JP2005261348A - New heat-resistant protein having aconitic acid hydratase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new heat-resistant protein having aconitic acid hydratase activity. <P>SOLUTION: The new protein having aconitic acid hydratase activity comprises a specific amino acid sequence. This protein, which is a hyperthermophilic archaebacterium, is obtained by cloning a gene estimated to encode a protein having aconitic acid hydratase activity from the gene sequence of Sulfolobus tokodaii strain 7(JCM10545), a kind of aerobic thermoacidophilic crenarchaeon, followed by expression using Escherichia coli. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel thermostable protein having aconitic acid hydratase activity. This application is an application related to the outcome of the commission of the government.

Aconitic acid hydratase (EC 4.2.1.3) is an enzyme that catalyzes the reaction of changing citric acid to cis-aconitic acid in the citric acid cycle, and is widely distributed in various organisms. In cells, it is localized in mitochondria. In addition, as an industrial utilization method of aconitic acid hydratase, there is L-lysine synthesis by an enzymatic method using a metabolic intermediate as a raw material (see, for example, Patent Document 1). When the enzyme is used industrially, a heat-resistant one is preferable, but conventionally known heat-resistant aconitic acid hydratase includes an enzyme derived from Thermus thermophilus (see, for example, Patent Document 1), There are enzymes derived from Sulfolobus acidocaldarius (for example, see Non-Patent Document 1) and enzymes derived from Sulfolobus solfataricus (for example, see Non-Patent Document 2). Such an aconitic acid hydratase derived from heat-resistant bacteria is expected to expand industrial applications, but those derived from the three kinds of bacteria are still insufficient.

On the other hand, there has been research on hyperthermophilic archaea other than the above-mentioned bacteria (for example, see Non-patent Document 3), Sulfolobus tokodaii (JCM10545) (JCM10545) (for example, non-patented) In Reference 4), the gene has already been analyzed (for example, see Non-Patent Document 5). Thus, if this hyperthermophilic archaeon produces aconitic acid hydratase, it is expected to have excellent heat resistance, which may further expand industrial applications.

Japanese Patent Laid-Open No. 2001-157276 Eur. J. Biochem., 268 (6), 1760-1771, 2001 Biochem. Soc. Trans., 30 (4), 685-687, 2002 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 aconitic acid hydratase activity.

In order to achieve the above object, the genome information of Sulfolobus tokodaii (JCM10545), a hyperthermophilic archaeon, was investigated, and it was found that this bacterium may produce aconite hydratase. It was. As a result of further research based on this finding, the inventors succeeded in expressing a novel heat-resistant protein having aconitate hydratase activity from the bacterial gene, and reached the present invention.
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: 1.
(B) A heat-resistant protein 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: 1 and having aconite hydratase activity.
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.

  The aconitic acid hydratase activity is a function of acting on citric acid to produce cisaconitic acid.

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: 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 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.

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

The present inventors have obtained aconitic acid hydratase activity 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. The novel gene (SEQ ID NO: 2) presumed to be expressed was cloned 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 its deduced amino acid sequence is as shown in SEQ ID NO: 1. As long as the thermostable protein of the present invention has aconitate hydratase 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 “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: 1, for a protein whose amino acid sequence is 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: 2. For example, a part of the base sequence represented by SEQ ID NO: 2 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: 2 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. Further, as a commercially available plasmid, for example, pET-11a (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.

  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, a terminator, a 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 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, a medium containing a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the microorganism can be used so long as the transformant can be cultured efficiently. 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. 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 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 10 mL of SSC solution (0.15 M NaCl, 0.015 M sodium citrate). 0.5 M EDTA, 100 mg / ml 0.1 ml chicken egg white lysozyme and 0.5 ml 10% nonionic surfactant Brij-58, left at 0 ° C. for 30 minutes, 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 the DNA was dissolved 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 aconite hydratase gene, and thermostable aconit by PCR using this primer. Restriction enzyme sites were introduced before and after the translation region of the acid hydratase gene. The DNA polymerase used was KOD Dash (manufactured by Toyobo).
Forward primer (SEQ ID NO: 3): 5'-ATATCATATGGTAGACTTGTTTATGAAATTTCAGATTTCT-3 '
Reverse primer (SEQ ID NO: 4): 5'-ATATGGATCCGCGGCCGCTTATTAACTTTCATATAATAATTT-3 '

  After the PCR reaction, deoxyadenosine was added to the 3 ′ end side 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 plasmid was purified from the culture solution by a modified alkaline SDS method. The presence of the expected size insert in the plasmid was confirmed by agarose electrophoresis. 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 aconitic acid hydratase 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 aconite hydratase gene was purified by agarose electrophoresis. pET-11a (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 A competent cell of Escherichia coli Rosetta-gami (DE3) (Novagen) was thawed and transferred to a Falcon tube by 0.1 mL. 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 LB medium (5 mL) containing ampicillin for 18 hours to express the thermostable aconitic acid hydratase 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 crushed liquid. 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 in FIG.
Samples in which the expression of thermostable aconite hydratase was observed were subjected to SDS polyacrylamide gel electrophoresis, transferred to a PVDF membrane by electroblotting, and the thermostable aconate hydratase band visualized by staining was excised. As a result of analyzing the amino terminal sequence using Model492Procise (Applied Biosystems), the 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 aconitic acid hydratase. This protein is composed of 855 amino acid residues, and its estimated molecular weight is 95.8 kD, which almost coincides with the result of FIG.

  According to the present invention, a novel thermostable protein having aconite hydratase 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.

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

Claims (7)

The following heat-resistant protein (a) or (b) (a) A heat-resistant protein comprising the amino acid sequence of SEQ ID NO: 1.
(B) A heat-resistant protein 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 having aconitic acid hydratase activity. The protein according to claim 1, wherein the aconitic acid hydratase activity has a function of acting on citric acid to produce cisaconitic acid. The protein according to claim 1 or 2, which is derived from a hyperthermophilic archaea. The protein according to claim 3, wherein the hyperthermophilic archaea is Sulfolobus tokodaii (JCM10545). A vector comprising the DNA encoding the protein of any one of claims 1 to 4 or the DNA of SEQ ID NO: 2. A transformant transformed with 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 recovering the protein expressed in the culturing step. .

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