JP3012919B2 - Thermostable enzyme having aminoacylase and carboxypeptidase activities, and gene encoding the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a thermostable enzyme having aminoacylase activity and carboxypeptidase activity, and a gene encoding the same. Since the enzyme of the present invention has aminoacylase activity, it is effective for resolving optically active isomers of synthetic amino acids (D-form L-form), and has carboxypeptidase activity, which is effective for analysis of carboxyl group terminal of peptides. is there.

[0002]

2. Description of the Related Art Conventionally, optically active isomers (D-form L-form) of synthetic amino acids are separated by releasing only the acyl group of L-form acylamino acid using an aminoacylase.
Is being done. Carboxyl group terminal analysis (amino acid sequence analysis) of proteins and peptides is performed using carboxypeptidase.

[0003]

Since the conventional enzyme is unstable at high temperatures, the above-mentioned reaction has to be performed at a relatively low temperature. However, by performing the above reaction at a high temperature, many advantages such as improvement of reaction efficiency and removal of contaminating microorganisms can be considered. Therefore, there is a need for an enzyme that can be used at a high temperature and that is stable at a high temperature. The present invention
It has been made under such a technical background, and an object of the present invention is to provide a thermostable aminoacylase and carboxypeptidase.

[0004]

Means for Solving the Problems In order to solve the above problems, the present inventors have focused on ultra-thermophilic bacteria growing at 90 to 100 ° C. and presumed to exhibit the enzyme activity from the gene sequence thereof. Gene found. In addition, Escherichia coli is used to produce an enzyme from the gene, which is then heated to a high temperature (90-95).
° C) and showed aminoacylase and carboxypeptidase activities, and the present invention was completed based on these findings.

That is, the present invention relates to an enzyme having the following properties. (1) Has aminoacylase activity and carboxypeptidase activity (2) Optimal temperature is 90-95 ° C (3) Optimal pH is 6.5-8.0 (4) Activity even after heating at 95 ° C for 3 hours (pH 7.5) (5) Molecular weight of 40,000 (SDS polyacrylamide gel electrophoresis) The present invention relates to a gene encoding the following protein (a) or (b). (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted or added; and Protein having peptidase activity

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The enzyme of the present invention has the following properties. (1) It has two types of enzyme activities, aminoacylase activity and carboxypeptidase activity. (2) Changes in enzyme activity with changes in temperature are as shown in FIG. As shown in this figure, the optimal temperature of the enzyme of the present invention is
90-95 ° C, optimal temperature is 90 ° C. (3) The optimum pH is 6.5 to 8.0, and the optimum pH is 7.5. (4) A thermostable enzyme that does not lose its activity even after heating at 95 ° C. for 3 hours (pH 7.5). (5) The molecular weight measured by SDS polyacrylamide electrophoresis is about 40,000.

[0007] The amino acid sequence of the enzyme of the present invention can be represented by the amino acid sequence described in SEQ ID NO: 1 or the amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1. here,
"One or several" refers to the site-specific mutation induction method (Nucl
eic Acid Research, Vol. 10, No. 20, p6487-6500).

[0008] Since the enzyme of the present invention is contained in microorganisms, it can be obtained therefrom. Specifically, it is obtained by crushing the microorganism cells, suspending the cells in a buffer, and purifying the supernatant obtained by centrifugation by various chromatography using the enzyme activity as an index. As a microorganism to be used, Pyrococcus horikoshi, a sulfur-metabolizing hyperthermic archaeon (RICM Microbial Strain Preservation Facility (JCM) No. 9974, hereinafter abbreviated as “JCM 9974”) can be used. Any microorganism can be used as long as it can produce the enzyme of the present invention. For example, a microorganism into which the gene of the present invention described below is introduced may be used.
The buffer used, the conditions for centrifugation, and the chromatography used may be those commonly used when purifying enzymes from microbial cells. The presence or absence of the enzyme activity can be determined, for example, by using a benzyloxycarbonylated amino acid and a benzyloxycarbonylated peptide as a substrate and detecting an amino acid generated by hydrolysis by a ninhydrin reaction.

Since the enzyme of the present invention has aminoacylase activity, the optically active isomer of a synthetic amino acid (D-form L
Can be used for the resolution of carboxyl group ends of peptides since it has carboxypeptidase activity. The gene of the present invention
A protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and which has an aminoacylase activity and a carboxypeptidase activity Encodes a protein. Here, “one or several” refers to a range that can be deleted or the like by the site-specific mutagenesis method as described above.

The gene of the present invention can be obtained, for example, as follows. First, DNA is extracted from a microorganism having the gene of the present invention, partially digested with a restriction enzyme, and inserted into a vector. This recombinant vector is introduced into a suitable host microorganism, and the DNA is sequenced.
The homology region of the present enzyme is examined from the obtained sequence data, and the structural gene of the present enzyme is found. A primer that binds to both ends of the enzyme structural gene is synthesized and PCR is performed.
Amplifies only this enzyme gene. Thereby, the gene of the present invention can be obtained. Here, examples of the microorganism used include JCM 9974, but are not limited thereto. Extraction of DNA does not need to use a special method, can be performed according to a conventional method, and a commercially available kit or the like can be used. The restriction enzymes used are not particularly limited, but include HindIII, EcoRI, XhoI
And the like. Examples of a vector to be used include, but are not limited to, pBAC108L, pFOS1, M13, pUC19, and the like. Examples of the host microorganism used include Escherichia coli and yeast, but are not limited thereto. The means for introduction into the host microorganism may be determined according to the vector to be used.For example, when pBAC108L is used, electroporation is preferable, and when pFOS1 is used, λ phage or the like is preferably used. . Escherichia coli BL21 containing the gene of the present invention
(DE3) has been deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-16051 (Deposit date: January 27, 1997).

[0011] Since the gene of the present invention encodes the enzyme of the present invention, the enzyme of the present invention can be produced in large quantities by introducing it into a microorganism and expressing it.
As a vector used for expressing a gene,
pET-11a, pET-15a, pET15b and the like can be exemplified,
E. coli BL21 (DE3)
Escherichia coli XL1-Blue MR and the like can be mentioned.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.

[0013]

【Example】

(Example 1) (Culture of bacteria) JCM9974 was cultured by the following method. 13.5g salt, 4g
Na ₂ SO ₄ , 0.7 g KCl, 0.2 g NaHCO ₃ , 0.1 g KB
r, 30 mg of H ₃ BO ₃ , 10 g of MgCl ₂ .6H ₂ O, 1.5 g of CaC
l ₂ , 25 mg SrCl ₂ , 1.0 ml resasulin solution (0.2 g /
L), 1.0 g of yeast extract, 5 g of bactopeptone in 1 L
, And the solution was adjusted to pH 6.8 and sterilized by pressure. Then, dry heat sterilized elemental sulfur was added to a concentration of 0.2%, the medium was saturated with argon to make it anaerobic, and JCM9974 was inoculated. Whether or not the medium became anaerobic was confirmed by adding a Na ₂ S solution and not coloring the pink color of the resasurin solution with Na ₂ S in the culture solution. This culture was cultured at 95 ° C. for 2 to 4 days, and then centrifuged to collect the bacteria.

(Example 2) Preparation of chromosomal DNA The chromosomal DNA of JCM9974 was prepared by the following method.
After completion of the culture, the cells were collected by centrifugation at 5000 rpm for 10 minutes. The cells were washed with 10 mM Tris (pH 7.5) -1 mM EDTA solution.
After washing twice, it was sealed in an In Cert Agarose (manufactured by FMC) block. This block was treated in a solution containing 1% N-lauroyl sarcosine and 1 mg / ml protease K, and chromosomal DNA was separated and prepared in an agarose block.

(Example 3) Preparation of library clone containing chromosomal DNA The chromosomal DNA obtained in Example 2 was partially digested with a restriction enzyme HindIII, and then a fragment of about 40 kb was prepared by agarose gel electrophoresis. This DNA fragment and the restriction enzyme HindIII
Vector pBAC108L (Stratage
ne) and pFOS1 (Stratagene) using T4 ligase. When the former vector was used, the DNA after the completion of ligation was immediately introduced into E. coli by electroporation. When the latter vector pFOS1 was used, the DNA after completion of the ligation was packed into λ phage particles in a test tube using GIGA Pack Gold (manufactured by Stratagene), and the particles were infected into E. coli by transferring the DNA into E. coli. Introduced. The E. coli population resistant to the antibiotic chloramphenicol obtained by these methods was isolated from BAC and Fosmid.
A library. Clones suitable for covering the chromosome of JCM9974 were selected from the library and clones were sorted.

(Example 4) Determining the nucleotide sequence of each BAC or Fosmid clone The nucleotide sequence of the aligned BAC or Fosmid clone was determined sequentially by the following method. The DNA of each BAC or Fosmid clone recovered from E. coli was fragmented by sonication, and DNA fragments of 1 kb and 2 kb in length were recovered by agarose gel electrophoresis. This fragment was inserted into the HincII restriction enzyme site of the plasmid vector pUC118 (manufactured by Takara), and the shotgun clone was inserted into each BAC or
500 clones were prepared per Fosmid clone. The nucleotide sequence of each shotgun clone was determined by PerkinElmer, ABI
The determination was made using an automatic nucleotide sequence reader 373 or 377 manufactured by the company. The base sequence obtained from each shotgun clone was linked and edited using base sequence automatic linking software Sequencher, and the entire base sequence of each BAC or Fosmid clone was determined.

Example 5 Identification of the Gene of the Present Invention The nucleotide sequence of each of the BAC or Fosmid clones determined above was analyzed by a large-scale computer to determine the nucleotide sequence of the gene of the present invention. The nucleotide sequence is shown in SEQ ID NO: 2, and the deduced amino acid sequence is shown in SEQ ID NO: 1.

Example 6 Construction of Expression Plasmid A restriction enzyme (Nd) was added before and after the structural gene region of the gene of the present invention.
The following DNA primers were synthesized for the purpose of constructing eI and XhoI) sites, and restriction enzyme sites were introduced before and after the gene by PCR. upper Primer: 5'-TTTTGAATTCTTTCATATGGATGAGTTTATAAT
TAAAAGAGC-3 'lower Primer: 5'-TTTTAGATCTTGATCACTCGAGTCAAAGGGAGA
After GGTAGTGATAAGTCAGAA-3 'PCR reaction, complete digestion with restriction enzymes (NdeI and XhoI) (37
(2 hours at C), the structural gene was purified.

PET-11a, (Novagen) was replaced with the restriction enzyme NdeI
After digestion and purification with XhoI, the above-described structural gene was ligated with T4 ligase at 16 ° C. for 2 hours. Ligated DNA
Was introduced into competent cells of E. coli-XL1-BlueMRF ′ to obtain transformant colonies. The expression plasmid was purified from the obtained colonies by an alkaline method.

Example 7 Expression of Recombinant Gene Using this expression plasmid, E. coli BL21 (DE
3), Novagen). The transformant was cultured in 2YT medium containing ampicillin until the absorbance at 600 nm reached 1, followed by IPTG (Isopropyl-bD-thiogala).
ctopyranoside) was added, and the cells were further cultured for 6 hours. After the culture, the cells were collected by centrifugation (6,000 rpm, 20 min).

Example 8 Purification of Heat-Stable Enzyme Twice the amount of alumina was added to the collected cells, and the cells were pulverized. Then, 5 times 10 mM Tris-HCl buffer (pH 8.0) was added. A suspension was obtained. The obtained suspension was heated at 85 ° C. for 30 minutes, centrifuged (11,000 rpm, 20 minutes), and the supernatant was adsorbed on a HiTrapQ (Pharmacia) column to obtain an active fraction. The obtained active fraction solution was concentrated with Centricon (manufactured by Amicon) and passed through a gel filtration column Superdex200 (manufactured by Pharmacia) to obtain a purified enzyme.

Example 9 Properties of Enzyme (1) Optimum pH The optimum pH of the enzyme activity was measured using a 50 mM sodium acetate buffer, a 50 mM phosphate buffer, and a 50 mM borate buffer at pH 4 to 4.
9 substrates (benzyloxycarbonyl-Gly-Gly-Ph
e, Synthetic substrate for carboxypeptidase) A 20 mM solution was prepared, and the initial rate of the hydrolysis activity of the enzyme was measured at 85 ° C. to determine the rate. Since the maximum initial velocity was obtained near pH 7.5, the optimum pH was concluded to be 7.5.

(2) Optimal temperature 20 mM benzyloxycarbonyl-Gly-Gly-P
Using he, a certain amount of the enzyme was added to a 50 mM phosphate buffer (pH 7.5) and reacted for 30 minutes, and the relative activity was examined. The maximum activity (optimal temperature) was 90 ° C. (FIG. 1). (3) Heat resistance The enzyme solution (0.1 mg / mL) was added to a 50 mM phosphate buffer (pH 7.
5) After heating at 95 ° C for 3 hours, the temperature was lowered to 85 ° C and the residual activity was examined, but no decrease in activity was observed. Circular dichroism (CD) was measured at 25 to 90 ° C., but no change was found in the spectrum.

[0024]

The present invention provides a thermostable enzyme having aminoacylase and carboxypeptidase activities. By this enzyme, D-form L of synthetic amino acid under high temperature
It will allow for body splitting and carboxyl terminal analysis of proteins and peptides. In addition, this enzyme can be expected to have improved organic solvent resistance because the enzyme molecule is stable.

[0025]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 388 Sequence type: Protein Topology: Unknown Origin: Organism name: Pyrococcus horikoshi Met Asp Glu Phe Ile Ile Lys Arg Ala Lys Glu Leu Gln Gly Tyr Ile 1 5 10 15 Val Glu Lys Arg Arg Asp Phe His Met Tyr Pro Glu Leu Lys Tyr Glu 20 25 30 Glu Glu Arg Thr Ser Lys Ile Val Glu Glu Glu Leu Lys Lys Leu Gly 35 40 45 Tyr Glu Val Val Arg Thr Ala Lys Thr Gly Val Ile Gly Ile Leu Lys 50 55 60 Gly Lys Glu Asp Gly Lys Thr Val Ala Leu Arg Ala Asp Met Asp Ala 65 70 75 80 Leu Pro Ile Gln Glu Glu Asn Asp Val Pro Tyr Lys Ser Leu Gly Phe 85 90 95 Pro Gly Lys Met His Ala Cys Gly His Asp Ala His Thr Ala Met Leu 100 105 110 Leu Gly Ala Ala Lys Ile Leu Ala Glu Met Lys Asp Glu Leu Gln Gly 115 120 125 Thr Val Lys Leu Ile Phe Gln Pro Ala Glu Glu Gly Gly Leu Gly Ala 130 135 140 Lys Lys Ile Val Glu Glu Gly His Leu Asp Asp Val Asp Ala Ile Phe 145 150 155 160 Gly Ile His Val Trp Ala Glu Leu Pro Ser Gly Ile Ile Gly Ile Lys 165 170 175 Ser Gly Pro Leu Leu Ala Ser Ala Asp Ala Phe Arg Val Leu Ile Lys 180 185 190 Gly Lys Gly Gly His Gly Ala Ala Pro His Leu Ser Ile Asp Pro Ile 195 200 205 Ala Leu Ala Val Asp Leu Val Asn Ala Tyr Gln Lys Ile Ile Ser Arg 210 215 220 Glu Val Asp Pro Leu Gln Pro Ala Val Leu Ser Val Thr Ser Ile Lys 225 230 235 240 Ala Gly Thr Thr Phe Asn Val Ile Pro Glu Ser Ala Glu Ile Leu Gly 245 250 255 Thr Ile Arg Thr Phe Asp Glu Glu Val Arg Asp Tyr Ile Val Arg Arg 260 265 270 Met Lys Glu Ile Thr Glu Asn Phe Ala Asn Gly Met Arg Cys Glu Gly 275 280 285 Lys Phe Glu Leu Thr Ile Glu His Ile Pro Pro Thr Ile Asn Asn Glu 290 295 300 Lys Leu Ala Asn Phe Ala Arg Asp Val Leu Lys Val Leu Gly Glu Ile 305 310 315 320 Arg Glu Pro Lys Pro Thr Met Gly Ala Glu Asp Phe Ala Phe Tyr Thr 325 330 335 Thr Lys Ala Pro Gly Leu Phe Ile Phe Leu Gly Ile Arg Asn Glu Glu 340 345 350 Lys Gly Ile Ile Tyr Pro His His His Pro Lys Phe Asn Val Asp Glu 355 360 365 Asp Ile Leu Trp Met Gly Ala Ala Ile His Ser Leu Leu Thr Tyr His 370 375 380 Tyr LeuSer Leu 385

SEQ ID NO: 2 Sequence length: 1176 Sequence type: Genomic DNA Topology: linear Origin: Organism name: Pyrococcus horikoshii Sequence ATG GAT GAG TTT ATA ATT AAA AGA GCA AAA GAG TTG CAG GGG TAT ATA GTT GAG AAG AGG AGA GAT TTC CAC ATG TAC CCA GAG CTG AAG TAT GAG GAA GAG AGG ACT TCT AAA ATA GTG GAG GAA GAA TTA AAA AAG CTC GGA TAT GAA GTT GTC AGA ACT GCA AAA ACA GGG GTT ATA GGT ATC CTT AAA GGA AAA GAA GAC GGA AAA ACC GTG GCT TTA AGG GCT GAT ATG GAT GCT TTG CCT ATT CAG GAG GAG AAT GAT GTT CCT TAT AAG TCC TTA GGG TTC CCT GGG AAG ATG CAT GCT TGT GGT CAC GAC GCC CAC ACA GCA ATG CTA CTA GGA GCA AAG ATA TTA GCG GAG ATG AAA GAT GAG TTG CAG GGA ACC GTT AAA TTA ATA TTC CAA CCT GCT GAG GAA GGA GGA CTT GGA GCA AAG AAG ATA GTT GAA GAA GGG CAC TTG GAT GAT GTA GAT GCT ATC TTT GGG ATC CAC GTAGGCC GAG CTA CCT TCG GGA ATC ATA GGA ATC AAG AGC GGG CCA CTA CTA GCG TCT GCA GAT GCA TTT AGG GTT TTG ATA AAG GGG AAG GGA GGT CAC GGA GCA GCA CCG CAC CTC TCG ATA GAC CCA ATA GCT TTA GCT GTA GAT TTA GTG AAT GCC TAT CAG AAG ATA ATA TCG AGG GAA GTA GAT CCA CTA CAA CCA GCG GTT CTT AGC GTT ACC TCG ATA AAA GCT GGA ACG ACA TTT AAT GTA ATT CCA GAA GCC GCG ATA CTT GGA ACC ATA AGG ACA TTC GAT GAG GAA GTT AGG GAT TAT ATA GTA AGG AGA ATG AAG GAG ATT ACT GAA AAC TTT GCC AAT GGG ATG AGG TGC GAG GGG AAG TTT GAA CTA ACG ATA GAG CAT ATA CCC ACA ATA ATA AAT GAA AAG CTG GCG AAT TTT GCG AGG GAT GTG CTA AAG GTT CTG GGC GAA ATT AGG GAG CCT AAA CCA ACG ATG GGA GCT GAA GAT TTT GCC TTT TAT ACA ACC AAA GCC CCA GGA CTG TTC ATA TTC CTG GGA ATA AGA AAG GAA AGA GGA ATA ATT TAC CCA CAC CAC CAT CCA AAG TTT AAT GTG GAT GAG GAC ATC TTG TGG ATG GGA GCA GCG ATC CAT TCT CTT CTG ACT TAT CAC TAC CTC TCC CTT TGATTTTTTCAC

[Brief description of the drawings]

FIG. 1 is a diagram showing a change in enzyme activity with a change in temperature.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:19) (C12N 9/80 C12R 1:19) (C12N 15/09 ZNA C12R 1:01) (72) Inventor Kosugi Kaji 1-3-1 Higashi, Tsukuba, Ibaraki Pref., Institute of Biotechnology, Industrial Technology Institute (72) Inventor Katsuhiko Higuchi 1-3-1, Higashi, Tsukuba, Ibaraki Pref., Institute of Biotechnology, Industrial Technology Institute (56) References JP-A-6-46866 (JP, A) JP-A-63-216481 (JP, A) JP-A-62-44181 (JP, A) JP-A-63-219379 (JP, A) -278086 (JP, A) JP-A-3-224483 (JP, A) Appl. Environ. Microbiol. , Vol. 59 (11), p. 3878-3888 (1993) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00 C12N 9/48 BIOSIS (DIALOG) CA (STN) WPIDS (STN) GenBank / EMBL / DDBJ (GENETYX) PIR / Swisprot Geneseq

Claims (3)

(57) [Claims] An enzyme having the following properties: (1) Has aminoacylase activity and carboxypeptidase activity (2) Optimal temperature is 90-95 ° C (3) Optimal pH is 6.5-8.0 (4) Activity even after heating at 95 ° C for 3 hours (pH 7.5) (5) Molecular weight is 40,000 (SDS polyacrylamide electrophoresis) 2. The enzyme according to claim 1, which is represented by the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence in which one or several amino acids have been deleted, substituted or added in SEQ ID NO: 1. 3. A gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted or added; and Protein having peptidase activity