JPH0646866A - Gene capable of coding thermoduric carboxypeptidase and production of thermoduric carboxypeptidase using the same - Google Patents

Abstract

PURPOSE:To obtain a new DNA sequence useful for producing a thermoduric carboxypeptidase. CONSTITUTION:The objective gene is a DNA sequence coding a thermoduric carboxypeptidase derived from a microorganism which is a highly thermophilic bacterium of the genus Thermus and capable of coding, e.g. an amino acid sequence of the formula. This gene is obtained by estimating and synthesizing a DNA sequence capable of coding the carboxypeptidase from 29 residues of an amino acid sequence on the N-terminal side of the thermoduric carboxypeptidase, using the resultant oligonucleotide as a probe, obtaining a DNA fragment containing the thermoduric carboxypeptidase from a chromosomic DNA of Thermus.aquaticus YT-1 strain which is a microorganism belonging to the genus Thermus and cloning the resultant DNA fragment.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a thermostable carboxypeptidase and a method for producing a thermostable carboxypeptidase using the same, and more specifically, it is produced by a microorganism belonging to the genus Thermus The present invention relates to a DNA sequence encoding a thermostable carboxypeptidase and a method for producing the thermostable carboxypeptidase using the DNA sequence.

The carboxypeptidase encoded by the DNA sequence of the present invention exhibits strong thermostability with an optimum temperature of 70 ° C. to 80 ° C. and is extremely stable. Therefore, this enzyme is extremely useful as a reagent for food processing or for analysis of carboxyl terminal of proteins, peptides and the like.

[0003]

2. Description of the Related Art Carboxypeptidase is an enzyme that releases an amino acid residue on the carboxyl terminal side of proteins, peptides, etc. This enzyme has been isolated and purified from microorganisms, plants, animals and the like. In addition, this enzyme is classified into serine carboxypeptidase, metallocarboxypeptidase, cysteine carboxypeptidase and the like according to the difference in expression of its catalytic activity. However, the DNA sequence of the gene has been determined in a few cases, and no report has been made particularly on the DNA sequence encoding thermostable carboxypeptidase.

[0004]

Carboxypeptidases derived from microorganisms belonging to the genus Thermus, which are present in only a trace amount in the microbial cells, require a great deal of labor for their isolation and purification, and thus are more difficult than those of other sources. It was economically disadvantageous.
Efficiently produce this thermostable carboxypeptidase,
In order to facilitate purification, production by a genetic engineering method is ideal. However, as described above, a DNA sequence encoding a carboxypeptidase derived from a microorganism belonging to the genus Thermus has not been reported yet. The object of the present invention is to determine the DNA sequence of a gene encoding a thermostable carboxypeptidase derived from a microorganism belonging to the genus Thermus, which is an extremely thermophilic bacterium, and use this to determine the production of carboxypeptidase by genetic engineering techniques. It is to establish a method for enabling efficient production of thermostable carboxypeptidase.

A microorganism belonging to another Thermus genus or an organism of another closely related genus using a DNA fragment or an oligonucleotide containing a DNA sequence encoding a thermostable carboxypeptidase revealed by the present invention as a probe (searcher). It becomes possible to clone the carboxypeptidase of.

[0006]

The present inventors have already succeeded in purifying thermostable carboxypeptidase and determining its properties (JP-A-63-21648). After that, as a result of further research, we succeeded in determining 29 residues of the amino acid sequence on the N-terminal side of thermostable carboxypeptidase. From this amino acid sequence, a DNA sequence encoding carboxypeptidase was deduced and an oligonucleotide was synthesized. Using this oligonucleotide as a probe, a microorganism belonging to the genus Thermus, Thermus aquaticus (T. aquaticus) YT-1
A DNA fragment containing a thermostable carboxypeptidase gene (hereinafter sometimes referred to as carboxypeptidase T) was successfully isolated from the chromosomal DNA of the strain.

It was also possible to determine the entire DNA sequence coding for carboxypeptidase T in the DNA fragment. Furthermore, referring to this elucidated DNA sequence, a method for producing a plasmid into which the carboxypeptidase T gene has been incorporated and using E. coli transformed with this plasmid to produce a heat-resistant carboxypeptidase extremely efficiently is provided. I found it. The present invention has been completed based on such findings.

That is, the present invention relates to a DNA sequence encoding a thermostable carboxypeptidase derived from a microorganism of the genus Thermus of the extreme thermophile and a DNA containing the DNA sequence.
The present invention provides a method for producing thermostable carboxypeptidase, which comprises using a transformant transformed with a plasmid having a fragment.

The microorganism belonging to the genus Thermus of the highly thermophilic bacterium used in the present invention is, for example, Thermus aquaticus YT-1 strain. This strain has been deposited with the American Type Culture Collection as ATCC 25104 and is available to many (American Type Cultur
e Collection, Catalogue of Bacteria and Phages17th
ed. 1989).

DN of carboxypeptidase T of the present invention
The A sequence can be determined as follows. (1) Cloning of gene for carboxypeptidase T Cloning techniques according to the present invention include, for example, J. Sambr
ook, EFFritsch, T. Maniatis (ed.), "Molecular Clon
ing: A Laboratory Manual, 2nd edition ", Cold Spring
It is introduced in detail in Harbor Laboratory Press, 1989, etc., and can be executed by any number of people. In addition, materials such as strains, plasmid vectors and enzymes used in the present invention are easily available.

First, Thermus aquaticus YT-1
The cells are cultured to collect the bacterial cells. Total bacterial cell DNA is extracted from the bacterial cells by a conventional method. Then, cut with an appropriate restriction enzyme,
Perform agarose gel electrophoresis. This was transferred to Southern transfer [EM. Southern, J. Biol. Chem. 98. 508].
(1975)] to transfer to the membrane. On the other hand, hybridization is carried out using an oligonucleotide synthesized with reference to the amino acid sequence on the N-terminal side of purified carboxypeptidase T as a probe, and a band to which the probe specifically hybridizes is detected. A DNA fragment corresponding to this specifically hybridized band is extracted from the gel, inserted into an appropriate plasmid vector, and this plasmid is introduced into Escherichia coli for transformation.
Next, by colony hybridization, colonies that specifically hybridize with the above-mentioned probe are inoculated, and a plasmid is extracted from them. These plasmids are analyzed to create a restriction map.

(2) Determination of DNA sequence of carboxypeptidase T gene The nucleotide sequence of the region containing the carboxypeptidase T gene in the plasmid obtained as described above is determined. Deduce the amino acid sequence from this sequence,
It is confirmed by comparing it with the amino acid sequence of carboxypeptidase T of natural product.

(3) Genetically engineered production of carboxypeptidase T Based on the knowledge of the gene sequence of carboxypeptidase T thus obtained, carboxypeptidase T
Carboxypeptidase T can be produced by incorporating the gene portion into an appropriate commercially available expression vector and using the transformant introduced with the plasmid.

[0014]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 <Purification of carboxypeptidase T and determination of N-terminal amino acid sequence> Purification of carboxypeptidase T is described in JP-A-63-216481 and Japanese Patent Application No. 3-208748.
It can be carried out by the method shown in the specification. The thus-purified carboxypeptidase T could be used to determine the amino acid sequence of 29 residues from the N-terminus using a protein sequencer (FIG. 1).

Example 2 <Synthesis of Oligonucleotide Probe> From the amino acid sequence determined in Example 1, a DNA sequence predicted to encode it was deduced, and two kinds of mixed oligonucleotides were synthesized using a DNA synthesizer. did. In the synthesis, the codon usage method of the Thermus bacteria reported so far was referred to (FIG. 1).

Example 3 <Southern hybridization> The DNA of Thermus aquaticus YT-1 was cleaved with each of three types of restriction enzymes, and Southern hybridization was carried out using the probe shown in Example 2 according to a conventional method. . as a result,
For both of the two types of synthesized probes, Hind III
A hybrid band was shown in a DNA fragment of 4.3 kbp and about 6.3 kbp of BamHI (Fig. 2).

Example 4 <Cloning of Fragment Containing Carboxypeptidase T Gene> Thermus aquaticus YT-1 DNA was cloned into Hi.
It was cut with nd III and subjected to agarose gel electrophoresis according to a conventional method. A DNA fragment of about 4.3 kbp was extracted from the gel and inserted into the HindIII site of vector plasmid pBR322 (commercial product). E. coli using the DNA thus obtained
The HB101 strain was transformed. Then, colony hybridization was performed on this E. coli, and as a result, about 75
Of the 0 colonies, 3 showed specific hybridization. Plasmids were extracted from these 3 strains and Hind I
After degradation with II, Southern hybridization was performed, and it was confirmed that two of them hybridized with both of the two types of synthesized plasmids. They have the same structure.
(Fig. 3).

Example 5 <Determination of nucleotide sequence of carboxypeptidase T gene>
The DNA fragment containing the carboxypeptidase T gene obtained in Example 4 was cleaved with various restriction enzymes and a restriction enzyme map was prepared according to a conventional method (FIG. 4). On the other hand, Southern hybridization was carried out using the synthetic oligonucleotide probe prepared in Example 2, and the nucleotide sequence of the DNA fragment was determined for the SmaI fragment of about 1 kbp that hybridizes with the probe. As a result, carboxypeptidase T
It was revealed that it encodes the N-terminal amino acid sequence of. The total length of the structural gene predicted from the molecular weight of carboxypeptidase T is about 1.8 kbp.
It was revealed that the 3 kbp HindIII fragment contained the entire region of the carboxypeptidase T gene.

Therefore, the total DNA sequence of the structural gene portion of carboxypeptidase T was determined by a conventional method. The DNA sequence of the structural gene of carboxypeptidase T and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 2 in the sequence listing. From the deduced amino acid sequence, the start codon is A
It was confirmed that it was GTG instead of TG, and that the amino acid sequence from the N-terminal to the first 29 residues was the same as the N-terminal amino acid sequence determined from the purified preparation of natural product carboxypeptidase T. . Further, it was revealed that carboxypeptidase T was encoded by a base corresponding to 511 amino acids. The estimated molecular weight was 57950 Daltons.

Example 6 <Expression of carboxypeptidase T gene in Escherichia coli>
Referring to the nucleotide sequence of the present enzyme shown in Example 5, an EcoRI site was introduced immediately upstream of the start codon, and at the same time, a 36-mer was used to replace the start codon with GTG instead of ATG.
Was synthesized and site-directed mutagenesis was performed by the Kunkel method. The mutated DNA obtained in this way was Ec
After cutting with oRI and HindIII, the resulting DNA fragment of about 3.3 kbp was inserted into the same restriction enzyme site of the expression plasmid pEXP7. As a result, an expression vector plasmid pCP7 of about 7 kbp having the gene for this enzyme immediately downstream of the tac promoter, which is expected to be highly expressed in Escherichia coli, was constructed (Fig. 5).

Using this plasmid pCP7, E. coli MV 118
Four strains were transformed. Escherichia coli having this plasmid pCP7 was inoculated into 1.5 liters of LB medium, cultured at 37 ° C for 3 hours, induced with IPTG (Isopropyl β-D-thiogalactopyranoside), and further cultured at 37 ° C for 3 hours. Then, centrifugation was performed to collect the cells, and the cells were disrupted by ultrasonic treatment. After that, heat treatment (70 ° C, 1 hour),
After further centrifugation, the supernatant was recovered. Proteins derived from Escherichia coli are heat-denatured and are mostly removed as a precipitate by centrifugation. The supernatant was purified by Butyl Toyopearl, Mono Q column chromatography. Table 1 shows the purification table for the fractions obtained at each stage of purification. Also,
The results of SDS electrophoresis of each fraction are shown in FIG.

[0022]

[Table 1]

The final-stage Mono Q fraction showed almost the same specific activity as that of the purified preparation from Thermus aquaticus YT-1 cells, and was obtained in a yield of 50% or more for 1.5 liters of culture solution. 2.8 mg of carboxypeptidase preparation was obtained.
Further, it was shown that when Escherichia coli, which is a room temperature bacterium, is produced as a host, most of the Escherichia coli-derived protein can be removed by heat treatment at 70 ° C. Also, this Mono Q fraction is SD
S-electrophoresis showed a single band at almost the same position as the purified standard product from Thermus aquaticus, which had the same molecular weight as the natural product and was of high purity.

[0024]

INDUSTRIAL APPLICABILITY The present invention has revealed the DNA sequence of a gene encoding a thermostable carboxypeptidase derived from Thermus aquaticus YT-1 belonging to the genus Thermus and the amino acid sequence deduced therefrom. Further, according to the present invention, a production method for mass-producing a thermostable carboxypeptidase by a genetic engineering method is provided for the first time, and the carboxypeptidase is extremely efficiently and economically isolated and isolated from conventional Thermus cells. It has become possible to produce.

It is also possible to use the DNA fragment having the DNA sequence of the present invention as a probe to clone the carboxypeptidase gene of other species of Thermus species or organisms of other genus closely related to Thermus genus.

[0026]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 511 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Thr Pro Glu Ala Ala Tyr Gln Asn Leu Leu Glu Phe Gln Arg Glu 1 5 10 15 Thr Ala Tyr Leu Gly Ser Leu Gly Ala Leu Ala Ala Trp Asp Gln Arg 20 25 30 Thr Met Ile Pro Arg Lys Gly His Gly His Arg Ala Arg Gln Met Ala 35 40 45 Ala Leu Ala Arg Leu Leu His Glu Arg Ala Thr Asp Pro Arg Ile Gly 50 55 60 Glu Trp Leu Glu Lys Val Glu Gly Ser Ser Leu Val Glu Asp Pro Leu 65 70 75 80 Ser Asp Ala Ala Val Asn Val Arg Ala Trp Arg Arg Ala Tyr Glu Arg 85 90 95 Ala Arg Ala Ile Pro Glu Arg Leu Ala Val Glu Leu Ala Gln Ala Arg 100 105 110 Ser Glu Gly Glu Thr Ala Trp Glu Ala Leu Arg Pro Arg Asp Asp Trp 115 120 125 Gln Gly Phe Leu Pro Tyr Leu Lys Arg Leu Phe Ala Leu Ala Lys Glu 130 135 140 Glu Ala Glu Ile Leu Met Ala Val Gly Pro Asp Pro Leu Asp Pro Pro 145 150 155 160 Tyr Gly Glu Leu Tyr Asp Ala Leu Leu Asp Gly Tyr Glu Pro Gly Ala 165 170 175 Arg Ala Arg Asp Leu Glu Pro Le u Phe Arg Glu Leu Ser Ser Gly Leu 180 185 190 Lys Gly Leu Leu Asp Arg Ile Leu Gly Ser Gly Arg Arg Pro Asp Val 195 200 205 Gly Val Leu His Arg His Tyr Pro Lys Glu Ala Gln Arg Ala Phe Ala 210 215 220 Leu Glu Leu Leu Gln Ala Cys Gly Tyr Asp Leu Glu Ala Gly Arg Leu 225 230 235 240 Asp Pro Thr Ala His Pro Phe Glu Ile Ala Ile Gly Pro Gly Asp Val 245 250 255 Arg Ile Thr Thr Arg Tyr Tyr Glu Asp Phe Phe Asn Ala Gly Ile Phe 260 265 270 Gly Thr Leu His Glu Met Gly His Ala Leu Tyr Glu Gln Gly Leu Pro 275 280 285 Glu Ala His Trp Gly Thr Pro Arg Gly Glu Ala Ala Ser Leu Gly Val 290 295 300 His Glu Ser Gln Ser Arg Thr Trp Glu Asn Leu Val Gly Arg Ser Leu 305 310 315 320 Gly Phe Trp Glu Arg Phe Phe Pro Arg Ala Lys Glu Val Phe Ser Ser 325 330 335 Leu Ala Asp Val Arg Leu Glu Asp Phe His Phe Ala Val Asn Ala Val 340 345 350 Glu Pro Ser Leu Ile Arg Val Glu Ala Asp Glu Val Thr Tyr Asn Leu 355 360 365 His Ile Leu Val Arg Leu Glu Leu Glu Leu Ala Leu Phe Arg Gly Glu 370 375 380 Leu Phe Leu Glu Asp Leu Pro Glu Al a Trp Arg Glu Lys Tyr Arg Ala 385 390 395 400 Tyr Leu Gly Val Ala Pro Arg Asp Tyr Lys Asp Gly Val Met Gln Asp 405 410 415 Val His Trp Ser Gly Gly Met Phe Gly Tyr Phe Pro Thr Tyr Thr Leu 420 425 430 Gly Asn Leu Tyr Ala Ala Gln Phe Phe Ala Lys Ala Gln Glu Glu Leu 435 440 445 Gly Pro Leu Glu Pro Leu Phe Ala Arg Gly Glu Phe Thr Pro Phe Leu 450 455 460 Asp Trp Thr Arg Arg Lys Ile His Ala Glu Gly Ser Arg Phe Arg Pro 465 470 475 480 Arg Ala Leu Val Glu Arg Val Thr Gly Ser Pro Pro Gly Ala Gln Ala 485 490 495 Phe Leu Arg Tyr Leu Glu Ala Lys Tyr Gly Ala Leu Tyr Gly Phe 500 505 510

SEQ ID NO: 2 Sequence length: 1536 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Thermus aquaticu
s) Origin: Features of YT-1 sequence Characteristic symbol: CDS Location: 1. ． 1533 Method of Characterizing E Sequence GTG ACG CCG GAA GCC GCT TAT CAG AAC CTG TTG GAG TTC CAG AGG GAA 48 Met Thr Pro Glu Ala Ala Tyr Gln Asn Leu Leu Glu Phe Gln Arg Glu 1 5 10 15 ACC GCC TAC CTG GGT TCC TTA GGG GCC CTG GCC GCC TGG GAC CAG CGC 96 Thr Ala Tyr Leu Gly Ser Leu Gly Ala Leu Ala Ala Trp Asp Gln Arg 20 25 30 ACC ATG ATC CCC AGA AAG GGG CAC GGG CAC CGG GCC CGG CAG ATG GCC 144 Thr Met Ile Pro Arg Lys Gly His Gly His Arg Ala Arg Gln Met Ala 35 40 45 GCT CTG GCC CGC CTC CTC CAC GAG CGG GCC ACC GAC CCC AGG ATC GGG 192 Ala Leu Ala Arg Leu Leu His Glu Arg Ala Thr Asp Pro Arg Ile Gly 50 55 60 GAG TGG CTG GAG AAG GTG GAG GGG TCG TCC CTG GTG GAG GAC CCC CTT 240 Glu Trp Leu Glu Lys Val Glu Gly Ser Ser Leu Val Glu Asp Pro Leu 65 70 75 80 TCC GAT GCC GCC GTC AAC GTG CGG GCC TGG CGG CGG GCC TAC GAG AGG 288 Ser Asp Ala Ala Val Asn Val Arg Ala Trp Arg Arg Ala Tyr Glu Arg 85 90 95 GCC CGG GCC ATT CCC GAG AGG CTG GCT GTG GAG CTG GCC CAG GCC AGG 336 Ala Arg Ala Ile Pro Glu Arg Leu Ala Val Glu Leu Ala Gln Ala Arg 100 105 110 AGC GAG GGG GAG ACC GCC TGG GAG GCC CTG CGC CCC AGG GAC GAC TGG 384 Ser Glu Gly Glu Thr Ala Trp Glu Ala Leu Arg Pro Arg Asp Asp Trp 115 120 125 CAG GGC TTC CTG CCC TAC CTC AAG CGC CTT TTC GCC CTG GCC AAG GAG 432 Gln Gly Phe Leu Pro Tyr Leu Lys Arg Leu Phe Ala Leu Ala Lys Glu 130 135 140 GAG GCG GAG ATC CTC ATG GCC GTG GGG CCA GAC CCC CTG GAC CCC CCC 480 Glu Ala Glu Ile Leu Met Ala Val Gly Pro Asp Pro Leu Asp Pro Pro 145 150 155 160 TAC GGG GAG CTT TAC GAC GCC CTC CTG GAC GGC TAC GAG CCC GGG GCC 528 Tyr Gly Glu Leu Tyr Asp Ala Leu Leu Asp Gly Tyr Glu Pro Gly Ala 165 170 175 AGG GCG AGG GAC CTC GAG CCC CTC TTC CGG GAG CTC TCC TCG GGC CTC 576 Arg Ala Arg Asp Leu Glu Pro Leu Phe Arg Glu Leu Ser Ser Gly Leu 180 185 190 AAG GGT CTT CTG GAC CGC ATC CTG GGA AGC GGG CGG AGG CCC GAC GTC 624 Lys Gly Leu Leu Asp Arg Ile Leu Gly Ser Gly Arg Arg Pro Asp Val 195 200 205 GGC GTC CTC CAC CGC CAC TAC CCT AAG GAG GCC CAG AGG GCC TTC GCC 672 Gly Val Leu His Arg His Tyr Pro Lys Glu Ala Gln Arg Ala Phe Ala 210 215 220 TTA GAG CTC CTT CAG GCC TGC GGG TAC GAC CTC GAG GCC GGC CGT CTG 720 Leu Glu Leu Leu Gln Ala Cys Gly Tyr Asp Leu Glu Ala Gly Arg Leu 225 230 235 240 GAC CCC ACC GCC CAC CCC TTT GAG ATC GCC ATC GGC CCC GGG GAC GTG 768 Asp Pro Thr Ala His Pro Phe Glu Ile Ala Ile Gly Pro Gly Asp Val 245 250 255 CGC ATC ACC ACC CGC TAC TAC GAG GAC TTC TTC AAC GCC GGC ATC TTC 816 Arg Ile Thr Thr Arg Tyr Tyr Glu Asp Phe Phe Asn Ala Gly Ile Phe 260 265 270 GGC ACC CTC CAC GAG ATG GGC CAC GCC CTC TAC GAG CAG GGC CTG CCC 864 Gly Thr Leu His Glu Met Gly His Ala Leu Tyr Glu Gln Gly Leu Pro 275 280 285 GAG GCC CAC TGG GGC ACC CCC CGG GGG GAG GCC GCC TCC TTG GGG GTC 912 Glu Ala His Trp Gly Thr Pro Arg Gly Glu Ala Ala Ser Leu Gly Val 290 295 300 CAC GAG TCC CAA AGC CGC ACC TGG GAG AAC CTG GTG GGC CGC TCC TTG 960 His Glu Ser Gln Ser Arg Thr Trp Glu Asn Leu Val Gly Arg Ser Leu 305 310 315 320 GGC TTC TGG GAG CGC TTC TTC CCC CGG GCC AAG GAG GTC TTT TCC AGT 1008 Gly Phe Trp Glu Arg Phe Phe Pro Arg Ala Lys Glu Val Phe Ser Ser 325 330 335 CTG GCC GAC GTG CGC CTG GAG GAC TTC CAC TTC GCC GTC AAC GCC GTG 1056 Leu Ala Asp Val Arg Leu Glu Asp Phe His Phe Ala Val Asn Ala Val 340 345 350 GAG CCC TCC CTG ATC CGG GTG GAG GCC GAC GAG GTT ACC TAT AAC CTC 1104 Glu Pro Ser Leu Ile Arg Val Glu Ala Asp Glu Val Thr Tyr Asn Leu 355 360 365 CAC ATC CTG GTG CGC CTG GAG CTG GAG CTG GCC CTA TTC CGG GGG GAG 1152 His Ile Leu Val Arg Leu Glu Leu Glu Leu Ala Leu Phe Arg Gly Glu 370 375 380 CTT TTC CTC GAG GAC CTG CCC GAG GCC TGG CGG GAA AAG TAC CGG GCC 1200 Leu Phe Leu Glu Asp Leu Pro Glu Ala Trp Arg Glu Lys Tyr Arg Ala 385 390 395 400 TAC CTG GGC GTG GCC CCA AGG GAC TAC AAG GAC GGG GTC ATG CAG GAC 1248 Tyr Leu Gly Val Ala Pro Arg Asp Tyr Lys Asp Gly Val Met Gln Asp 405 410 415 GTC CAC TGG TCG GGG GGG ATG TTC GGC TAC TTC CCC ACC TAC ACC CTG 1296 Val His Trp Ser Gly Gly Met Phe Gly Tyr Phe Pro Thr Tyr Thr Leu 420 425 430 GGC AAC CTC TAC GCT GCC CAG TTC TTC GCC AAG GCC CAG G AG GAG CTT 1344 Gly Asn Leu Tyr Ala Ala Gln Phe Phe Ala Lys Ala Gln Glu Glu Leu 435 440 445 GGG CCC TTG GAG CCC CTC TTC GCC CGC GGG GAG TTC ACC CCC TTC CTG 1392 Gly Pro Leu Glu Pro Leu Phe Ala Arg Gly Glu Phe Thr Pro Phe Leu 450 455 460 GAC TGG ACC AGG CGC AAG ATC CAC GCC GAG GGG AGC CGC TTC CGC CCC 1440 Asp Trp Thr Arg Arg Lys Ile His Ala Glu Gly Ser Arg Phe Arg Pro 465 470 475 480 CGG GCC CTG GTG GAG AGG GTG ACG GGA AGC CCC CCC GGC GCT CAA GCC 1488 Arg Ala Leu Val Glu Arg Val Thr Gly Ser Pro Pro Gly Ala Gln Ala 485 490 495 TTC CTC AGG TAC CTG GAG GCC AAG TAC GGG GCC CTT TAC GGC TTC TGA 1536 Phe Leu Arg Tyr Leu Glu Ala Lys Tyr Gly Ala Leu Tyr Gly Phe 500 505 510

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of 29 residues at the N-terminal side of carboxypeptidase T used in the present invention and the sequence of a mixed 28mer and 23mer oligonucleotide used as a probe.

FIG. 2 shows the results of Southern hybridization. The arrows indicate the positions of about 4.36 kbp and 6.55 kbp bands hybridized with the probe shown in FIG.

FIG. 3 shows the structure of plasmid pCP (about 8.7 kbp). The thick line is DN containing the carboxypeptidase T gene.
The A fragment portion is shown, and the thin line portion is the vector plasmid pBR3.
22 parts are shown.

FIG. 4 shows a restriction enzyme map and a nucleotide sequencing strategy of an approximately 4.3 kbp Hind III fragment containing the carboxypeptidase T gene derived from Thermus aquaticus YT-1 in the plasmid pCP. In particular, the carboxypeptidase T gene coding region is shown in detail. The black arrow indicates the structural gene portion of carboxypeptidase T. GT
G indicates the start codon. Trm indicates a stop codon.

FIG. 5: Sequence of 36-mer primer used to mutate start codon GTG to ATG by site-directed mutagenesis and plasmid constructed by site-directed mutagenesis
1 shows the structure of pCP7 The white arrow shows the structural gene portion of carboxypeptidase T. Ptac indicates the position of the tac promoter.

FIG. 6: Carboxypeptidase T produced in E. coli
FIG. 3 is a diagram of SDS electrophoresis showing the purification process of.

[Explanation of symbols]

Lane symbols HB, HP, BH, H, B, P, M in FIG.
Is as follows. HB: Hind III / BamHI HP: Hind III / PstI BP: BamHI / PstI H: Hind III B: BamHI P: PstI M: Molecular weight marker M, V, C, H, B, Q, T of lane symbols in FIG. Is as follows. M: Molecular weight marker V: E. coli whole cells (without pCP7) C: E. coli whole cells (with pCP7) H: Supernatant after heat treatment B: Butyltoyopearl purified fraction Q: Mono Q purified fraction Fraction T: Natural product carboxypeptidase T purified preparation

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:01) (C12N 9/48 C12R 1:19)

Claims (5)

[Claims] 1. A DNA sequence encoding a thermostable carboxypeptidase derived from an extremely thermophilic bacterium, Thermus sp. 2. The DNA according to claim 1, wherein the DNA encodes the amino acid sequence of SEQ ID NO: 1 in the sequence listing or the amino acid sequence having substantially the same function.
Array. 3. The DNA sequence according to claim 1, wherein the DNA has the nucleotide sequence of SEQ ID NO: 2 in the sequence listing or the nucleotide sequence having substantially the same function. 4. A DNA sequence hybridizing to the DNA sequence according to claim 2 and encoding a polypeptide having thermostable carboxypeptidase activity. 5. A DNA containing the DNA sequence according to claim 1.
A method for producing thermostable carboxypeptidase, which comprises using a transformant transformed with a plasmid having a fragment.