RIKEN Biological Infrastructure Research Department, and can be distributed according to 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 heat-resistant protein consisting of the amino acid sequence of SEQ ID NO: 2.
(B) a heat-resistant amino acid sequence having the 6-N-hydroxyaminopurine metabolic activity and NTPase activity, comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 2 Sex protein.

According to the present invention, a novel thermostable protein having 6-N-hydroxyaminopurine metabolic activity can be provided.
The protein of the present invention has a function of catalyzing a reaction for converting xanthosine triphosphate (XTP) into xanthosine monophosphate (XMP). However, this function has not been known so far and cannot be estimated by ordinary search. Yes, this is the first function discovered by the present inventors. The optimum temperature for the reaction is 95 ° C.

As described above, the novel thermostable protein of the present invention is derived from a hyperthermophilic archaea, specifically from Sulfolobus tokodaii (JCM10545). 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: 1.

  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 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 metabolically degrading 6-N-hydroxyaminopurine by the enzyme of the present invention is a method in which the enzyme reaction is performed at a temperature of 95 ° C. using the protein of the present invention as the enzyme.

  Further, the production method of the present invention is a method for producing xanthosine monophosphate (XMP) from xanthosine triphosphate (XTP) by an enzyme, wherein the protein of the present invention is used as the enzyme and the temperature is 95 ° C. This is a method of performing an enzyme reaction.

  As described above, when the protein of the present invention is used, an enzyme reaction can be carried out in a high temperature range of 95 ° C., and 6-N-hydroxyaminopurine can be metabolized or decomposed even under industrial conditions. Can be implemented. In these methods, the enzyme reaction preferably has a pH of 7 or more.

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

The present inventors have obtained a 6-N- from the gene sequence of Sulfolobus tokodaii species 7 (JCM10545), which is a hyperthermophilic archaea collected from the ocean floor and is a kind of aerobic thermoacidophilic crenarchaeon. By cloning a gene (SEQ ID NO: 1) presumed to exhibit hydroxyaminopurine metabolic activity and expressing it using E. coli, the novel thermostable protein of the present invention was obtained. The gene cloning method was performed as described in Example 1 described later. The nucleotide sequence of the cloned gene is as shown in SEQ ID NO: 1, and its deduced amino acid sequence is as shown in SEQ ID NO: 2. Note that the heat-resistant protein of the present invention lacks one or more or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 as long as it has 6-N-hydroxyaminopurine metabolic activity and NTPase activity. Quality, substitution, addition or insertion. The “amino acid deletion, substitution, addition or insertion” in this amino acid sequence can be performed according to a method known to those skilled in the art (for example, 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: 2, 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 according to

An example of a gene encoding the protein of the present invention is the gene shown in SEQ ID NO: 1. For example, a part of the base sequence represented by SEQ ID NO: 1 is used as a primer from the genome of the hyperthermophilic archaeon Sulfolobus tokodaii (JCM10545) as shown in Example 2 described later. It can be prepared by a PCR method or a hybridization method using the DNA fragment as a probe. Further, based on the base 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 DNA of SEQ ID NO: 1 into an appropriate vector. The vector for inserting the gene 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 plasmid, for example, a pET-11a (manufactured by Novagen) or 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.

  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 to the gene of the present invention, a promoter, terminator, ribosome binding sequence and the like may be incorporated in the expression vector of the present invention so that the function of the gene of the present invention is exhibited. Furthermore, the gene of the present invention may be inserted by fusing sequences encoded by other proteins.

  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 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 is isolated and purified from the culture by using general biochemical methods such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination. can do. In addition, when using a culture solution as it is, proteins other than the protein of the present invention are inactivated by heat treatment, so that it can be used substantially as a solution containing only the protein of the present invention.

  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 Sulfolobus tokodaii (JCM10545) was cultured overnight in L medium at 37 ° C. and collected into an SSC solution (0.15 M NaCl, 0.015 M sodium citrate) 10 mL , 0.5 M EDTA, 100 mg / ml Chicken egg white lysozyme 0.1 mL and 10% nonionic surfactant Brij-58 0.5 mL were added and allowed to stand at 0 ° C. for 30 minutes, and then proteinase K (manufactured by Merck) 5 mg Was dissolved in 0.2 mL of 10% SDS and 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 the mixture was 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 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. And allowed to stand overnight, and then precipitated with ethanol. Next, after dissolving the DNA in 5 mL of 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 plasmids 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 6-N-hydroxyaminopurine metabolizing enzyme gene. The restriction enzyme sites were introduced before and after the translation region of the thermostable 6-N-hydroxyaminopurine metabolizing enzyme gene by PCR. The DNA polymerase used was KOD Dash (manufactured by Toyobo).
Forward primer (SEQ ID NO: 3): 5'-ATATCATATGTTGAAAAGCGTAGAAGTAAACGTAATAACG-3 '
Reverse primer (SEQ ID NO: 4): 5'-ATATGGATCCGCGGCCGCTTATTAAGAGATATAAGTAAGAAA-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 a competent cell of E. 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 solution 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 base sequence of the insert of the purified plasmid was determined using BigDye Terminator kit (registered trademark: Applied Biosystems) and ABI PRISM 3700 DNA Analyzer (registered trademark: Applied Biosystems). The correct sequence of the 6-N-hydroxyaminopurine metabolizing enzyme gene was confirmed. A part of the plasmid having the correct sequence was completely digested with restriction enzymes NdeI and BamHI (2 hours at 37 ° C.), and then the thermostable 6-N-hydroxyaminopurine metabolizing enzyme structural gene was purified by agarose electrophoresis. pET-11a (manufactured by Novagen) was cleaved and purified with restriction enzymes NdeI and BamHI, and then ligated by reacting with the above structural gene and T4 ligase. 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 plasmid was purified from the culture solution by a modified alkaline SDS method.

2. Recombinant Gene Expression Competent cells of Escherichia coli Rosetta-gami (DE3) (Novagen) were thawed and transferred to 0.1 mL of a falcon tube. Among them, the above 1. After adding 0.002 mL of the purified expression plasmid solution and leaving it on ice for 20 minutes, heat shocking at 42 ° C. for 90 seconds, leaving it on ice for 1 minute, and then an LB agar plate containing chloramphenicol and ampicillin An appropriate amount was spread 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 6-N-hydroxyaminopurine metabolizing enzyme gene. After incubation, the cells were collected by centrifugation (13,000 G, 10 minutes).

  To the collected cells, 0.2 mL of a disruption solution (20 mM Tris-HCl, 100 mM KCl, pH 7.5) is added, and the cells are disrupted with an ultrasonic generator. 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 disrupted solution. The other sample tube was heat-treated (70 ° C., 10 minutes) and then centrifuged (13,000 G, 10 minutes) to separate the supernatant and the precipitate. The precipitate was suspended in 0.1 mL of the disrupted solution. . 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.

  This is a target recombinant protein visualized by staining after SDS polyacrylamide gel electrophoresis for a sample with thermostable 6-N-hydroxyaminopurine metabolizing enzyme expression, electrophoretic blotting to a PVDF membrane A band of thermostable 6-N-hydroxyaminopurine metabolizing enzyme was cut out and analyzed for amino terminal sequence using protein sequencer Model 492Procise (manufactured by Applied Biosystems). The sequence was determined. This sequence confirmed that the expressed protein was a thermostable 6-N-hydroxyaminopurine metabolizing enzyme. This expressed protein is composed of 189 amino acid residues, and its estimated molecular weight is 21.7 kD, which almost coincides with the molecular weight in SDS-PAGE shown in FIG.

Mass culture of recombinant Escherichia coli Escherichia coli DH5α strain was transformed according to a conventional method using plasmid DNA (pET-11a vector) prepared in the same manner as in Example 2. Plasmid DNA was extracted from the transformed E. coli DH5α using the alkaline SDS method. This plasmid DNA was used to transform Escherichia coli Rosetta-Gami (DE3) strain. 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 precultured at 37 ° C. for about 6 hours until just before the start of the culture. The total amount of this preculture was added to 3 L of 4 × LBL medium (4% peptone, 2% yeast extract, 2% NaCl, 50 μg / mL ampicillin), and 37 ° C. in a high-density culture tank (manufactured by ABLE). The computer program was controlled at pH 7.2, pressure 0.02 Pa, and cultured. 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 18 hours before the collection, an autoclaved 300 mL expression induction solution (10% lactose, 20% glycerol) was added. The cells were collected using a large centrifuge (AvantiHP-30I manufactured by Beckman) when the growth of E. coli entered the stationary phase (45 hours after the start of culture). The collected cells were stored at -30 ° C. At this time, a small amount of bacterial cells are taken separately, dissolved and suspended in 150 mM NaCl, 20 mM Tris-HCl (pH 8), 5 mM β-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.), and an ultrasonic crusher (UDY, manufactured by TOMY). 201). This solution is divided into two equal parts, one is centrifuged at 9100G for 10 minutes at 4 ° C, separated into supernatant and precipitate, and the other is kept at 75 ° C for 10 minutes in a thermostatic bath (DryThermounit DTU-1C manufactured by TAITEC) After heating, the mixture was centrifuged at 9100 G at 4 ° C. for 10 minutes, and separated into a supernatant and a precipitate. These four types of supernatant and precipitate (precipitate is resuspended in the cell disruption solution) and denaturant (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 2.4% β-mercaptoethanol, 0.005% bromophenol blue (manufactured by Wako Pure Chemical Industries, Ltd.) was 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, Ltd.), the gel after electrophoresis was stained and decolored to confirm the expression of the target protein.

Purification of recombinant protein In Example 3, the bacterial cells stored at -30 ° C were dissolved and suspended in 150 mM NaCl, 20 mM Tris-HCl (pH 8), 5 mM β-mercaptoethanol, and an ultrasonic crusher ( The mixture was crushed with TOMY UD-201), heated in a thermostat set at 75 ° C. (TAITEC, DryThermoUnit DTU-1C) for 10 minutes, and then quickly cooled. Next, this crushed 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 20 mM MES (2-Morpholinoethanesulfonic acid) pH 6.0, 5 mM β-mercaptoethanol using a protein purification apparatus (AKTA explorer manufactured by Amersham Biosciences), and then a cation. The sample was passed through an exchange column (RESOURCE ^™ S manufactured by Amersham Biosciences). Elution was performed with sodium chloride, and each eluted fraction was confirmed by SDS-PAGE electrophoresis, and the fraction of the target protein was collected.

  Next, the collected fraction was replaced with a 10 mM phosphate buffer solution and then passed through a hydroxyapatite column (Ceramic Hydroxyapatite, Type I Column, CHT-101 manufactured by Bioscale). Elution was performed with a high-concentration phosphate buffer solution, and each eluted fraction was confirmed by SDS-PAGE electrophoresis, and the fraction of the target protein was collected.

  Next, the collected fraction was replaced with a buffer solution of 10 mM Tris-HCl, pH 8.0, 5 mM β-mercaptoethanol, and then concentrated by centrifuging using a centrifugal concentration tube (VIVASPIN10000 manufactured by Millipore) to measure the activity. It was used for.

Activity measurement Measurement Method The protein purified in Example 4 has a function of reducing the variability due to 6-N-hydroxyaminopurine, and its primary sequence homolog is xanthosine triphosphate (XTP), deoxyinosine triphosphate (dITP) and the like as deoxyribonucleotides. Has activity to decompose into pyrophosphate. In this activity evaluation, the activity against XTP, which is considered to be the most specific substrate, was measured under the following various conditions. The measuring method is as follows. That is, a 50 mM buffer solution containing 10 mM MgCl ₂ (manufactured by Nacalai Tesque), 1 mM XTP (manufactured by JENA BIOSCIENCE), and 4 nM of the purified protein of Example 4 (varies depending on the measurement, as described later). ) After reacting 100 μL for 10 minutes, the reaction was stopped by adding 100 μL of 100 mM Pi. For quantification, HPLC (Amersham Biosciences, AKTA purifier) using an anion exchange column (manufactured by Tosoh Corporation, TSK-GEL DEAE-2SW) was used, and the relative value of the integrated value of the peak of the reaction product and the product (product) The product of (integral value of product) / (integral value of product + integral value of reactant) and the substrate concentration was used as the product concentration to quantify the progress of the reaction.

2. The above measurements were performed at optimum temperatures of 45, 60, 80, and 95 ° C., and k _app was determined. As a buffer solution, Tris (manufactured by Nacalai Tesque) -HCl (pH 7.3 at each temperature) was used. The measurement results are shown in the graph of FIG. As shown in the figure, the highest activity was shown at 95 ° C.

3. Prior to the thermal stability the measurement, the protein purified in Example 4, at 80 ° C. and 95 ° C., were pretreated 0.5,1,1.5,2,2.5,3 hours. Using the pre-treated product, the measurement was performed to obtain k _app . The buffer solution includes Tris (Nacalai Tesque) -HCl (pH 7.2 at 80 ° C.) at a pretreatment temperature of 80 ° C., and Glycine (Nacalai Tesque) at a pretreatment temperature of 95 ° C.—NaOH (pH 7 at 95 ° C.). .3) and the reaction temperature was 80 ° C. Note that the pre-processing time of 0 hour is the time when no pre-processing is performed. The measurement results are shown in the graph of FIG. As shown in the figure, it was found that by performing the pretreatment, a slight decrease in the activity was observed, but the activity was maintained after the treatment at a high temperature.

4). PH optimum
Each condition of pH 5.84, 6.0, 6.4, 6.84, 7.25, 7.49, 8.03, 8.34, 8.93, 9.48, 10.0, 10.5 Then, the measurement was performed to obtain k _app . The buffer solution was MES (manufactured by Dojindo Laboratories) at pH 5.89, Tris (manufactured by Nacalai Tesque) -HCl at pH 6.0, 6.4, 6.84, and 7.25, pH 7.49. , 8.03, 8.34 using glycine (Nacalai Tesque) -NaOH, and pH 8.93, 9.48, 10.0, 10.5 using CAPS (Dojindo Laboratories). Was 80 ° C. The measurement results are shown in the graph of FIG. As shown in the figure, a decrease in activity is observed around pH 8-9, which is considered to be affected by isoelectric point precipitation because it is near the isoelectric point. Excluding this point, it can be seen that strong activity is exhibited in a high pH region (pH 7 or more).

  According to the present invention, a novel thermostable protein having 6-N-hydroxyaminopurine metabolic 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 agarose electrophoresis photograph of purified plasmid insert DNA 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 the relationship of temperature-k _app in one embodiment of the present invention. FIG. 4 is a graph showing the relationship of pretreatment time-k _app at 80 ° C. and 95 ° C. in one example of the present invention. FIG. 5 is a graph showing the relationship of pH-k _app in one example of the present invention.

SEQ ID NO: 1: Base sequence of 6-N-hydroxyaminopurine metabolic active enzyme SEQ ID NO: 2: Amino acid sequence of 6-N-hydroxyaminopurine metabolic active enzyme SEQ ID NO: 3: Structure of 6-N-hydroxyaminopurine metabolic active enzyme The forward primers for introducing restriction enzyme sites NdeI, BamHI and NotI at the end of the gene are shown.
SEQ ID NO: 4 shows a reverse primer for introducing restriction enzyme sites NdeI, BamHI and NotI at the ends of the structural gene of 6-N-hydroxyaminopurine metabolizing enzyme.
SEQ ID NO: 5: N-terminal amino acid sequence

Claims (1)

A method for producing xanthosine monophosphate (XMP) from xanthoseine triphosphate (XTP) by an enzyme, wherein heat resistance of the following (a) or (b) derived from the hyperthermophilic archaeon Sulfolobus tokodaii (JCM10545) The manufacturing method including performing the said enzyme reaction on the conditions of temperature 80-95 degreeC and pH 7-10.5 using sex protein.
(A) A heat-resistant protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) A heat-resisting 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, and catalyzing a reaction of converting XTP to XMP. Sex protein.

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