JPH10165181A - Heat-resistant dehydrogenase gene of isocitric acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene comprising a DNA coding a heat- resistant isocitric acid dehydrogenase having a specific amino acid sequence and capable of inexpensively and readily producing the dehydrogenase enzyme useful as a reagent, etc., for measuring D-isocitric acid in high amount of production. SOLUTION: This new DNA codes heat-resistant isocitric acid dehydrogenase comprising an amino acid sequence represented by the formula or an amino acid sequence obtained by adding, loosing or replacing one or plural amino acids in this amino acid sequence and having an amino acid sequence bringing about isocitric acid dehydrogenase activity, and the DNA can inexpensively produce a heat-resistant isocitric acid dehydrogenase derived from Thermus aquaticus and catalyzing a reaction converting isocitric acid to α-ketoglutaric acid and useful as a reagent, etc., for measuring D-isocitric acid in high productivity. The DNA is obtained by screening chromosomal DNA library of Thermus aquaticus YT1 (ATCC 25104).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA encoding a thermostable isocitrate dehydrogenase derived from Thermus aquaticus,
The present invention relates to a recombinant vector containing the DNA, a transformant transformed with the recombinant vector, and a method for producing isocitrate dehydrogenase using the transformant.

[0002]

BACKGROUND ART Isocitrate dehydrogenase is an enzyme that catalyzes a reaction of producing α-ketoglutarate by oxidative decarboxylation of a substrate isocitrate with a coenzyme NADP and an essential metal Mg ^2+. It is useful as a reagent for measuring isocitrate. However, the isocitrate dehydrogenase known so far is generally easily deactivated by heat, lacks stability when used as an enzyme reagent in the above-mentioned measurement system, and has a complicated production method, and is high. There was a problem that the production amount could not be reduced at a low price.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a heat-resistant isocitrate dehydrogenase which does not inactivate even at a high temperature, and which is a source of the heat-resistant isocitrate dehydrogenase in order to easily produce the enzyme in a high production amount. DN encoding isocitrate dehydrogenase
A: To provide a recombinant vector containing the DNA, and a method for producing a thermostable isocitrate dehydrogenase using a transformant transformed with the recombinant vector.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors isolated and determined the structure of a thermostable isocitrate dehydrogenase gene from the thermophile Thermos aquaticus. In addition, the inventors have succeeded in efficiently producing thermostable isocitrate dehydrogenase using genetic engineering techniques, and have completed the present invention.

That is, the present invention relates to an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids have been added, deleted or substituted, and has an isocitrate dehydrogenase activity. Is a DNA encoding a thermostable isocitrate dehydrogenase encoding an amino acid sequence that results in The present invention is further characterized in that a recombinant vector containing the DNA, a transformant transformed with the recombinant vector, and culturing the transformant in a medium to collect thermostable isocitrate dehydrogenase. Is a method for producing a thermostable isocitrate dehydrogenase. Hereinafter, the present invention will be described in detail.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The DNA encoding the thermostable isocitrate dehydrogenase of the present invention is obtained, for example, from a microorganism which is a donor of the thermostable isocitrate dehydrogenase gene that produces the thermostable isocitrate dehydrogenase. Can be Such microorganisms include those belonging to Thermus Aquaticus,
Specifically, Thermus Aquaticus YT1 (ATCC 251
04) can be used.

The DNA encoding the thermostable isocitrate dehydrogenase of the present invention can be obtained through the following steps. (i) DNA that is isolated and purified from a microorganism that is a donor of a thermostable isocitrate dehydrogenase gene that produces a thermostable isocitrate dehydrogenase, and that is obtained by cutting the DNA with a restriction enzyme, The DNA is ligated and closed at the cohesive ends of both DNAs with DNA ligase or the like to obtain a recombinant DNA vector.
The A vector is transferred to a replicable host microorganism, and colony hybridization is performed using a synthetic DNA probe prepared based on a partial amino acid sequence derived from a thermostable isocitrate dehydrogenase to retain the recombinant DNA vector. The microorganism is isolated, and (iii) the recombinant DNA vector is separated and purified from the cultured microorganism cells, and the target DNA is cut out with a restriction enzyme.

[0008] The above acquisition method will be described in detail below.
First, the above-mentioned bacteria, which are donor microorganisms, are aerated and stirred in a liquid medium, for example, for about 1 to 2 days, and the resulting culture is collected by centrifugation, and then lysed to obtain heat-resistant isocitrate dehydrogenation. A lysate containing the enzyme gene can be prepared. As a lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme, and other enzymes such as a protease and a surfactant such as sodium lauryl sulfate are used in combination as necessary, and further, freeze-thawing which is a method of physically destroying the cell wall. Alternatively, a French press treatment may be performed in combination with the lysis method described above.

From the lysate thus obtained, DNA
Can be separated and purified by a conventional method, for example, by appropriately combining methods such as protein removal treatment by phenol extraction, protease treatment, ribonuclease treatment, and alcohol precipitation centrifugation.

[0010] The method of cutting the DNA of the separated and purified microorganism can be carried out by, for example, ultrasonic treatment or treatment with a restriction enzyme. However, in order to facilitate the binding of the obtained DNA fragment to a vector, treatment with the restriction enzyme is carried out. It is preferable to carry out. Restriction enzymes which act, inter alia, on specific nucleotide sequences, such as HindIII, B
Type II restriction enzymes such as amHI and SacI are suitable.

As the vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous propagation in a host microorganism is suitable. Examples of phage include Escherichia coli (Escherichia coli).
ia coli) as the host microorganism, M13m
p18, λgt10, λgt11 and the like can be used. As the plasmid, for example, when Escherichia coli is used as a host microorganism, pBR322, pACYC1
84, pUC118, pBluescript, and the like. It is desirable to obtain a vector fragment by cleaving such a vector with the same restriction enzyme used for cutting the microorganism DNA which is the thermostable isocitrate dehydrogenase gene donor described above.

The method of linking the microbial DNA fragment and the vector fragment may be any method using a known DNA ligase. For example, after annealing the cohesive end of the microbial DNA fragment and the cohesive end of the vector fragment, an appropriate DNA
A recombinant DNA of a microbial DNA fragment and a vector fragment is prepared by using ligase. If necessary, after annealing, the DNA can be transferred to a host microorganism, and a recombinant DNA can be prepared using in-vivo DNA ligase.

As the host microorganism, any recombinant DNA can be stably and autonomously proliferated. For example, Escherichia coli XL1-Blue MRF ', Escherichia
Cory JM109, Escherichia Cory HB101 etc. can be used.

As a method for transferring the recombinant DNA to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, the recombinant DNA is transferred using a commercially available competent cell, and then the microinjection method is performed. May be used.

Selection of the presence or absence of transfer of the target recombinant DNA to the host microorganism is performed by a method for searching for a microorganism capable of simultaneously expressing the drug system marker of the vector carrying the target recombinant DNA and thermostable isocitrate dehydrogenase. Alternatively, there is a method in which colonies grown on a selective medium are searched by performing hybridization with a DNA probe synthesized based on the amino acid sequence.

The nucleotide sequence of the thermostable isocitrate dehydrogenase gene obtained by the above-described method is the same as that of Science (Sc.
issue), 214, 1205-1210 (198)
It is decoded by the dideoxy method described in 1) (SEQ ID NO: 2), and the entire amino acid sequence of the thermostable isocitrate dehydrogenase is deduced from the base sequence (SEQ ID NO: 1). On the other hand, many methods can be used to obtain a DNA encoding a sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1. For example, a method of mutagenizing a gene to produce a point or deletion mutation; a method of selectively cleaving a gene, then removing or adding selected nucleotides, and ligating the gene; Mutagenesis and the like can be mentioned. The recombinant D containing the thermostable isocitrate dehydrogenase gene once selected in this way
NA can be easily removed from transformed microorganisms and transferred to other host microorganisms. Further, a vector fragment obtained by cutting a heat-resistant isocitrate dehydrogenase gene DNA from a recombinant DNA having a heat-resistant isocitrate dehydrogenase gene by a restriction enzyme or the like, and cutting the same by a similar method. And transfer to a host microorganism can be easily carried out.

The culture form of the host microorganism, which is a transformant, may be selected by taking into consideration the nutritional and physiological properties of the host. Usually, liquid culture is used in many cases. It is advantageous to perform As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose,
Molasses, pyruvic acid and the like are used. As a nitrogen source,
Any available nitrogen compound, such as peptone,
Meat extract, yeast extract and the like are used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

The cultivation temperature can be appropriately changed within a range in which the bacteria grow and produce thermostable isocitrate dehydrogenase. In the case of Escherichia coli, the temperature is preferably about 20 to 42 ° C. The cultivation time varies somewhat depending on the conditions, but the culture may be terminated at an appropriate time in consideration of the time when the thermostable isocitrate dehydrogenase reaches the maximum yield.
It is about 12 to 36 hours. The pH of the medium can be appropriately changed within a range in which the bacteria grow and produce a thermostable isocitrate dehydrogenase, but the pH is particularly preferably about 6.0 to 8.0.

The thermostable isocitrate dehydrogenase in the culture is obtained by collecting the cells by means such as filtration or centrifugation of the culture and then destroying the cells by a mechanical method or an enzymatic method such as lysozyme. And, if necessary, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and / or
Alternatively, a surfactant is added to solubilize the thermostable isocitrate dehydrogenase, and separated and collected as an aqueous solution.

The heat-resistant isocitrate dehydrogenase thus obtained is concentrated, for example, under reduced pressure, membrane concentrated, and further subjected to salting out treatment with ammonium sulfate, sodium sulfate, or the like, or a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. May be precipitated by a fractional precipitation method according to the method described above. Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[0021]

【Example】

[Example 1] (Separation of chromosomal DNA) The chromosomal DNA of Thermus Aquaticus YT1 (ATCC 25104) was isolated by the following method. The strain was transformed into 200 ml of 0.5% polypeptone, 0.3% yeast extract, 0.2%
70 in a liquid medium consisting of sodium chloride (pH 7.5)
After shaking culture at ℃ overnight, centrifuge (4000 rpm, 10 minutes)
And collected. 0.15M sodium chloride, 0.1M
The cells are washed once with EDTA and centrifuged again (400
(0 rpm, 10 minutes). Some of the cells (0.
6 g) in 12 ml of Tris-hydrochloric acid (pH 9.0), 1%
The cells were suspended in a solution containing sodium lauryl sulfate and 0.1 M sodium chloride, freeze-thawed twice, and lysed. 1 mg of proteinase K was added to this solution,
For 3 hours. Then add an equal amount of phenol,
The mixture was stirred slowly, and the aqueous layer and the oil layer were separated by centrifugation (4000 rpm, 10 minutes), and the aqueous layer was collected. This operation was repeated twice more, and twice the amount of ethanol was gently added to the finally collected aqueous layer. The precipitated DNA is wound up with a glass rod and washed with 70% ethanol, and then 2 m
1 mM of 10 mM Tris-hydrochloric acid and 1 mM EDTA. Escherichia coli XL1-Blue MRF 'was purchased from Stratagene, and competent cells were prepared by the method of Hanahan and used as a host for library preparation.

Example 2 (Preparation of DNA Fragment Containing Gene Encoding Thermostable Isocitrate Dehydrogenase and Recombinant Vector Having the DNA Fragment) 1 μg of the DNA obtained in Example 1 was digested with the restriction enzyme BamHI at 37 ° C. C. for 2 hours to completely decompose, then use restriction enzyme B
pBluesc, a vector completely digested with amHI
1 μg of riptIISK (+) (Stratagene)
And T4 DNA ligase at 16 ° C. overnight, and ligated to the vector. A portion of the ligated DNA is
The cells were introduced into competent cells of Collie XL1-Blue MRF 'to obtain about 4000 transformant colonies.
On the other hand, the N-terminal amino acid sequence of the thermostable isocitrate dehydrogenase directly purified from the cells of Thermus aquaticus was determined, and a degenerate synthetic DNA probe (5′-ATCCAGATCCC [CG] CAGGA) based on this sequence was determined.
GGG [CG] GAGAAGATCAC [CG] ATC
CAGGAGGG-3 ′, a portion surrounded by [] indicates a degenerated portion). A replica of the above-mentioned emerged colony was copied to a nitrocellulose membrane by a known method, lysed by a known method, DNA was fixed on the membrane, and a probe labeled with a synthetic DNA probe labeling kit manufactured by Amersham was used. To perform colony hybridization. As a result, two positive clones were obtained, and both clones had the same BamH of about 3.0 kb.
It had an I insert. One of these plasmids is called pT
It was ac31-1.

[Example 3] (Determination of base sequence) The base sequence of the 3.0 kb BamHI inserted fragment of pTac31-1 was determined using an AutoRead sequencing kit manufactured by Pharmacia. The determined nucleotide sequence is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2.

Example 4 (Construction of Expression Plasmid) An NdeI restriction enzyme site was introduced near the start codon of the thermostable isocitrate dehydrogenase gene of pTac31-1 by site-directed mutagenesis, and restriction was carried out from the obtained plasmid. The coding region is cut out using enzymes NdeI and XhoI,
It was ligated to the separately prepared expression vector ptKSNd (-). ptKSNd (-) is obtained by modifying the lac promoter of the vector pBluescriptIIKS (-) manufactured by Stratagene to a tac promoter,
This is one in which an NdeI restriction enzyme site has been introduced so as to overlap the start codon of the cZ gene. Thus, a high expression plasmid pEXAC1 of thermostable isocitrate dehydrogenase was prepared. Using this pEXAC1, Escherichia
coli). The obtained transformant was 1996
November 28, FE joined the Institute of Biotechnology and Industrial Technology
Deposited as RM P-15962.

Example 5 (Expression of Recombinant Thermostable Isocitrate Dehydrogenase Gene) The high expression plasmid pEXAC1 of the thermostable isocitrate dehydrogenase obtained in Example 4 was transformed into Escherichia coli XL1.
-BlueMRF 'was introduced into competent cells to obtain a transformant. The transformant was treated with 2 L of 1.6% tryptone, 1.0% yeast extract, 0.5% sodium chloride,
After shaking culture at 37 ° C. overnight in a liquid medium containing 0.1 mM isopropyl-β-D-thiogalactopyranoside and 100 μg / μl ampicillin (pH 7.5), centrifugation (400
(0 rpm, 10 minutes). 3 times the amount of cells
0 mM Tris-HCl (pH 7.6), 0.5 mM ED
The suspension was suspended in a solution containing TA and disrupted by sonication. The suspension was subjected to ultracentrifugation at 45,000 rpm for 30 minutes to obtain a cell extract, and the supernatant was transferred to another tube.
Heat treatment was performed at 30 ° C. for 30 minutes. The precipitated denatured protein is removed by centrifugation, and then the supernatant is subjected to DEAE Toyopearl (Tosoh).
For purification. The above-mentioned heat-treated protein solution is adsorbed to DEAE Toyopearl previously equilibrated with a solution containing 10 mM Tris-hydrochloric acid (pH 7.6) and 0.5 mM EDTA, non-adsorbed substances are washed away with the equilibration solution, and further added to the equilibration solution. A concentration gradient with a solution containing 1.0 M sodium chloride was applied, and 100 mg of the eluted thermostable isocitrate dehydrogenase was recovered.

[Example 6] (Properties of thermostable isocitrate dehydrogenase) (1) Optimum temperature
200 μl of activity measurement solution containing 0 mM Tris-hydrochloric acid (pH 7.6), 0.5 mM magnesium chloride, 0.5 mM NADP, and 0.5 mM isocitrate, warmed to each temperature
Was performed by adding 4 μl of the enzyme solution to the sample. The activity was measured by following the increase in absorption at 340 nm that occurs when the coenzyme NADP was converted to NADPH, and the initial rate was used as the activity value. As shown in FIG. 1, the optimal temperature of the activity was 85 ° C.
It was near.

(2) Optimum pH The optimum pH of the activity of the thermostable isocitrate dehydrogenase was measured using 0.5 mM magnesium chloride, 0.5 mM NAD.
P, in a solution containing 0.5 mM isocitrate, to a final concentration of 50
Bistrispropane of various pHs to give mM was added to prepare a solution having a total volume of 200 μl, and 4 μl of an enzyme solution was added. The measurement of the activity is the same as described above. The activity measurement temperature was unified to 60 ° C. As shown in FIG. 2, the optimum pH for the activity was around 8 to 9.5.

(3) Thermostability The thermostability of isocitrate dehydrogenase is measured by 50
This was performed by adding 4 μl of the enzyme solution to 200 μl of an activity measurement solution containing mM Tris / hydrochloric acid (pH 7.6), 0.5 mM magnesium chloride, 0.5 mM NADP, and 0.5 mM isocitrate. After heating 10 µl of the enzyme solution at each temperature for 10 minutes, 4 µl of the enzyme solution was collected and used for activity measurement. As shown in FIG. 3, the thermostable isocitrate dehydrogenase has an activity of about 2/3 even after a heat treatment at 90 ° C. without impairing the activity even at a heat treatment at 80 ° C. for 10 minutes. Was.

[0029]

According to the present invention, there are provided a gene encoding a thermostable isocitrate dehydrogenase and a method for efficiently producing the thermostable isocitrate dehydrogenase by genetic engineering using the gene. You.

[0030]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 426 Sequence type: amino acid Number of chains: double-stranded Topology: linear Sequence type: peptide sequence Met Ala Tyr Gln Arg Ile Gln Ile Pro Gln Glu Gly Glu Lys Ile Thr 1 5 10 15 Ile Gln Glu Gly Val Leu Lys Val Pro Asp Gln Pro Ile Ile Gly Phe 20 25 30 Ile Glu Gly Asp Gly Thr Gly Pro Asp Ile Trp Arg Ala Ala Gln Pro 35 40 45 Val Leu Asp Ala Ala Val Ala Lys Ala Tyr Gly Gly Gln Arg Arg Ile 50 55 60 Val Trp Val Glu Leu Tyr Ala Gly Glu Lys Ala Asn Gln Val Tyr Gly 65 70 75 80 Glu Pro Ile Trp Leu Pro Glu Glu Thr Leu Glu Phe Ile Arg Glu Tyr 85 90 95 Leu Val Ala Ile Lys Gly Pro Leu Thr Thr Pro Val Gly Gly Gly Ile 100 105 110 Arg Ser Ile Asn Val Ala Leu Arg Gln Glu Leu Asp Leu Tyr Ala Cys 115 120 125 Val Arg Pro Val Arg Trp Phe Gln Gly Val Pro Ser Pro Val Lys His 130 135 140 Pro Glu Leu Val Asn Met Val Ile Phe Arg Glu Asn Thr Glu Asp Ile 145 150 155 160 Tyr Ala Gly Ile Glu Trp Pro Ala Gly Ser Glu Glu Val Lys Lys Val 165 170 175 Leu Asp Ph e Leu Lys Arg Glu Phe Pro Lys Ala Tyr Ala Lys Ile Arg 180 185 190 Phe Pro Glu Thr Ser Gly Leu Gly Leu Lys Pro Ile Ser Lys Glu Gly 195 200 205 Thr Glu Arg Leu Val Glu Ala Ala Ile Glu Tyr Ala Ile Lys Glu Asp 210 215 220 Leu Pro Ser Val Thr Leu Val His Lys Gly Asn Ile Met Lys Phe Thr 225 230 235 240 Glu Gly Ala Phe Arg Glu Trp Gly Tyr Ala Leu Ala Arg Glu Lys Tyr 245 250 255 Gly Ala Thr Pro Leu Asp Gly Gly Pro Trp His Val Leu Lys Asn Pro 260 265 270 270 Arg Thr Gly Arg Glu Ile Val Ile Lys Asp Met Ile Ala Asp Asn Phe 275 280 285 Leu Gln Gln Ile Leu Leu Arg Pro Asp Glu Tyr Ser Val Ile Ala Thr 290 295 300 Met Asn Leu Asn Gly Asp Tyr Ile Ser Asp Ala Leu Ala Ala Gln Val 305 310 315 320 Gly Gly Ile Gly Ile Ala Pro Gly Ala Asn Ile Asn Tyr Lys Thr Gly 325 330 335 His Ala Val Phe Glu Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Gly 340 345 350 Gln Asp Lys Val Asn Pro Ser Ser Val Ile Leu Ser Gly Glu Met Met 355 360 365 Leu Arg Tyr Leu Gly Trp Asn Glu Ala Ala Asp Leu Ile Ile Arg Ala 370 375 380 380 Met Glu Arg Th r Ile Ser Lys Gly Leu Val Thr Tyr Asp Phe His Arg 385 390 395 400 Leu Leu Val Ala Glu Gly Lys Pro Ala Thr Leu Leu Lys Thr Ser Glu 405 410 415 Phe Gly Gln Ala Leu Ile Gln His Met Asp *** 420 425

SEQ ID NO: 2 Sequence length: 2872 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: chromosomal DNA Origin: Thermus aquaticus Strain: YT1 (ATCC25104) Sequence GCTCCTTCCC GTGGACCCCT GGGCGAAGGT GGCCTCCTTC TCCAAAAGGA GGACCTTAAG 60 CCCCGCCTCC GCTAGCCGGT AGGCCGAGGC CGCCCCTACG ATCCCCGCCC CCACCACCAG 120 CACATCCGCC ACCTCCCCAG TTTAGGAAGC CGGGAGTATG CTAGGCCCCG GAGGTACCT 179 ATG GCC TAC CAG CGC ATC CAG ATT CCC CAG GAG GGC GAA AAG ATC ACC 227 Met Ala Tyr Gln Arg Ile Gln Ile Pro Gln Glu Gly Glu Lys Ile Thr 1 5 10 15 ATC CAA GAG GGC GTC CTG AAG GTG CCG GAC CAG CCC ATC ATC GGC TTC 275 Ile Gln Glu Gly Val Leu Lys Val Pro Asp Gln Pro Ile Ile Gly Phe 20 25 30 ATT GAG GGG GAT GGG ACC GGC CCT GAC ATC TGG AGA GCG GCC CAA CCC 323 Ile Glu Gly Asp Gly Thr Gly Pro Asp Ile Trp Arg Ala Ala Gln Pro 35 40 45 GTC CTA GAC GCC GCC GTG GCC AAA GCC TAC GGC GGG CAA CGG CGC ATC 371 Val Leu Asp Ala Ala Val Ala Lys Ala Tyr Gly Gly Gln Arg Arg Ile 50 55 60 GTC TGG GTG GAG CTT TAC GCC GGG GAA AAG GCC AAC CAG GTC TAC GGG 419 Val Trp Val Glu Leu Tyr Ala Gly Glu Lys Ala Asn Gln Val Tyr Gly 65 70 75 80 GAG CCC ATC TGG CTC CCC GAG GAG ACC CTG GAG TTC ATC CGG GAG TAC 467 Glu Pro Ile Trp Leu Pro Glu Glu Thr Leu Glu Phe Ile Arg Glu Tyr 85 90 95 CTG GTG GCC ATC AAG GGC CCC CTG ACC ACG CCG GTG GGC GGC GGC ATC 515 Leu Val Ala Ile Lys Gly Pro Leu Thr Thr Pro Val Gly Gly Gly Ile 100 105 110 CGG AGC ATC AAC GTG GCC CTC AGG CAG GAG CTG GAC CTC TAC GCC TGC 563 Arg Ser Ile Asn Val Ala Leu Arg Gln Glu Leu Asp Leu Tyr Ala Cys 115 120 125 GTG CGC CCC GTG CGC TGG TTC CAG GGG GTG CCC AGT CCG GTG AAG CAC 611 Val Arg Pro Val Arg Trp Phe Gln Gly Val Pro Ser Pro Val Lys His 130 135 140 CCG GAG CTG GTC AAC ATG GTC ATC TTC CGG GAG AAC ACC GAG GAC ATC 659 Pro Glu Leu Val Asn Met Val Ile Phe Arg Glu Asn Thr Glu Asp Ile 145 150 155 160 TAC GCC GGG ATT GAG TGG CCG GCG GGG AGC GAG GAG GTA AAG AAG GTC 707 Tyr Ala Gly I le Glu Trp Pro Ala Gly Ser Glu Glu Val Lys Lys Val 165 170 175 CTA GAC TTC TTG AAG CGG GAG TTC CCC AAG GCC TAC GCC AAG ATC CGC 755 Leu Asp Phe Leu Lys Arg Glu Phe Pro Lys Ala Tyr Ala Lys Ile Arg 180 185 190 TTC CCC GAG ACC TCG GGC CTG GGC CTG AAG CCC ATC TCC AAG GAG GGC 803 Phe Pro Glu Thr Ser Gly Leu Gly Leu Lys Pro Ile Ser Lys Glu Gly 195 200 205 ACG GAG CGC CTG GTG GAG GCG GCC ATT GAG TAC GCC ATC AAG GAG GAC 851 Thr Glu Arg Leu Val Glu Ala Ala Ile Glu Tyr Ala Ile Lys Glu Asp 210 215 220 CTC CCC AGC GTG ACC CTG GTC CAC AAA GGC AAC ATC ATG AAG TTC ACC 899 Leu Pro Ser Val Thr Leu Val His Lys Gly Asn Ile Met Lys Phe Thr 225 230 235 240 GAA GGG GCC TTC CGG GAG TGG GGC TAC GCC CTG GCC CGG GAA AAG TAC 947 Glu Gly Ala Phe Arg Glu Trp Gly Tyr Ala Leu Ala Arg Glu Lys Tyr 245 250 255 GGG GCC ACG CCC CTG GAC GGC GGG CCC TGG CAC GTC CTC AAA AAC CCC 995 Gly Ala Thr Pro Leu Asp Gly Gly Pro Trp His Val Leu Lys Asn Pro 260 265 270 CGC ACC GGC AGG GAG ATC GTT ATC AAG GAC ATG ATC GCC GAC AAC TTC 1043Arg Thr Gly Arg Glu Ile Val Ile Lys Asp Met Ile Ala Asp Asn Phe 275 280 285 CTG CAG CAG ATC CTC CTC CGC CCC GAC GAA TAC TCG GTG ATC GCC ACC 1091 Leu Gln Gln Ile Leu Leu Arg Pro Asp Glu Tyr Ser Val Ile Ala Thr 290 295 300 ATG AAC CTG AAC GGG GAC TAC ATC TCC GAT GCC CTG GCC GCC CAG GTG 1139 Met Asn Leu Asn Gly Asp Tyr Ile Ser Asp Ala Leu Ala Ala Gln Val 305 310 310 315 320 GGG GGC ATC GGC ATC GCC CCC GGG GCC AAC ATC AAC TAC AAG ACG GGC 1187 Gly Gly Ile Gly Ile Ala Pro Gly Ala Asn Ile Asn Tyr Lys Thr Gly 325 330 335 CAC GCC GTC TTT GAG GCC ACC CAC GGC ACC GCC CCC AAG TAC GCT GGC 1235 His Ala Val Phe Glu Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Gly 340 345 350 CAG GAC AAG GTG AAC CCC AGC AGC GTC ATC CTC TCC GGG GAG ATG ATG 1283 Gln Asp Lys Val Asn Pro Ser Ser Val Ile Leu Ser Gly Glu Met Met 355 360 365 CTT CGC TAC CTG GGC TGG AAC GAG GCG GCG GAC CTC ATC ATC AGG GCC 1331 Leu Arg Tyr Leu Gly Trp Asn Glu Ala Ala Asp Leu Ile Ile Arle Ala 370 375 380 ATG GAG AGG ACC ATC AGC AAG GGC CTG GTC ACC TAC GA C TTC CAC CGC 1379 Met Glu Arg Thr Ile Ser Lys Gly Leu Val Thr Tyr Asp Phe His Arg 385 390 395 400 CTC CTG GTG GCC GAG GGC AAG CCC GCC ACG CTT CTT AAG ACC AGC GAG 1427 Leu Leu Val Ala Glu Gly Lys Pro Ala Thr Leu Leu Lys Thr Ser Glu 405 410 415 TTC GGC CAG GCC CTG ATC CAG CAC ATG GAC TGA AAACGTTTGG GGCCCCCGCC 1480 Phe Gly Gln Ala Leu Ile Gln His Met Asp *** 420 425 GTGGCAAAAG CCACGGCGGG GTGCTGGAC GGCGCCGAGGCC GG GGCGTTGAGG GCAAGAGGCC CGGAGAGGGC 1600 CACCTGGGCG GAAAGCCCGA GCTCCGGGAG GAGGAAGACC CCCTGTCCCC CCGGCCTCTC 1660 CACCAGGACG CCCGGGCCCT CGTAGCCCTT TTCCATCAGG TAGAGGAGGG TCCAGTGGAG 1720 CTTGCTCCGC CTCTCCCCTT CCCGCACCAG GTCGGCCACC GCCTCCGCCG CCCCCACCCG 1780 CTCCAGGACC TCCCCCTGGG AAAGGGGCCT TTCCCCCTTG AGCCAGGCCC TGAGCTGCTG 1840 GTGAGCCACC AGGTCCAGGT AGCGCCTTAA GGGGCTCGTC ACCTGGGCGT AGAGGGGAAG 1900 GCCGAGGCCC CGGTGGGGGG CGGGGACGGCC TTGAGCTGCG CCCTCTTCA GGGTCTTCCG 1960 CTGCGCCCAC ATGGCGGCGA GGCCCTCCCCC TCCACCCGGT GGGAAGGGGC CTCCTGGGT 2020 GGCGAAGGGA AAGGGAAGGC CCTCCCTCAGG GCCAGGTGGG CGGCGCGTAG GCGAAGGAG 2080 CATGGCCTCC CGCACCCAGA CCCGGCTTTCA TAGGGGGGAA GGGGGGTGAT CCGGATCTC 2140 CTCCCCCTCC ACCCGAACCT TGACCTCGGGC AGGGCGATGT CCAAAGCCCC CTGGGCCAG 2200 GCGCTTCCGA AAAAAGTCCC CCGCCAAGGCC TTCATGGGCG CCAGGGCCTC CACCTCGAG 2260 GGCTCCCGGT AGAAAGCCGC CTCACCCGCAC CCAGGAGAGG TAAAGGTCCT CCCGCAAAA 2320 GGCTCCTTCC GGGGAGACCA GAAGCTCAAAG GTGAGGGCTG GGGAGACCTC CTTAAGCCC 2380 CAGCCCAGGG CCTCGGTCAC CGCCAGGGGGA GCATGGGCAC CGTGCCCTCG GGCAGATAG 2440 AGGTTGGCCC CCCGGCGGAG GGCCTCCTGGT CCAGGGGGCT TCCCGGCCCG ACCAAAGCG 2500 GCCACATCGG CCACATGGAC GAAAAGGTGGA AGCCCTCCTC CACCCTTTCG GCGTAAAGG 2560 GCGTCGTCCG GGTCCTGGCT CCCCTCGTCGT CAATGGCGAA GGCGGGGAGG TGGGTGAGG 2620 TCCACCCGCT CCTCCTCGGG CAGGGGGGGGA CGGGGAGGTC CGGCGGGGCC AGGGGAAGG 2680 CCAAGCCGCC TGGGGTGGGG GTTTTCCCGCC GCCAGAGGCC CAGGCGGAGA AGGAGCCGT 2740 GGGCGGCCTC GGGGGTTTCG GGAAGGCCCAG GGCCTTGAGA AGCCGGCTTT CCTTCCTCT 2800 CGCCGTGGGC CAGAGCCTCC ACCTCGGCCAG GAGGGGGCGG TCCTCGAGGG AGGGCCGTC 2860 CCTGGCGGAT CC 2872

[Brief description of the drawings]

FIG. 1 shows thermostable isocitrate dehydrogenase activity at each temperature.

FIG. 2 shows the thermostable isocitrate dehydrogenase activity at each pH.

FIG. 3 shows the thermostability of thermostable isocitrate dehydrogenase activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:01) (C12N 1/21 C12R 1:19) (C12N 9/04 C12R 1:19)

Claims (7)

[Claims] 1. An amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence, and which has an isocitrate dehydrogenase activity. DNA encoding thermostable isocitrate dehydrogenase. 2. A DNA encoding the thermostable isocitrate dehydrogenase having the nucleotide sequence of SEQ ID NO: 2. 3. The DNA according to claim 2, which is derived from Thermus Aquaticus. 4. A recombinant vector obtained by inserting the DNA according to claim 1 into a vector DNA. A transformant transformed with the recombinant vector according to claim 4. 6. A method for producing a thermostable isocitrate dehydrogenase, comprising culturing the transformant according to claim 5 in a medium, and collecting thermostable isocitrate dehydrogenase from the culture. 7. An amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence, and which has an isocitrate dehydrogenase activity. Thermostable isocitrate dehydrogenase.