JP3032779B2 - Thermophilic dehydrogenase, DNA encoding the same, method for producing the same, and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a thermophilic dehydrogenase, a DNA encoding the same, a method for producing the same, and a use thereof. Specifically, the present invention relates to the use of hydroxyamino acids or alcohols at temperatures above 70 ° C. for NAD or NADP.
As an enzyme capable of performing a dehydrogenation reaction.

[0002]

2. Description of the Related Art Dehydrogenase is a kind of oxidoreductase and is a general term for enzymes that catalyze a dehydrogenation reaction. This enzyme catalyzes, for example, the oxidation of alcohols to ketones, the oxidation of aldehydes to carboxylic acids, oxidative deamination, the formation of unsaturated bonds, many of which are NAD (H)
And NADP (H) as the acceptor (or donor).

Conventionally, dehydrogenase enzymes of normal temperature bacteria have been used to regenerate coenzymes consumed in oxidoreductase reactions involving NAD or NADP, which are coenzymes. It could only be used at relatively low temperatures (N. Katayama, I. Urabe and H. Okada, Eur. J. Bi
ochem. 132 , 403 (1983)). At present, yeast dehydrogenase and the like, which are considered to be relatively thermophilic, are known, for example, yeast dehydrogenase (N. Katayama et al., Supra), but all are unstable at temperatures exceeding 70 ° C. If industrially dehydration reactions of alcohols and aldehydes can be carried out at higher temperatures, not only will it be possible to handle enzymes stably, but also to improve reaction efficiency and prevent contaminating microorganisms. It is believed that many advantages can be provided.

[0004] For the individual quantification of hydroxyamino acids such as serine and threonine, for example, a method in which Cl-C≡N produced by periodate-chloramine T treatment is colored with hirazolone or an aldehyde produced by periodate oxidation is used. Although a method of quantifying by developing color with chromotropic acid is known, these reactions have low specificity and are complicated. If such a quantitative reaction can be performed enzymatically, there is a possibility that a specific and simple measurement method can be provided.

[0005]

SUMMARY OF THE INVENTION
It is an object of the present invention to provide a thermophilic dehydrogenase enzyme capable of dehydrogenating a hydroxyamino acid or an alcohol at a high temperature exceeding the above. This enzyme has a so-called threonine dehydrogenase activity and is more stable at higher temperatures than previously known activities. In addition, this enzyme is specific for hydroxyamino acids, and the amount of hydroxyamino acids can be determined by colorimetric determination of coenzyme.

[0006] Another object of the present invention is to provide a DNA encoding the above dehydrogenase enzyme. Still another object of the present invention is to provide the DNA
An object is to provide an expression vector into which NA has been introduced, and a host cell transformed with this expression vector.

[0007] Another object of the present invention is to provide a method for producing the above dehydrogenase enzyme using the above host cells. Still another object of the present invention is to use the above enzyme for enzymatic dehydrogenation of a hydroxy compound or for quantification of a hydroxy compound utilizing the reaction.

[0008]

Means for Solving the Problems The present inventors have proposed 90-102.
Focusing on hyperthermophilic bacteria that can grow at ℃, find a gene that is presumed to show the enzyme activity of the present invention from their gene sequence, and further produce the enzyme using genetic recombination technology,
It has been confirmed that this enzyme is stable at a high temperature of 90 to 95 ° C. and shows an enzymatic activity for dehydrogenating hydroxyamino acids or alcohols at 90 ° C. or higher, and thus completed the present invention. By incorporating the gene obtained here into another microorganism, it is also possible to produce the desired recombinant enzyme in large quantities. That is, the present invention provides an enzyme capable of dehydrogenating a hydroxyamino acid or alcohol at a temperature exceeding 70 ° C.

This enzyme is characterized in that it is active at a temperature of 90 ° C. or higher. In an embodiment of the present invention, the enzyme has an optimal pH of 7.5 to 10.0 and an optimal temperature of about 95 ° C. The enzyme of the present invention can be obtained from a hyperthermophilic bacterium, particularly a sulfur-metabolizing thermophilic bacterium, preferably Pyrococcus horikoshii (Accession No. JCM9974) by a gene cloning technique (J. Sambrook et al., Molecular C
loning, A Laboratory Manual, Second Ed., Cold Sprin
g Harbor Laboratory Press (1989)) to prepare a cDNA encoding the enzyme, further integrate this into an appropriate vector, and then transform into an appropriate host cell.
It can be produced by expressing the DNA.

[0010] Specifically, the enzyme of the present invention has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or lacks one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Includes those with lost, substituted or added amino acid sequences and having dehydrogenase activity at temperatures above 70 ° C. The present invention also provides
A DNA encoding the enzyme as defined above is provided. Specifically, this DNA has an enzyme having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence, and DNA encoding an enzyme having a dehydrogenase activity at a temperature higher than ℃. More specifically, the DNA of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.

Alternatively, the DNA of the present invention encodes an enzyme that hybridizes under stringent conditions to DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing and has dehydrogenase activity at a temperature exceeding 70 ° C. DNA containing the DNA. Here, stringent conditions means that hybridization occurs only when at least 80% identity, preferably at least 90% identity, and most preferably at least 95% identity exists between the sequences. I do. Generally, hybridization is about 5 to 25 above the melting temperature (Tm) of a perfect hybrid.
It is performed at a temperature lower by ° C. J. Sambrook et al., Molecular Clon
ing, A Laboratory Manual, Second Ed., Cold Spring H
arbor Laboratory Press (1989) can be referred to.

The present invention further provides an expression vector containing the above DNA. Specifically, it is a plasmid containing a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. The vector contains a regulatory sequence such as a promoter on the 5 ′ side of the DNA so that the DNA of the present invention can be expressed. Vectors can further include genetic elements such as origins of replication, ribosome binding sites, transcription termination sequences, selectable markers (eg, antibiotic resistance, auxotrophy, etc.).

[0013] The present invention further provides a host cell transformed with the above expression vector. Host cells are derived from prokaryotes or eukaryotes. Examples of prokaryotes include bacteria (such as Escherichia coli and Bacillus subtilis) and fungi. Examples of eukaryotes include yeasts, plants, and insects. And mammals. The vector of the present invention can be arbitrarily selected according to the host, and such selection is well known to those skilled in the art.

[0014] The present invention also provides a method for producing a target cell, comprising culturing a host cell capable of expressing a DNA encoding the present enzyme as defined above in a suitable medium, and recovering the resulting enzyme. Provided is a method for producing an enzyme capable of performing a dehydrogenation reaction at a temperature exceeding 70 ° C. The present invention further provides the use of the enzyme as defined above in the enzymatic dehydrogenation of a hydroxy compound in the presence of NAD or NADP or the quantification of the hydroxy compound utilizing the reaction. Specific examples of the hydroxy compound include hydroxy amino acids (such as serine and threonine), primary, secondary and tertiary alcohols, and sugar alcohols.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme of the present invention is a thermophilic enzyme which exists stably even at a temperature usually exceeding 70 ° C., especially at a temperature exceeding 90 ° C., and has a dehydrogenase activity. As described above, this enzyme is capable of dehydrogenating hydroxyamino acids or alcohols at a temperature exceeding 70 ° C., particularly at a temperature exceeding 90 ° C. In an embodiment of the present invention, the enzyme is genomic D of Pyrococcus horikoshii, a sulfur-metabolizing thermophile (Accession No. JCM9974).
It can be obtained as a recombinant from NA using polymerase chain reaction (PCR) and gene recombination technology. The resulting enzyme has the amino acid sequence shown in SEQ ID NO: 1 (estimated molecular weight: 37,742), has an optimal pH of 7.5 to 10.0, an optimal temperature of about 95 ° C., and has a 20 mM phosphate buffer (pH 7). .5) Medium, 95 ° C
90% or more activity after heating for 3 hours.

The DNA encoding the enzyme of the present invention may have the nucleotide sequence shown in SEQ ID NO: 2, and based on this sequence, 10 or more, preferably 15
As described above, more preferably, a hybridization probe comprising about 20 to about 50 bases is prepared, and using this, a genomic gene library derived from a thermophilic bacterium such as Pyrococcus genus and the species and subspecies thereof is used. Thus, it is also possible to screen for a DNA encoding the enzyme of the present invention. Alternatively, even for bacteria having low heat resistance, a chemical mutagen, subjected to mutation treatment with ultraviolet light, etc.,
Bacteria that can withstand an environment exceeding 70 ° C. can be screened to obtain a target DNA or enzyme. The gene library includes, for example, λZAPII, pBluescript (registered trademark) cloning vector (Stratagene Cloning Sys
tems) using commercially available vectors.

The thermophilic enzyme obtained as described above comprises
It has dehydrogenase activity at a temperature exceeding 70 ° C., but has an amino acid sequence in which one or more amino acids are deleted, substituted or added (including insertion) in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing . These enzymes are so-called variants of the enzyme having the amino acid sequence shown in SEQ ID NO: 1, and usually have at least 70% homology, preferably at least 80% homology, more preferably at least 90% homology at the amino acid level. It can have the above homology. In general, in a region that does not excessively affect the enzyme activity, it is considered that the enzyme activity is maintained to a greater or lesser degree even if amino acid deletions, substitutions, additions, and other modifications are made in that region. The amino acid represented by SEQ ID NO: 1 is genetically engineered by using well-known techniques such as site-directed mutagenesis and PCR (SNHo, HDHunt, RMHorton, JK Pullen and LRPease, Gene 77 , 51 (1989)). It is also possible to introduce mutations in the sequence. Examples of substitutions include hydrophobic amino acids (Ala, Val, Le
u, Ile, etc.), substitution between Ser and Thr, Asp and Glu
Substitution between Asn and Gln, substitution between Lys and Arg,
Substitutions between Phe and Tyr are mentioned. Examples of additions are:
Addition of Met to the N-terminus of the mature protein found in the expression product in a bacterial host, N to facilitate inducing the mature protein
Addition of a His tag or a Met-Lys or Met-Arg sequence to the end may be mentioned. In addition, the carboxyl-terminal or amino-terminal amino acid residues of the enzyme protein may be truncated as long as the enzyme activity is not lost.

[0018]

The present invention also provides a DNA encoding the enzyme as defined above. That is, the DNA of the present invention includes those encoding all enzymes capable of dehydrogenating hydroxyamino acids or alcohols at a temperature exceeding 70 ° C. In an embodiment of the invention, the DNA comprises the nucleotide sequence shown in SEQ ID NO: 2. The DNA
Is a DN consisting of the nucleotide sequence shown in SEQ ID NO: 2.
It can encode an enzyme that hybridizes with A under stringent conditions and has dehydrogenase activity at a temperature above 70 ° C., especially above 90 ° C.
In the present specification, the stringent condition means at least
It means that hybridization occurs only when there is 80% identity, preferably at least 90% identity, most preferably at least 95% identity between the sequences. This condition can vary depending on the length of the DNA strand,
Generally, hybridization is performed at a temperature of about 5 to 25 ° C. below the melting temperature (Tm) of a perfect hybrid .

Based on the DNA sequence shown in SEQ ID NO: 2,
A probe having a sequence consisting of 10 or more complementary thereto, preferably 15 or more, more preferably about 20 to about 50 bases and all possible degenerate sequences is prepared by a DNA synthesizer or the like, and this probe is used. The DNA of the present invention can be screened from genomic genes of organisms such as thermophilic bacteria. The selected DNA uses, as a primer, a sequence complementary to a sequence of at least 10 bases, preferably at least 15 bases, more preferably about 20 to about 100 bases at the 5 ′ end and 3 ′ end shown in SEQ ID NO: 2. PCR reaction (eg, denaturation 94 ° C, 1 minute; annealing 57 ° C, 2 minutes; extension 70)
The conditions of 35 cycles can be used with 3 minutes at 1 ° C. as one cycle. ). Or,
The selected DNA can be cloned by incorporating it into a cloning vector.

The present invention further provides an expression vector containing the above DNA, a host cell transformed or transfected with the vector, and the production of the enzyme of the present invention by recombinant techniques. The vector of the present invention may be in the form of a plasmid, phage, virus, or the like. For example, bacterial plasmids (pBR322, pKC30, pCFM536 and the like), phage DNA (lambda phage and the like), yeast plasmids (pG-1 and the like), baculovirus, vaccinia virus, adenovirus and other viral DNAs as vectors for mammalian cells, Agrobacterium as a vector for a plant cell, such as a derivative of SV40, may be mentioned, but any other vector can be used as long as it is replicable and viable in a host cell.

The vector may contain a replication origin, a selection marker, a promoter, a ribosome binding site, and the like, if necessary. The eukaryotic cell vector may further contain an RNA splice site, a polyadenylation signal, and the like, if necessary. It may be added. As an origin of replication, for E. coli vectors, for example, ColE1, R factor, those derived from F factor,
For yeast vectors, for example, 2 μm DNA, those derived from ARS1, and for mammalian cell vectors, for example, SV4
0, those derived from adenovirus or bovine papilloma virus can be used.

[0023] Promoters are regulatory sequences to direct the synthesis of mRNA encoding DNA of the present invention, representative examples include adenovirus or SV40 promoter, the E. coli lac or trp promoter, phage lambda P _L promoter, ADH, PHO5, GPD, PGK, and AOX1 promoters for yeast, nuclear polyhedrosis virus-derived promoter for silkworm cells, and CaMV promoter for plant cells.

The selection marker is a gene for imparting a phenotype for selecting a transformed host cell to the host. For example, a vector for Escherichia coli includes a kanamycin resistance gene, an ampicillin resistance gene, and a tetracycline resistance gene. Etc., for yeast vectors, Leu2, Trp
1, Ura3 gene and the like, and mammalian cell vectors include neomycin resistance gene, thymidine kinase gene, dihydrofolate reductase gene and the like.

As the vector, commercially available vectors can be used. For example, bacterial vectors such as pQE70, pQE60, pQE-9 (Qiage
n), pBluescript II KS, ptrc99a, pKK223-3, pDR5
40, pRIT2T (Pharmacia), pET-11a (Novagen); and eukaryotic pXT1, pSG5 (Stratagene), pSV
K3, pBPV, pMSG, pSVL SV40 (Pharmacia).
The DNA of the present invention can be introduced into a vector by any means. Desirably, the vector contains a polylinker with various restriction sites therein, or contains a single restriction site. A specific restriction site in the vector is cleaved by a specific restriction endonuclease, and the DNA of the present invention is inserted into the cleavage site.

The expression vector containing the DNA and the regulatory sequence of the present invention can be used for transforming an appropriate host to cause the host to express and produce the enzyme of the present invention. Examples of hosts include bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis, and the like; fungal cells, such as strains of the genus Aspergillus;
Yeast cells such as baker's yeast and methanol-assimilating yeast; insect cells such as Drosophila S2 and Spodoptera Sf9
And the like; mammalian cells such as CHO, COS, BHK, 3T3, and C127; plant cells such as dicotyledonous plants.

Transformation or transfection can be performed by a known method such as a calcium phosphate method, DEAE-dextran mediated transfection, or electroporation. The enzyme of the present invention expresses the host cell transformed or transfected as described above under the control of a promoter, and recovers the produced target enzyme. For expression, after the host cells are grown or grown to an appropriate cell density, the promoter is induced by means of a temperature shift or chemical induction (such as IPTG), and the cells are further cultured for a certain period of time.

When the enzyme of the present invention is excreted out of the cell, it is directly from the medium, and when it is present in the cell, it is physically (ultrasound-destruction, mechanical destruction) or chemical (cells). After disrupting the cells with a lysing agent), the enzyme is purified. Enzymes can be obtained from recombinant cell culture medium by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, HPLC, electrophoresis, chromatofocusing, etc. Can be partially or completely purified by a combination of conventional techniques.

The present invention further relates to a dehydration method comprising contacting the enzyme of the present invention with a hydroxy compound in the presence of NAD or NADPH to perform a dehydrogenation reaction of the hydroxy compound and recovering a dehydrogenated product. A method for producing an elemental product is provided. The hydroxy compound can be quantified by utilizing this dehydrogenation reaction. In particular, it can be used for quantification of hydroxy amino acids (serine, threonine, etc.).

Examples of hydroxy compounds include hydroxy amino acids such as threonine and serine; primary, secondary and tertiary alcohols such as methanol, ethanol, propanol, butanol, glycerol and erythritol; sugar alcohols such as mannitol, arabitol and myoinositol However, all substrates that can be dehydrogenated by the enzyme of the present invention are intended to be included. The enzyme of the present invention can be used as it is or immobilized on an immobilized carrier. The immobilization is performed by incorporation of the enzyme into an insoluble carrier such as polysaccharide derivative, amino acid copolymer, polyacrylamide, styrene resin, ethylene-maleic acid copolymer, porous glass, etc. Can be done by

[0031]

The present invention will be further described by the following examples, which should not be construed as limiting the invention. <Example 1> Pyrococcus horikoshii (registration number JC
M9974) culture and separation of chromosomal DNA 13.5 g of NaCl, 4 g of Na ₂ SO ₄ , 0.7 g of KCl, 0.2 g of NaH
CO ₃ , 0.1 g of KBr, 30 mg of H ₃ BO ₃ , 10 g of MgCl ₂ .6H
Dissolve ₂ O, 1.5 g CaCl ₂ , 25 mg SrCl ₂ , 1.0 mL resasulin solution (0.2 g / L), 1.0 g yeast extract, and 5 g bactopeptone in 1 L water, and adjust the pH of this solution to 6.8. After being adjusted and saturated with carbon dioxide to make it anaerobic, JCM9974 was inoculated. Whether the medium became anaerobic was confirmed by adding a Na ₂ S solution and not coloring the pink color of the resasurin solution due to Na ₂ S in the culture solution. This culture was incubated at 95 ° C for 2 hours.
After culturing for ４4 days, the cells were collected by centrifugation at 5,000 rpm for 10 minutes.

The cells were cultured in 10 mM Tris (pH 7.5) -1 mM EDTA
After washing twice with the solution, it was sealed in an InCert Agarose (FMC) block. This block was treated in a 1% N-lauroyl sarcosine-1 mg / mL protease K solution to separate and prepare chromosomal DNA in an Agarose block.

<Example 2> Cloning, sequencing and construction of an expression plasmid of a thermophilic threonine dehydrogenase gene From the chromosomal DNA obtained in Example 1, a desired gene was cloned and sequenced. In order to introduce restriction enzyme sites NdeI and BamHI before and after the structural gene region of the dehydrogenase gene, two sets of primers in which a mutation was introduced into the NdeI cleavage sequence in the structural gene:

5'-TTTT GAATTC GTG CATATG TCAGAAAAGATGGTA-
3 '(SEQ ID NO: 3) (where the single underline before is EcoRI, and the single underline after is NdeI
Is shown. ) And 3'-TACGAATTTACTATGGAAATGATT CCTAGG GGG CCATGG TTTT-5 '
(SEQ ID NO: 4) (where the single underline before is BamHI and the single underline below is KpnI
Is shown. ); And 5'-TCTTGCAACCAG T ATATGTGGAACTGATCTTCATAT-3 '(SEQ ID NO: 5) (Here, the single underline indicates a mutation site. That is, T is substituted for C at position 484 in the structural gene sequence.) And 3'-ATTCCAAGAACGTTGGTCATATACACCTTGACTAGA-5 '(SEQ ID NO: 6)

The desired restriction enzyme sites were introduced before and after the gene by PCR. After the PCR reaction, the restriction enzyme Nde
The cells were treated with I and BamHI at 37 ° C. for 2 hours to completely degrade, and the structural gene was purified. Sequence analysis revealed that the thermophilic threonine dehydrogenase gene had the nucleotide sequence shown in SEQ ID NO: 2 and that the enzyme had the amino acid sequence shown in SEQ ID NO: 1.

The plasmid pET-11a (manufactured by Novagen) was digested with restriction enzymes NdeI and BamHI, purified and then reacted with the above structural gene at 16 ° C. for 2 hours using T4 ligase.
Connected. A part of the ligated DNA was subjected to E. coli-XL2-BlueMRF '.
Into competent cells (Novagen) to obtain transformant colonies. The expression plasmid was purified from the obtained colonies by an alkaline method (using a Plasmid Maxi Kit manufactured by Qiagen). This expression plasmid is transformed into E. coli strain E. coli.
Transfer to BL21 (DE3) (Novagen) and "Escherichia coli TDH"
Was deposited with the Research Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry on August 7, 1998, and given the accession number FERM P-16931.

Example 3 Expression of Recombinant Gene Competent cells of Escherichia coli (E. coli BKL21 (DE3); manufactured by Novagen) were thawed and 0.1 mL thereof was transferred to a Falcon tube. 0.005 mL of the expression plasmid solution obtained in Example 2 was added to the tube, and the mixture was allowed to stand on ice for 30 minutes.
Heat shock for 2 seconds, 2YT medium (10 g yeast extract, 16 g bactotryptone, 5 g NaCl in 1 liter of medium)
0.9 mL of a solution containing) was added, and the mixture was cultured at 37 ° C for 1 hour. Thereafter, an appropriate amount was spread on a 2YT agar plate containing ampicillin and cultured at 37 ° C. overnight to obtain a transformant. The obtained transformant was cultured for 600 n in a 2YT medium containing ampicillin.
After culturing until the absorption of m reaches 1, IPTG (isopropyl-β-D-thiogalactopyranoside) was added.
The cells were cultured for 6 hours. After the culture, the cells were collected by centrifugation at 6,000 rpm for 20 minutes.

<Example 4> Purification of thermophilic threonine dehydrogenase enzyme To the transformed Escherichia coli obtained in Example 3, twice the amount of alumina was added, and the cells were pulverized, and then 5 times 10 mM Tris-HCl was added.
A buffer (pH 8.0) was further added to obtain a suspension. The suspension was heated at 85 ° C. for 30 minutes, centrifuged at 11,000 rpm for 20 minutes, the supernatant was adsorbed to a HiTrap Q (Pharmacia) column, the fraction was monitored by electrophoresis, and the activity was monitored. Fractions were obtained. The obtained active fraction solution was concentrated with Centricon (manufactured by Amicon), and then subjected to gel filtration column Superdex 200 (Pharmacicon).
(a company) to obtain a purified enzyme. The obtained enzyme had the following properties.

(1) Optimum pH The optimum pH of the enzyme activity was 50 mM sodium acetate buffer, 50 mM
M4 phosphate buffer and 50 mM Tris-HCl buffer were used to make a buffer having a pH of 4 to 8, and the activity was determined by the following activity measurement. That is, the specific substrate L-threonine 10 mM, NAD 1m
M, 1 mM solution of ZnSO _4, and the reaction solution containing the purified enzyme solution 10μL
One mL was prepared and determined by measuring the initial rate of hydrolysis activity of the enzyme at 65 ° C. Spectrophotometer (JASCO V550)
Was used to measure the increase in absorbance at 340 nm. as a result,
The optimum pH of the purified enzyme was 7.5 to 10.0.

(2) Optimal temperature Using 10 mM L-threonine as a specific substrate,
A certain amount of enzyme was added to s buffer (pH 8) and allowed to react, and the relative activity was examined. The optimum temperature was about 95 ° C. (3) Heat resistance The purified enzyme solution (0.45 mg / mL) was heated at 95 ° C. for 3 hours, and the remaining activity was examined. The decrease in the activity was 10% or less. Circular dichroism (CD) was measured at 25-90 ° C, and the results at 25 ° C and 90 ° C were obtained by dissolving 0.1 g / mL enzyme in 100 mM NaCl in 50 mM phosphate buffer. In comparison, it was observed that slight thermal denaturation occurred at 90 ° C.
According to the calorimeter (DSC), the main peak of denaturation was observed at 95, 107 and 114 ° C.

(4) Molecular weight and metal content Although electrophoresis of the purified enzyme showed a molecular weight of 30,000 to 40,000 daltons, the gel filtration eluate showed activity at an elution position twice as large as 30,000 to 40,000. Eluted, indicating that this enzyme was usually present as a homodimer. In addition, atomic emission analysis (Inductively coupled plasma atomic emission)
spectrometry) revealed that the enzyme contained one zinc molecule.

[0042]

According to the present invention, there is provided a thermophilic enzyme capable of dehydrogenating a hydroxyamino acid or alcohol at a temperature exceeding 70 ° C, particularly 90 ° C. This enzyme can be used for analysis such as enzymatic dehydrogenation of hydroxy compounds at high temperature, regeneration of coenzyme, and specific quantification of hydroxy amino acids such as threonine and serine.

[0043]

[Sequence List] SEQUENCE LISTING <110> Agency of Industrial Science and Technology <120> Heat-resistant dehydrogenase, DNA encoding the same, process for preparing the same, and use of the same <130> 11900223 <160> 6 <210 > 1 <211> 348 <212> PRT <213> Pyrococcus horikoshii <400> 1 Met Ser Glu Lys Met Val Ala Ile Met Lys Thr Lys Pro Gly Tyr Gly 1 5 10 15 Ala Glu Leu Val Glu Val Asp Val Pro Lys Pro Gly Pro Gly Glu Val 20 25 30 Leu Ile Lys Val Leu Ala Thr Ser Ile Cys Gly Thr Asp Leu His Ile 35 40 45 Tyr Glu Trp Asn Glu Trp Ala Gln Ser Arg Ile Lys Pro Pro Gln Ile 50 55 60 Met Gly His Glu Val Ala Gly Glu Val Val Glu Ile Gly Pro Gly Val 65 70 75 80 Glu Gly Ile Glu Val Gly Asp Tyr Val Ser Val Glu Thr His Ile Val 85 90 95 Cys Gly Lys Cys Tyr Ala Cys Arg Arg Gly Gln Tyr His Val Cys Gln 100 105 110 Asn Thr Lys Ile Phe Gly Val Asp Thr Asp Gly Val Phe Ala Glu Tyr 115 120 125 Ala Val Val Pro Ala Gln Asn Ile Trp Lys Asn Pro Lys Ser Ile Pro 130 135 140 Pro Glu Tyr Ala Thr Leu Gln Glu Pro Leu Gl y Asn Ala Val Asp Thr 145 150 155 160 Val Leu Ala Gly Pro Ile Ser Gly Lys Ser Val Leu Ile Thr Gly Ala 165 170 175 Gly Pro Leu Gly Leu Leu Gly Ile Ala Val Ala Lys Ala Ser Gly Ala 180 185 190 Tyr Pro Val Ile Val Ser Glu Pro Ser Asp Phe Arg Arg Glu Leu Ala 195 200 205 Lys Lys Val Gly Ala Asp Tyr Val Ile Asn Pro Phe Glu Glu Asp Val 210 215 220 Val Lys Glu Val Met Asp Ile Thr Asp Gly Asn Gly Val Asp Val Phe 225 230 235 240 Leu Glu Phe Ser Gly Ala Pro Lys Ala Leu Glu Gln Gly Leu Gln Ala 245 250 255 Val Thr Pro Ala Gly Arg Val Ser Leu Leu Gly Leu Tyr Pro Gly Lys 260 265 270 Val Thr Ile Asp Phe Asn Asn Leu Ile Ile Phe Lys Ala Leu Thr Ile 275 280 285 Tyr Gly Ile Thr Gly Arg His Leu Trp Glu Thr Trp Tyr Thr Val Ser 290 295 300 Arg Leu Leu Gln Ser Gly Lys Leu Asn Leu Asp Pro Ile Ile Thr His 305 310 315 320 Lys Tyr Lys Gly Phe Asp Lys Tyr Glu Glu Ala Phe Glu Leu Met Arg 325 330 335 Ala Gly Lys Thr Gly Lys Val Val Phe Met Leu Lys 340 <210> 2 <211> 1047 <212> DNA <213> Pyrococcus horikoshii <400> atgtcagaaa agatggtagc tatcatgaag acaaagcctg ggtatggtgc tgagcttgtt 60 gaggtggacg ttccaaagcc tggacctgga gaagttctca ttaaggttct tgcaaccagc 120 atatgtggaa ctgatcttca tatatacgag tggaatgagt gggcacagag caggattaag 180 ccacctcaga taatggggca tgaagttgcg ggagaagtcg ttgaaatcgg gccaggtgtg 240 gaaggaattg aagttggtga ctacgtttcc gttgaaacgc acatagtttg tggcaagtgt 300 tacgcctgca gaaggggcca gtaccacgtc tgccagaata ccaagatatt tggcgtcgat 360 acagatggcg tattcgcaga gtatgccgtt gttccagccc aaaatatctg gaagaatcct 420 aaatcaattc caccagaata tgcaaccctc caggagccct tgggtaatgc tgttgatacc 480 gttttggcag gaccaatttc tggtaagagc gttttaataa ccggagcggg gccccttgga 540 ttattgggaa ttgcagtggc aaaagcgtct ggcgcatacc ctgtgatagt ctcagaacct 600 agtgacttca gaagggagtt ggccaagaag gtaggtgctg attacgttat caatccattc 660 gaagaggatg tcgttaagga ggtaatggat atcacagatg gaaatggtgt tgacgtattt 720 ctggaattca gtggagctcc taaggctcta gagcagggtc ttcaagcagt tacacctgct 780 ggaagggtat ctctcctggg tctataccca ggaaaggtta ccattgactt taacaatcta 840 ataatcttca aggcgct aac aatctatgga ataactggaa ggcacctctg ggaaacttgg 900 tatacagttt caaggctcct tcagagcgga aagctcaact tagatcctat tataactcat 960 aaatataagg gattcgacaa gtacgaggaa gcctttgagc taatgagggc cggcaaaacg 1020 ggtaaagttg tttttatgct taaatga 1047 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <400> 3 ttttgaattc gtgcatatgt cagaaaagat ggta 34 <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <400> 4 tacgaattta ctatggaaat gattcctagg gggccatggt ttt 43 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <400> 5 tcttgcaacc agtatatgtg gaactgatct tcatat 36 <210> 6 <211> <212> DNA <213> Artificial Sequence <400> 6 attccaagaa cgttggtcat atacaccttg actaga 36

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:19) (C12N 9/04 C12R 1:19) (C12N 15/09 ZNA C12R 1:01) (72) Inventor Matsui Ikuo 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, Institute of Biotechnology and Industrial Technology Research Institute (72) Inventor Hiroyasu Ishida 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, Institute of Biotechnology and Industrial Technology Research Institute (72) Inventor Kazuhiko Ishikawa 1-3-1 Higashi, Tsukuba City, Ibaraki Pref., Institute of Industrial Technology, Biotechnology and Industrial Technology Research Institute (72) Inventor Susumu Ando 1-3-1, Higashi 1-3-1 Higashi, Tsukuba, Ibaraki Pref., Institute of Biotechnology, Industrial Technology Institute ( 72) Inventor Aniseur Rahman Khan, Bangladesh, Dhaka-1216, Mirpur, House-9, Road-4, Block -G, Section -1 examiner Susumu Hikichi (56) Reference Biochem. J. 316 (1996) p. 115-122 Extremophiles 2 (1998, May) p. 123-130 DNA Res. 5 (1998, May) p. 55-76 (58) Field surveyed (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) GenBank / EMBL / DDBJ / Geneseq JICST file (JOIS) WPI (DIALOG)

Claims (15)

(57) [Claims] (1) an optimum pH of 7.5 to 10.0 and a molecular weight of 30,000;
~ 40,000 Dalton (electrophoretic measurement) and NAD
Or Japanese that serine and Toreoni down in the presence of NADP may be dehydrogenation reaction at temperatures above 90 ° C.
A thermophilic threonine dehydrogenase enzyme that is characteristic. 2. The enzyme according to claim 1, which is derived from the genus Pyrococcus. 3. The enzyme according to claim 2, wherein the optimum temperature is about 95 ° C. 4. The enzyme according to claim 1, which is a recombinant. 5. An amino acid sequence having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or having one or more amino acids deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. The enzyme according to any one of claims 1 to 4, wherein the enzyme has threonine dehydrogenase activity at a temperature exceeding 90 ° C. 6. A DN encoding the enzyme according to claim 5.
A. 7. The DNA according to claim 6, which comprises the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 8. Hybridization with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing under stringent conditions, and threonine dehydration at a temperature exceeding 90 ° C.
7. Encoding an enzyme having a drogenase activity.
DNA according to 1. 9. An expression vector comprising the DNA according to any one of claims 6 to 8. 10. The expression vector according to claim 9, which is a plasmid containing a DNA having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. [11] A host cell transformed with the expression vector according to [9] or [10]. 12. The host cell according to claim 11, which is Escherichia coli. (13) culturing the host cell according to (11) or (12), resulting in an optimum pH of 7.5 to 10.0 produced as a result of expression.
Has a molecular weight of 30,000 to 40,000 daltons (electrophoresis measurement).
And serine and tritium in the presence of NAD or NADP.
A method for producing the enzyme, comprising recovering a threonine dehydrogenase enzyme capable of dehydrogenating leonine at a temperature exceeding 90 ° C. 14. Use of the enzyme according to any one of claims 1 to 5 in enzymatic dehydrogenation of a hydroxy compound in the presence of NAD or NADP, or in quantification of the hydroxy compound using the reaction. (15) the hydroxy compound is serine or threo;
15. Use according to claim 14, which is nin .