JP2004261141A - Cathepsin c-like enzyme and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology to practically produce a heat-resistant enzyme that has cathepsin C-like action that is useful as a component for dentifrice with high heat resistance because of its structural stability. <P>SOLUTION: Cathepsin C (dipeptidyl peptidase)-like enzyme has been found from a microorganism in Pyrococcus to provide the process for production, isolation and purification of the enzyme. In addition, the gene of the enzyme is elucidated to provide the biogenetic production process. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００３５】
精製酵素の反応ｐＨを表２に示す。ｐＨ７−８で１００％の高活性に達した。したがって反応至適条件は、ｐＨ７−８、８０℃である。
【００３６】
【表２】

【００３７】
精製酵素のカテプシンＣの蛍光基質に対する生産物の相対活性を示す。表３のように本酵素は各種のカテプシンＣ基質を分解する。
【００３８】
【表３】

【００３９】
また、本精製酵素は、インシュリンＢ鎖のような長鎖に対する分解活性を有する。
精製酵素の阻害剤との挙動は表４に示す。
【００４０】
【表４】

【００４１】
上記阻害剤は、セリン酵素の阻害剤（ＰＭＳＦ（ｐｈｅｎｙｌｍｅｔｈｙｌｓｕｌｆｏｎｙｌ ｆｌｕｏｒｉｄｅ）， ＴＬＣＫ（Ｎ−ｔｏｓｙｌ−Ｌ−ｐｈｅｎｙｌａｌａｎｉｎｅｃｈｏｌｏｍｅｔｈｙｌ ｋｅｔｏｎｅ），ＤＦＰ（ｄｉｉｓｏｐｒｏｐｙｌ ｆｌｕｏｒｏｐｈｏｓｐｈａｔｅ），金属酵素阻害剤（ｏ−ｐｈｅｎａｎｔｈｒｏｌｉｎ）， ＥＤＴＡ （ｅｔｈｙｌｅｎｅｄｉａｍｉｎｅｔｅｔｒａａｃｅｔｉｃ ａｃｉｄ），ＳＨ酵素阻害剤（２−ＭＥ（２−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ），及びシステイン酵素阻害剤（ＮＥＭ（Ｎ−ｅｔｈｙｌｍａｌａｉｍｉｄｅ）， ＭＩＡ（ｍｏｎｏｉｏｄｏａｃｅｔｉｃ ａｃｉｄ），Ｃｙｓｔａｔｉｎである。
【００４２】
以上の結果から、セリン酵素阻害剤、金属酵素阻害剤、システイン酵素阻害剤ともに阻害したので、本件カテプシンＣ様酵素は、セリン、金属、システインが活性発現に関与していることは推定される。
【００４３】
［実施例４］ カテプシンＣ様酵素の遺伝子工学的製造
（１）染色体ＤＮＡの調製
Ｐｙｒｏｃｏｃｃｕｓ ｈｏｒｉｋｏｓｈｉｉ（（ＪＣＭ９９７４、ＪＣＭ微生物カタログ第７版（１９９９）を実施例１のごとく培養し、培養菌体を集菌し緩衝液で洗浄後、ＳＤＳ存在下プロテイナーゼＫで菌体を分解後クロロホルムフェノール溶液で変性タンパク質を除去した後、核酸をアルコール沈澱させた。粗精製核酸をＲＮａｓｅを働かせＲＮＡを除去し、パイロコッカスホリコシ菌のＤＮＡを得た。
【００４４】
（２）カテプシンＣ様酵素遺伝子を含むプラスミドの調製
カテプシンＣ様酵素遺伝子と推定されるＯＲＦを調製するためのＰＣＲ反応のプライマーを設計した。そのプライマーを用いて、上記のＤＮＡを鋳型としてＰＣＲ反応条件を検討したところ次ぎの条件が適していることが判明した。
Ｔｈｅ ｕｐｐｅｒ ｐｒｉｍｅｒ（５’ＧＴＧ ＡＴＡ ＣＣＣ ＡＴＡ ＴＧＡ ＣＴＴ ＣＴＡ ＴＣＧ ＡＡＴ Ｇ３’下線部はＮｄｅ Ｉ ｓｉｔｅ）
；ｔｈｅ ｌｏｗｅｒ ｐｒｉｍｅｒ（５’ＧＣＴ ＣＧＧ ＡＴＣ ＣＡＧ ＴＣＡ ＡＴＡ ＡＧＡ３’下線部はＢａｍ ＨＩ ｓｉｔｅ）アニーリング温度５９度
調製した遺伝子は、ベクタープラスミド（ｐＥＴ−１１ａ ｎｏｖａｇｅｎ社製）に結合させカテプシンＣ様酵素遺伝子を含むプラスミドを調製した。
【００４５】
（３）酵素の大腸菌への導入
上記調製したプラスミドをコンピテンシーの高い大腸菌（ＸＬ２−Ｂｌｕｅ ＭＲＦ’ ｓｔｒａｔａｇｅｎｅ社製）に形質導入した。
【００４６】
（４）酵素の生産と精製
上記大腸菌から精製したプラスミドを酵素生産用の大腸菌（ＢＬ２１−Ｃｏｄｏｎ Ｐｌｕｓ（ＤＥ３）ＲＩＬ ｓｔｒａｔａｇｅｎｅ社製）に導入し、その大腸菌を３７℃で培養した。一夜培養後，ＩＰＴＧを加え３７℃で４時間培養した。培養液を遠心分離して菌体を集め、凍結融解してカテプシンＣ酵素を抽出した。抽出液は、８５℃、３０分間熱処理し、熱変性しやすいタンパクを沈澱除去し上清の活性を測定した。上述のように調製したプラスミドで形質転換した形質転換体の上清を酵素液としＨ−Ｇｌｙ−Ｐｈｅ−ｐ−ｎｉｔｒｏａｎｉｌｉｄｅに反応させた。生産量は、培養液ｍＬあたり３１単位の活性が生産された。本酵素は、通常のように３７℃で培養してもインクルージョンボデーにならず、活性のあるカテプシンＣ様酵素が短時間に高活性で生産された。
【００４７】
［実施例４］ カテプシンＣ様酵素活性に対する界面活性剤の影響
実施例２で調製した本件精製酵素に対する界面活性剤の影響を、濃度を変えて測定した。その結果を下記表に示す。
【００４８】
【表５】

【００４９】
【発明の効果】
反応動力学的解析によると、この酵素のＧｌｙ−Ｐｈｅ−ｐ−ｎａｐｈｔｈｙｌａｍｉｄｅに対する最大反応速度は、１．５５ｘ１０^５／ｓｅｃであり、非常に大きな反応速度を示した。非常に効率の良い酵素であり、かつ高温でも使用できることから、種々の洗浄などに用いることが可能で、極めて有用な酵素である。
【００５０】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the production of novel proteases. More specifically, a novel cathepsin C-like enzyme derived from an archaebacterium belonging to the genus Pyrococcus having cathepsin C activity, a DNA encoding a novel cathepsin C-like enzyme derived from an archaea belonging to the genus Pyrococcus, a hyperthermophilic bacterium The present invention relates to the production of a cathepsin C-like enzyme derived from an archaebacterium belonging to the genus Pyrococcus using E. coli.
[0002]
[Prior art]
Many types of cathepsins are known as proteases found in lysosomal fractions of various animal cells. Cathepsin C is a papain-like cysteine protease that acts on a polypeptide to release a dipeptide from the N-terminus, and is also called dipeptidyl peptidase. Cathepsin C is located in the cytoplasmic granule lysosome of bone marrow cells and lymphocytes, and cleaves and activates a serine protease necessary for an immune / inflammatory response (Non-Patent Document 1: McGuire, MJ et al., J. Biol. Chem. 268). , 2458 (1993) Cathepsin C deficiency is one of the causes of periodontal disease (Non-Patent Document 2: Nature Genet. 23, 421-424 (1999).
[0003]
Cathepsin C is a gene derived from a higher animal or a gene encoding cathepsin C derived from a higher animal, such as yeast (Non-Patent Document 3: Appl. Microbiol. Biotechnol. (2002) 59, 252-258), insect cells, etc. Non-patent document 4: Biochemistry (2001) 40, 1671-1678) is only reported, and cathepsin C derived from other than animal cells is not known.
[0004]
[Non-Patent Document 1] Biol. Chem. 268, 2458 (1993)
[Non-Patent Document 2] Nature Genet. 23, 421-424 (1999)
[Non-Patent Document 3] Appl. Microbiol. Biotechnol. (2002) 59, 252-258.
[Non-Patent Document 4] Biochemistry (2001) 40, 1671-1678
[0005]
[Problems to be solved by the invention]
Cathepsin C of higher animals, which has been found so far, is sensitive to chemicals such as surfactants, and required chloride ions for expression of the activity. Many of the enzymes present in lysosomes have an optimum pH in the acidic region, and no enzyme having an optimum reaction temperature of 80 ° C. or higher has been found. An object of the present invention is to find a technique for industrially producing a structurally stable thermostable enzyme having a cathepsin C-like action, which is useful for dentifrices, detergents, amino acid analysis and the like.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the genome information sequence was analyzed, and a microorganism belonging to the genus Pyrococcus, which is a source of many thermostable enzymes and grows at 90 ° C. or higher, was identified. After culturing in a jar fermenter, the high activity of cathepsin C (dipeptidyl peptidase) was found in the culture solution, particularly in the supernatant, and its production, isolation and purification methods, and its properties were clarified, and this enzyme was useful for dentifrices, etc. A structurally stable cathepsin-like enzyme, the gene of which was cloned and expressed in Escherichia coli to confirm cathepsin C activity, thereby completing the present invention.
[0007]
As the microorganism belonging to the genus Pyrococcus, Pyrococcus horikoshi (JCM9974, JCM Microorganism Catalog, 7th edition (1999)) can be preferably used.
[0008]
In the present invention, cathepsin C-like means having a dipeptidyl peptidase activity that generates a dipeptide from the N-terminus.
[0009]
The cathepsin C-like enzyme of the present invention has almost no amino acid sequence homology with the conventionally known cathepsin C, and furthermore, the optimal pH is acidic because cathepsin C acts on lysosomes. Is an enzyme which is completely different from conventionally known cathepsin C because it is neutral and active.
[0010]
Furthermore, although conventional cathepsin C is a cysteine protease, the cathepsin C-like enzyme of the present invention may not be inhibited by a cysteine enzyme inhibitor in some cases and cannot be determined as cysteine protease.
[0011]
In the present invention,
(1) a cathepsin C-like enzyme having a low polymer structure and not requiring chloride ions for activity expression;
(2) a cathepsin C-like enzyme having an optimal reaction temperature in the range of 75 to 85 ° C and an optimal reaction pH of 7 to 8,
(3) the above cathepsin C-like enzyme having a molecular weight of 160 ± 20 kD,
(4) Cathepsin C-like enzyme derived from a microorganism belonging to the genus Pyrococcus, and (5) Cathepsin C-like enzyme in which the microorganism belonging to the genus Pyrococcus is Pyrococcus horikoshii.
[0012]
The cathepsin C-like peptide is obtained by culturing and growing a microorganism belonging to the genus Pyrococcus, preferably Pyrococcus horikoshii, under appropriate culture conditions (eg, 95 ° C.). For example, after the supernatant is concentrated by a membrane filter to prepare an enzyme solution, the enzyme solution can be purified by appropriately combining hydrophobic chromatography, anion exchange resin chromatography, and / or gel filtration chromatography.
[0013]
For example, alkyl agarose and Phenyl Sepharose can be used as hydrophobic chromatography, DEAE Sepharose and Q Sepharose HP can be used as anionic chromatography, and Sephadex 200 and Sepharose HP can be used as gel filtration chromatography.
[0014]
Further, the present invention
(6) a cathepsin C-like enzyme having the amino acid sequence of SEQ ID NO: 1 in the sequence listing,
(7) Cathepsin C-like containing an amino acid sequence in which one or several, preferably 1 to 10 amino acid residues are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing enzyme,
(8) Cathepsin C-like having 70% or more identity, preferably 85% or more identity, more preferably 95% or more identity with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. enzyme,
(9) a nucleic acid such as an isolated DNA molecule comprising a polynucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;
(10) A polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 in which one or several, preferably 1 to 10 amino acid residues are deleted, substituted, or added. A nucleic acid such as an isolated DNA molecule,
(11) an isolated nucleic acid such as a DNA molecule comprising the polynucleotide sequence of SEQ ID NO: 2 in the sequence listing, and (12) a stringent sequence complementary to the sequence of the polynucleotide sequence of SEQ ID NO: 2 in the sequence listing. Includes nucleic acids, such as isolated DNA molecules, that hybridize under conditions and encode a polypeptide having cathepsin C activity.
[0015]
In addition, the identity of an amino acid sequence and the identity of a polynucleotide sequence mean the degree of the relationship between sequences determined by comparing two or more amino acid sequences or polynucleotide sequences. As a program for determining identity, there are programs such as ClustalW and DNASIS for amino acid sequences, but the value of identity in the present invention means a value when ClustalW is used under default conditions.
[0016]
As stringent conditions, well-known stringent conditions can be used. For example, (a) low ionic strength, high temperature washing conditions, for example, 0.015 M NaCl, 0.0015 M sodium citrate, 0 4. Conditions for washing at 50 ° C. with 1% SDS, (b) 50% formaldehyde, 5 × SSC (0.75 M NaCl, 0.075 M citric acid), X Denhardt's solution, salmon sperm DNA (50 g / ml), 0.1% SDS, and 10% dextran sulfate, 42 ° C., and 0.2X SSC, 0.1% DS, washing at 42 ° C.
[0017]
Furthermore, the present invention also includes a recombinant vector containing the DNA molecule according to any one of the above (9) to (12).
[0018]
For example, a polypeptide comprising a DNA molecule according to any one of the above (9) to (12) under the control of a promoter for expression of a thermostable proline dipeptide degrading enzyme, and having a cathepsin C activity in a host cell. An expression vector having the ability to express is included. Specifically, as the promoter, a T7 promoter (Novagen), an mFDH1 promoter (Non-Patent Document 3), and an AUG1 promoter (Invitrogen) can be used. As an expression vector, a known expression vector can be used in each host. In Escherichia coli, for example, a pET11-a vector (T7 promoter (Novagen)), and in a methanol yeast, a pMFXU4 vector (mFDH1 promoter (mFDH1 promoter)) Non-patent document 3)) or an expression vector can be prepared based on the pMETα vector (AUG1 promoter (Invitrogen)). Needless to say, the nucleic acid sequence of the present cathepsin C-like enzyme may be modified in accordance with the codon usage in each host, and then loaded on a recombinant vector.
[0019]
Further, the present invention includes a transformant transformed by the vector. As the host, microbial cells, insect-derived cells, animal cells, or plant cells can be used. For example, E. coli, Candida. bondini, COS-7 cells.
[0020]
As a means for introducing the vector into a host, a known method suitable for each host can be used.For example, a calcium phosphate method, an electroporation method, and further, in Escherichia coli, a high-temperature shock method, and in a methanol yeast, , Lithium acetic acid method, and the like.
[0021]
As a transformant, specifically, a gene encoding the present cathepsin C-like enzyme was transformed with, for example, Escherichia coli transformed with a vector incorporated into pET11-a, or a vector incorporated into pMFXU4 having an FDHI promoter. There are transformants of insect-derived cell lines that have been introduced by incorporating them into vectors using methanol yeast and baculovirus promoters.
[0022]
Further, the present invention includes a step of culturing the transformant, a step of recovering a cathepsin C-like enzyme from the obtained culture, a method for producing a transformant of an enzyme protein having a cathepsin C-like enzyme activity, and The cathepsin C-like enzyme obtained by the production method is also included.
[0023]
Furthermore, the present invention also includes a cathepsin C-like enzyme particularly suitable for a toothpaste whose optimal temperature has been changed to around 37 ° C. by an evolutionary engineering technique.
[0024]
【Example】
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
[0025]
Example 1 Cultivation of Pyrococcus sorghum and detection of cathepsin C activity 13.5 g of NaCl, 4 g of Na ₂ SO ₄ , 0.7 g of KCl, 0.2 g of NaHCO ₃ , 0.1 g of KBr, 30 mg of H ₃ BO ₃ , 10 g MgCl ₂ .6H 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, 5 g bacto 15 L of a culture solution obtained by dissolving peptone in 1 L of distilled water was pasteurized for 30 minutes. After returning to normal pressure, dry heat sterilized powdered sulfur was added to a concentration of 0.1-0.2%. Next, 10 mL of a 25% sodium sulfide solution saturated with nitrogen gas and then autoclaved was added, and after confirming that the pink color of the resasurin solution was not colored, the solution was made anaerobic. 0.5 L was inoculated and cultured at 95 ° C. for 22 hours with stirring at 100 rpm.
[0026]
The activity of the culture solution precipitate was determined by centrifuging the medium, suspending the precipitate in a 50 mM Tris buffer of the medium, freeze-thawing, and sonicating to extract a cell-binding portion of 2.97 units / mL. There was activity. On the other hand, the activity of the culture supernatant could not be detected as it was, but was measured after concentration by a factor of 2 with a membrane filter and found to be 237.84 units / mL. Cathepsin C activity was measured using H-Gly-Phe-p-nitroanilide (manufactured by Bachem), and the amount that released 1 micromol of paranitroanilide per minute was defined as 1 unit. The activity per mg of protein was 154.8.
[0027]
Example 2 Purification of Cathepsin C-Like Enzyme The enzyme solution obtained in Example 1 was subjected to hydrophobic chromatography (Hiload 16/10 Phenyl Sepharose, manufactured by Amersham Bioscience) in 100 mM sodium phosphate buffer, pH 7.5, and ammonium sulfate concentration of 1 mol. The active portion was eluted at a concentration of 0 mol of ammonium sulfate. The specific activity was 6,200, and the yield was 31%. This enzyme is a hydrophobic protein adsorbed on a hydrophobic column.
[0028]
The above-mentioned hydrophobic chromatographic eluate was adsorbed on an anion exchange resin (HiTrap Q Sepharose HP Amersham Bioscience) and washed with 10 M Tris buffer pH8. The activity eluted at a salt concentration of 0.3-0.4M. The specific activity was 7,137, and the yield was 10.7%. This enzyme is an acidic protein adsorbed on an anion exchange resin.
[0029]
It was applied to a gel filtration chromatograph (Hiload 26/60 Sephadex 200 prepgrade, manufactured by Amersham Bioscience). When eluted with a 50 mM sodium phosphate buffer, an activity peak was present at a position corresponding to a molecular weight of 160 ± 20 kD in 234 mL of the eluate. The specific activity was 12,578, and the yield was 6.5%. When this fraction was subjected to SDS electrophoresis, a band of a main component of 62 ± 2 kD and a band of a minor component of 41 ± 1 kD were detected. Therefore, this enzyme was a protein having a low polymer structure.
[0030]
N-terminal analysis of the protein purified by electrophoresis revealed that it was TSIEWDEKTF, which was an amino acid sequence. The amino acid sequence is the sequence of SEQ ID NO: 1, and the nucleic acid sequence encoding the amino acid sequence is the sequence of SEQ ID NO: 2.
[0031]
Example 3 Enzymatic Properties of Enzyme Cathepsin C activity was determined by mixing 1 μL of 200 mM H-Gly-Phe-p-nitroanilide (manufactured by Bachem), 949 μL of 50 mM sodium phosphate buffer pH 7.5 and 50 μL of enzyme solution. Was reacted at 80 ° C. and the change in absorbance at 406 nm was observed. The molecular absorbance of paranitroanilide was 9.91 mM, and the amount of 1 μmol of paranitroanilide released per minute was defined as 1 unit.
[0032]
The activity was measured by adding 10 mM, 20 mM, 30 mM, and 40 mM salt to an enzyme solution containing no chloride ions, and no activity increase was observed due to the addition of chloride ions. Therefore, salt is not required for expressing the activity of the enzyme, and the enzyme can be used without undergoing a change in taste due to the addition of salt.
[0033]
The optimum reaction temperature of the purified enzyme was 80 ° C. (Table 1). In the case of the crude enzyme, although there were many variations, the optimum reaction temperature was 90-100 ° C, and at 37 ° C, the activity was 28% of the optimum temperature.
[0034]
[Table 1]

[0035]
Table 2 shows the reaction pH of the purified enzyme. A high activity of 100% was reached at pH 7-8. Therefore, the optimal conditions for the reaction are pH 7-8 and 80 ° C.
[0036]
[Table 2]

[0037]
The relative activity of the product with respect to the fluorescent substrate of the purified enzyme cathepsin C is shown. As shown in Table 3, this enzyme decomposes various cathepsin C substrates.
[0038]
[Table 3]

[0039]
In addition, the purified enzyme has a degrading activity for long chains such as insulin B chain.
Table 4 shows the behavior of the purified enzymes with the inhibitors.
[0040]
[Table 4]

[0041]
The above-mentioned inhibitors include serine enzyme inhibitors (PMSF (phenylmethylsulfonyl fluoride), TLCK (N-tosyl-L-phenylalanine chloromethyl ketone), DFP (diisopropyl thiophene, thiophene thiophene, thiophene, thiophene, thiophene, thiophene) , SH enzyme inhibitors (2-ME (2-mercaptoethanol)), and cysteine enzyme inhibitors (NEM (N-ethylmalaimide), MIA (monoiodoacetic acid), and Cystatin.
[0042]
From the above results, it was presumed that serine, metal, and cysteine are involved in the expression of the activity of the cathepsin C-like enzyme because the serine enzyme inhibitor, the metalloenzyme inhibitor, and the cysteine enzyme inhibitor were all inhibited.
[0043]
[Example 4] Genetic engineering production of cathepsin C-like enzyme (1) Preparation of chromosomal DNA Pyrococcus horikoshii ((JCM9974, JCM Microorganism Catalog 7th edition (1999) was cultured as in Example 1, and cultured cells were collected) After the cells were washed with a buffer, the cells were digested with proteinase K in the presence of SDS, denatured proteins were removed with a chloroform phenol solution, and the nucleic acids were precipitated with alcohol. Coccus horikoshii DNA was obtained.
[0044]
(2) Preparation of Plasmid Containing Cathepsin C-like Enzyme Gene Primers for a PCR reaction for preparing an ORF presumed to be a cathepsin C-like enzyme gene were designed. When the PCR conditions were examined using the primers and the above DNA as a template, the following conditions were found to be suitable.
The upper primer (5'GTG ATA CC C ATA TG A CTT CTA TCG AAT G3 ' underlined the Nde I site)
The lower primer (5 ′ GCT C GG ATC CAG TCA ATA AGA 3 ′ underlined is Bam HI site) Annealing temperature 59 ° C. The gene prepared at the annealing temperature of 59 ° C. is ligated to a vector plasmid (manufactured by pET-11a Novagen) to produce a cathepsin C-like enzyme. A plasmid containing the gene was prepared.
[0045]
(3) Introduction of enzyme into Escherichia coli The above-prepared plasmid was transduced into Escherichia coli (XL2-Blue MRF 'stratagene) having high competence.
[0046]
(4) Production and Purification of Enzyme The plasmid purified from the E. coli was introduced into E. coli (BL21-Codon Plus (DE3) RIL Stratagene) for enzyme production, and the E. coli was cultured at 37 ° C. After overnight culture, IPTG was added and the cells were cultured at 37 ° C for 4 hours. The culture was centrifuged to collect the cells, which were freeze-thawed to extract the cathepsin C enzyme. The extract was subjected to a heat treatment at 85 ° C. for 30 minutes to remove the protein which was easily denatured by heat, and the activity of the supernatant was measured. The supernatant of the transformant transformed with the plasmid prepared as described above was used as an enzyme solution and reacted with H-Gly-Phe-p-nitroanilide. The production amount was 31 units of activity per mL of the culture solution. This enzyme did not become an inclusion body even when cultured at 37 ° C. as usual, and an active cathepsin C-like enzyme was produced with high activity in a short time.
[0047]
Example 4 Effect of Surfactant on Cathepsin C-Like Enzyme Activity The effect of the surfactant on the purified enzyme prepared in Example 2 was measured at different concentrations. The results are shown in the table below.
[0048]
[Table 5]

[0049]
【The invention's effect】
According to the reaction kinetic analysis, the maximum reaction rate of this enzyme for Gly-Phe-p-naphthylamide was 1.55 × 10 ⁵ / sec, indicating a very large reaction rate. Since it is a very efficient enzyme and can be used even at high temperatures, it can be used for various washings and the like, and is a very useful enzyme.
[0050]
[Sequence list]

Claims (14)

A cathepsin C-like enzyme having the following properties:
(1) The optimal reaction temperature is 75-85 ° C,
(2) The optimum reaction pH is pH 7-8,
(3) Chloride ion is not required for activity expression The cathepsin C-like enzyme according to claim 1, which has a molecular weight of 160 ± 20 kD. The cathepsin C-like enzyme according to claim 1, which is obtained by culturing a bacterium belonging to the genus Pyrococcus. The cathepsin C-like enzyme according to claim 3, wherein the microorganism belonging to the genus Pyrococcus is Pyrococcus horikoshi. The cathepsin C-like enzyme according to claim 1, which has the amino acid sequence of SEQ ID NO: 1 in the sequence listing. The cathepsin C-like enzyme according to claim 1, which comprises an amino acid sequence in which one or several amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. An isolated nucleic acid comprising a polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. An isolated nucleic acid comprising a polynucleotide sequence encoding an amino acid sequence in which one or several amino acid residues have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. The isolated nucleic acid according to claim 7, comprising the polynucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing. An isolated nucleic acid capable of hybridizing under stringent conditions with a complementary strand of the polynucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing and encoding a polypeptide having cathepsin C activity. A method for producing a cathepsin C-like enzyme, comprising culturing a microorganism belonging to the genus Pyrococcus, and recovering the cathepsin C-like enzyme according to any one of claims 1 to 6 from the culture medium. An expression vector comprising the nucleic acid according to any one of claims 7 to 10. A method for producing a cathepsin C-like enzyme, comprising transforming a host cell with the expression vector according to claim 12. A cathepsin C-like enzyme produced by transforming a host with the expression vector according to claim 12.