JP2957246B2 - Microbial carboxypeptidase B-like enzyme - Google Patents

Description

The present invention relates to a novel microbial carboxypeptidase B of microbial origin.
Like enzyme.

(Prior art) Carboxypeptidase B (hereinafter referred to as CPase B)
Is already known [JEFolk, The Enzymes (3rd e
d.) 3 57~79 (1971)], this CPase B, in order to cut the arginine or lysine at the C-terminus of the peptide, is widely used as a biochemical reagent for primary structural analysis of proteins. Further, since CPase B has, for example, a kinin (Kinin) inactivating effect, it is expected to be used as a pharmaceutical.

CPase B is derived from human, bovine, porcine, rat pancreas, shark,
Those that are present in starfish and the like and are made from porcine pancreas extract as a raw material are commercially available. However, since pancreatic extracts contain other enzymes, such as kallikrein and trypsin, the separation of both enzymes from CPase B on an industrial scale is very expensive. Furthermore, animal organs have the disadvantage that they are not suitable for industrial-scale production as resources for producing oxygen.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and an object of the present invention is to provide a CPase B-like enzyme of microbial origin replacing CPase B derived from animal organs. It is in.

(Means for Solving the Problems) In order to achieve the above object, the present inventors searched for various microorganisms and found that Chaetomium sp. Belonging to Chaetomium (ketomium) collected from soil in Tondabayashi City, Osaka Prefecture. .
It has been found that F-33 produces oxygen with the desired carboxypeptidase properties. Then, the enzyme was purified and its properties were examined in detail. As a result, it was confirmed that the enzyme was a new type of CPase B-like enzyme, and the present invention was completed.

 The CPase B-like enzyme of the present invention has the following properties.

(1) Action: Arginine or lysine at the C-terminal of a peptide having arginine or lysine is released by hydrolysis.

(2) Substrate specificity: glycyl-glycyl-tyrosyl-arginine, bradykinin, neurotensin fragment 1-8
To release arginine at the C-terminus by hydrolysis.

(3) Optimum pH: about 8.5 (in Tris-HCl buffer).

(4) Optimum temperature: 55 ° C (5) pH stability: pH 7 ~
Stable at 10.

(6) Thermal stability: When kept at 4 ° C. and pH 7.5, it shows 100% activity for at least one year.

It shows 90% residual activity when heated at 55 ° C. for 5 hours.

(7) Molecular weight: The molecular weight measured by the gel filtration method is about 450,000.

(8) Isoelectric point: 5.49 The CPase B-like enzyme of the present invention is a Chaetomium spp.
omium sp. F-33. This strain is a new strain isolated from the soil collected by the inventors from Tondabayashi, Osaka Prefecture. The bacteriological properties of this strain are shown below.

(A) Growth state in medium Broth agar medium Growth at broth agar doubled value does not occur at 5%. 30
At 7 ° C., colonies reach a diameter of 4.7-5.7 cm in 7 days.
The appearance is white floc. After 14 days of culture, the colonies reach a diameter of 7-8 cm. After 21 days of solvent, the surface becomes dense and moist wool-like, but no coloration is observed and no ascus is formed. At 37 ° C, it grows poorly and is not skillful.

Czapek Agar Medium No growth takes place at 5 ° C. on Tzapek agar medium. At 30 ° C., colonies reach a diameter of 4-5 cm in 7 days. The properties are white floc and are wet compared to the colonies on the gravy agar medium. After 14 days of culture, the colonies reach a diameter of 7 to 8 cm and become partially black-green. After 30 days of cultivation, it becomes almost entirely black-green. The formation of ascocarcass is not seen.

Oatmeal agar medium Growth on oatmeal agar medium does not occur at 5 ° C. At 30 ° C., the diameter of the colony reaches 5 to 6 cm in 7 days, and becomes wool-like. Colony diameter is 7 in 14 days
It reaches ~ 8cm and the back side becomes dark olive green to green yellow.

Potato-glucose agar medium Growth on potato-glucose agar medium does not occur at 5 ° C. At 23 ° C., the colonies reach a diameter of 4 to 5 cm in 9 days, and are white flocculent. In 14 days, the colonies reach 6.5-7.5 cm in diameter. The inside of the middle portion is black-green, leaving the center and the circumference, and the back surface of the center is black-brown. As a result, the whole becomes moist, and an oil ball is formed on the colored portion. Ascospores are common but few ascospores. The formation of acacia is observed in the black-green area.

(B) Physiological and ecological properties Optimal growth conditions pH 5-7 Temperature 23 ° C-27 ° C Range of growth pH 4-8 Temperature 15 ° C-37 ° C Other remarkable features Cultivation in potato-glucose medium Produces a peptidase B-like enzyme in the cells.

(C) Microscopic findings Ascomycetes: Translucent when young and black-green when mature. Oval to subspherical, 200-240 × 150-200μ in size
m.

Top hair: dark olive brown to almost black with partition walls and straight hair. Branches almost at right angles, forming a complex mesh. Ends on a blunt tip without tapering. 7-7.5 μm width at base.

Side hair: similar to top hair, but narrow. Width at base 5-6
μm.

Ascospores: club-shaped 8 spores Ascospores: Colorless at first and dark olive brown later. Broad elliptical to circular. 6-7 × 4-6 μm.

Based on the above mycological properties, "A monograph of the genus chaetomiu
m) "(1970) [HKSeth]," Chaetomiaceae "(1963) [Ames (LMAme
s)] and “Mycobacterium Illustration (bottom)” (1978) (Keisuke Tsubaki et al.), and when these were searched, the strain was determined to belong to ketomium. This strain resembles Ketomium spinosum, but it differs from Ketomium spinosum in that its ascospores are oval (as described in the above-mentioned literature), and this strain has a broad elliptic to circular shape. In addition, ketomium
Spinosum does not grow at 37 ° C and turns gray-yellowish brown on oatmeal agar medium (described in the above literature),
In this strain, the reverse side is dark olive green to green yellow, 37 ° C
It is different in that it is viable but can grow.

Therefore, the present inventors named this strain Chaetomium (ketomium) sp. F-33. This strain was provided to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology
17).

In the present specification, designations of abbreviations for amino acids, peptides, protecting groups, and the like follow the conventional symbols in the art.

Culture conditions The medium for culturing the production strain for producing the CPase B-like enzyme of the present invention does not need to be exceptional, and a usual medium is used. Glucose, sucrose, glycerol, starch and the like are used as carbon sources. As a nitrogen source, meat extract, yeast extract, malt extract, polypeptone, casamino acid, various amino acids, synthetic amino acids, soy protein, fish meal and the like are used. As salts, Na
Cl, KCl, potassium tartrate, sodium nitrate, monopotassium phosphate, magnesium sulfate, calcium chloride and the like are used. In addition, bran, rice bran, rapeseed meal, molasses, corn steep liquor, potato extract, vitamins and the like are also useful.

In particular, the present carboxypeptidase B-like enzyme (hereinafter referred to as CP
Potato extract / sucrose medium is preferably used as a medium for culturing (including seed culture and production culture) of a producing bacterium (referred to as ase B-like enzyme) (described in detail in Examples). . Cultivation is at pH 5-7, preferably at pH 6.
It is carried out at around 25 ° C. to 37 ° C., preferably at 30 ° C., under shaking or aeration and stirring. The culture time is, for example, when using a Sakaguchi flask, 40 to 100 hours is appropriate,
When a fermenter is used, 45 to 50 hours is appropriate.

Enzyme collection method Since this CPase B-like enzyme is mainly present in the cells, after culturing, the cells are collected by filtration or centrifugation, etc.
The cells are crushed to extract the enzyme. For example, a method of grinding cells using a mortar, a ball mill, or the like in the presence of glass beads, sea sand, etc. (Dynomill A, Ba
chofen), freeze-thaw method, enzyme treatment method. The method for disrupting the cells is not particularly limited, but a method with a high recovery rate is preferred. After the disruption of the cells, a crude enzyme solution is obtained by adding a buffer solution and filtering or centrifuging.

In order to obtain a purified enzyme from the crude enzyme solution, the methods usually used for purifying the enzyme can be used alone or in combination. Examples thereof include salting out, solvent precipitation, various ion exchange column chromatography, hydrophobic chromatography, gel filtration, affinity chromatography, centrifugation, and electrophoresis. For example, the crude enzyme solution is fractionated with ammonium sulfate, then fractionated with ethanol, subjected to chromatography, and subjected to gel filtration to obtain a purified enzyme. As the above-mentioned chromatography treatment, for example, first, an ion exchange column (Q-Sepharose, DEAE Toyopearl, etc.) is applied, and then, a hydrophobic column (butyl Toyopearl, etc.)
Method. Examples of the gel filtration carrier include Toyopearl HW55S.

Activity measurement method 30 mM Bz-Gly-Arg 0.1 ml and 100 mM Tris-HCl (pH 8.
5) Add 0.05 ml of enzyme solution to 0.85 ml of the mixture, and start the reaction at 55 ° C. After reacting for 15 minutes, the mixture is immediately cooled on ice, 1 ml of 200 mM citrate buffer (pH 5.0) and 1 ml of ninhydrin reagent are sequentially added, and the mixture is rapidly stirred to stop the reaction. The obtained reaction solution is heated in boiling water for 5 minutes and then cooled on ice. To this, 2 ml of 60% (v / v) ethanol is added and mixed well, and then the absorption at 570 nm is measured. One unit (U) of the enzyme is an amount of the enzyme capable of decomposing 1 mol of the substrate (releasing 1 mol of Arg) per minute at 55 ° C.

The ninhydrin reagent used in this measurement was ninhydrin 15
g solution in 300 ml of methyl cellosolve, 0.01 M KCN
Solution B obtained by dissolving 250 ml of methyl cellosolve in 5 ml is 1: 5
It is prepared by mixing at a ratio of

Properties of oxygen Among the properties of the following enzymes, substrate specificity, inhibition, influence of various ions, molecular weight, isoelectric point, and Km value were measured using purified samples obtained in Examples described later. . For the optimum pH and stable pH range, the optimum temperature and the thermal stability, partially purified samples obtained as follows were used. First, ammonium sulfate was added to the cell extract at 80% saturation, and the obtained precipitate was dialyzed against 100 mM Tris-hydrochloric acid (pH 7.5). After the dialysate was adsorbed on Q-Sepharose equilibrated with the same buffer, the eluate was eluted by linearly increasing the NaCl concentration of the eluate to 0 to 1 M to obtain a partially purified sample.

Action The arginine or lysine at the terminal of a peptide whose C-terminus is arginine or lysine is released by hydrolysis. It works best with arginine-terminated peptides.

Substrate specificity Degradability of the synthetic peptides shown in Table 1 was examined. First, 5
Each substrate (synthetic peptide) was added to 100 mM Tris-HCl (pH 8.5) containing 1% ethanol at a ratio of 1 mM, and reacted at 55 ° C. for a predetermined time. The substrate degradation rate was calculated by quantifying the free amino acids generated by the ninhydrin method (according to the activity measurement method). Table 1 shows the results.

From Table 1, it can be seen that the CPase B-like enzyme of the present invention degrades a peptide having a C-terminal Arg best. Like Arg, peptides having a basic amino acid Lys at the C-terminus also degrade relatively well, but at a rate about 1/9 that of Arg. In this regard, the enzyme is a CPase B, for example, The E
CPase B described in nzyme, 3rd ed., 3, 57-79 (1971) (this enzyme degrades Arg-terminal and Lys-terminal peptides at similar rates). In the case of a peptide in which the second amino acid residue from the C-terminal is Phe (aromatic amino acid), the same level of resolution is observed,
It can be seen that in the case of aliphatic amino acids such as Gly, Ala, Ile, Leu, etc., the resolution differs depending on the type of C-terminal amino acid.

Next, the degradation of various natural peptides by this enzyme was examined. In a 10 mM phosphate buffer (pH 7.5) containing 1 mM substrate, 55
The enzyme was allowed to react for a predetermined time at ° C. Amino acid analyzer (Hitachi High Speed Amino Acid Analyzer 835)
Shape). Table 2 shows the results.

Similar to the results in Table 1, when the second amino acid residue from the C-terminal was an aromatic amino acid such as Tyr or Phe, a high degradation activity was exhibited. In contrast, the activity of Ala decreased to 1/3 to 1/4, and the degradation of Leu did not occur at all. Furthermore, the present CPase B-like enzyme was relatively well degraded even when the second amino acid residue from the C-terminal was Pro. This is because CPase B described in The Enzyme above is 2
This is a significant difference from the fact that the peptide with Pro is hardly degraded.

As described above, the CPase B-like enzyme of the present invention is similar to the conventional CPase B in that it degrades a peptide having a basic amino acid such as Arg or Lys at the C-terminus well, but differs in the following points. Is evident.

(I) Degradation of the peptide having Arg at the C-terminus is nearly 10 times faster than that of the peptide having Lys at the C-terminus.

(Ii) Degradation of the peptide in which the second amino acid from the C-terminal is Pro is also performed relatively favorably.

(Iii) When the second amino acid from the C-terminus is an aromatic amino acid, the decomposition is performed relatively favorably.

Optimum pH and stable pH Using this enzyme, an enzymatic reaction was carried out under pH conditions in the range of pH 6 to 10 according to the activity measurement method. FIG. 1 shows the relative activity of the enzyme at each pH. In FIG. 1, ● represents Tris-HCl, Δ represents KH ₂ PO ₄ —Na ₂ HPO ₄ , □ represents boric acid / KCl—Na ₂ CO ₃ ,
And ○ indicates the results obtained using each buffer solution of Na ₂ HPO ₄ -citrate. FIG. 1 shows that the optimum pH is about 8.5 at 55 ° C.

The enzyme was added to Britton-Robinson buffer at the specified pH for 2.76.
After dissolving at a rate of × 10 ⁻⁷ U / ml and keeping the mixture at 25 ° C. for 9 hours, the residual activity was measured. The result is shown in FIG. FIG. 2 shows that the stable pH range is pH 7.0 to 10.0.

Range of appropriate temperature for action Using this enzyme, Bz-Gly-Arg was used as a substrate, and an enzyme reaction was carried out under a temperature condition of 30 to 70 ° C according to the activity measurement method. Its relative activity is shown in FIG. FIG. 3 shows that the optimum temperature of the present enzyme is about 55 ° C.

Thermostability When this enzyme was kept at pH 7.5 and 4 ° C, it was not inactivated for at least one year. Even after maintaining at pH 7.5 and 55 ° C. for 4 hours, 90% residual activity was exhibited.

The enzyme was maintained at a concentration of 2.76 × 10 ⁻⁷ U / ml at pH 7.5 at the temperature shown in FIG. 4 (55 ° C. to 100 ° C.) for 15 minutes, and its residual activity was determined. The result is shown in FIG. FIG. 4 shows that the enzyme is stable up to 55 ° C. when kept at pH 7.5 for 15 minutes and completely inactivated at 80 ° C. or higher.

Inhibition 100 mM Tris containing the compounds shown in Table 3 at a ratio of 0.7 mM
After incubating the enzyme (3.8 × 10 ⁻⁹ U / ml) for 10 minutes in hydrochloric acid (pH 8.5), the residual activity was measured. Table 3 shows the results.

As shown in Table 3, this enzyme was similar to CPase B in EDTA, o
-Strongly inhibited by metal chelators such as phenanthroline. From this, it is clear that the present enzyme is a metal enzyme. However, since this enzyme is also inhibited by DTT and PCMB, it is considered that -SS bond and -SH group are necessary for the expression of activity. Therefore, it is considered that the enzyme is different from CPase B, which is also a metal enzyme.

Next, the effects of various ions on the activity of the present enzyme were examined. First, the enzyme solution was dialyzed against 100 mM Tris-HCl (pH 7.5) containing 1 mM EDTA at 4 ° C. for 12 hours, and then dialyzed again against 100 mM Tris-HCl (pH 7.5).
EDTA was removed. To this, the compounds shown in Table 4 were added at a concentration such that the desired ion became 1 mM, and the activity of the enzyme was measured.

As shown in Table 4, no activation was observed for the ionic species within the range tested, but rather it was inhibited. In particular, when CoCl ₂ was added, the activation was significantly reduced. This is the traditional
This is a feature that is significantly different from CPase B.

Molecular weight The molecular weight by gel filtration using Toyopearl HW55S is about 450,000.

Using a fast system (manufactured by Pharmacia) using the isoelectric point fast gel IEF3-9, pI carburetion kit pH2.
The isoelectric point of this enzyme was measured using 5-6.5 as a standard. As a result, the isoelectric point of this enzyme was 5.49.

Km value for Bz-Gly-Arg 100 mM Tris-HCl (pH 8.
The reaction was performed in 5), and the Km value was calculated. As a result, Km
The value was 0.59 mM.

Comparison with known enzymes Comparison of this enzyme with previously reported CPase B revealed the following differences.

Both enzymes convert basic amino acids Arg or Lys to C
Arg or Lys at the end of the peptide relative to the end is specifically released by hydrolysis. The CPase B-like enzyme of the present invention acts more liberally on Arg-containing substrates, and its degradation rate is about 10 times faster for Arg-containing substrates than for Lys-containing substrates. In contrast, conventional
CPase B acts to the same extent on substrates having Arg at the C-terminus and substrates having Lys at the C-terminus.

This enzyme acts on a peptide in which the second amino acid residue from the C-terminal is Phe and a peptide in which Pro is
Decomposes this relatively well. In contrast, conventional CP
ase B has low degradation of these.

Both enzymes are metalloenzymes, but in this case CoCl
_No activation by ₂ or ZnSO ₄ is observed. Further DTT
Or it is also inhibited by PCMB. In contrast, conventional CPase B is activated by CoCl ₂ or ZnSO ₄ and is less susceptible to inhibition by DTT or PCMB.

As described above, the enzyme of the present invention comprises
Or the property of specifically releasing Lys,
Same as conventional CPase B. However, it is a novel enzyme that differs entirely in terms of substrate specificity, sensitivity to inhibitors and ionic effects. Therefore, this enzyme is CPase
It was named B-like enzyme.

(Examples) Hereinafter, the present invention will be described with reference to examples.

(A) Culture of bacterial cells Peel the potatoes and cut them into 1 cm squares.
After adding 00 ml, the mixture was heated and boiled to perform extraction for 1 hour. After cooling, filtration was carried out by blistering, and finally distilled water was added to a volume of 1000 ml. 2% (w / v) sucrose was added to the potato extract thus obtained to obtain a potato extract / sucrose medium.

This potato extract / sucrose medium is sterilized and
aetomium sp. F-33 strain was inoculated and seed-cultured at 30 ° C. for 3 days. Separately, put the above potato extract / sucrose medium 3.5 in a 5-volume fermenter, and add
Sterilization was performed for minutes. 300 ml of the seed culture
, And culturing was performed under the conditions of 30 ° C., a stirring speed of 100 rpm, and an aeration rate of 1 / min. After 45 hours of culture in which the enzyme activity is maximized, the cells are collected by suction filtration using bleached filter paper,
70 g of cells were obtained.

(B) Isolation and Purification of Enzyme Approximately the same amount of sea sand was added to 70 g of the cells, and the cells were crushed in a mortar. To this, 100 mM Tris-HCl (pH 7.5) 20
Add 0 ml, leave for a while, perform suction filtration using a bleached filter paper, and filter 200 ml of the filtrate (crude enzyme extract) (total activity).
7.73 × 10 ⁻⁴ U, specific activity 0.0232). The above extraction operation was performed under ice cooling.

Using this crude enzyme extract, purification was performed as described below. First, the crude enzyme extract was fractionated with ammonium sulfate at 40-60% saturation, and the enzyme was recovered as a precipitate. 100 m of this precipitate
After dissolving in M Tris-hydrochloric acid (pH 7.5), dialysis was performed overnight at 4 ° C., followed by ethanol fractionation (40 to 60% v / v), and the enzyme was recovered as a precipitate again ( All activities 4.18 ×
10 ^-4 U, specific activity 7.64; yield 54%).

The precipitate was dissolved in 100 mM Tris-hydrochloric acid (pH 6.0) and dialyzed against the same buffer. The dialysis solution was diluted with Q-Sepharose (1.6 × 1
3cm; manufactured by Pharmacia), then thoroughly wash the column with 100mM Tris-HCl (pH 6.0; containing 0.1M NaCl), and linearly increase the NaCl concentration from 0.1M to 0.4M. To elute the enzyme. This enzyme has NaCl concentration of eluent.
Eluted from around 0.3M (total activity 3.39 × 10 ^-4 U, specific activity 4
0.8; yield 44%).

Next, the active fraction was dialyzed against 100 mM Tris-hydrochloric acid (pH 7.5), and then adsorbed on a DEAE Toyopearl 650M (1 × 9 cm; Tosoh Corporation) column previously equilibrated with the same buffer. 100mM Tris-HCl containing 0.1mM NaCl (pH7.5)
After sufficient washing with, elution was performed at 2 ml per fraction with a linear concentration gradient of 0.1 M to 0.15 M NaCl. Book C
The Pase B-like enzyme was eluted when the NaCl concentration was around 0.12 M (total activity 2.60 × 10 ⁻⁴ U, specific activity 144; yield 34%).

The eluted active fraction was washed with 100 mM Tris-HCl (pH 7.5, 3M
After dialysis against NaCl, butyltoyopearl 650 (1 × 9) previously equilibrated with the same buffer
cm; manufactured by Tosoh Corporation) and washed with the same buffer solution.
The enzyme was eluted by linearly decreasing the NaCl concentration from M to 0M. This CPase B-like enzyme has a NaCl concentration of about 1
It was eluted around M (total activity 1.78 × 10 ⁻⁴ U, specific activity 278, yield 23%).

The eluted active fraction was sufficiently dialyzed against 100 mM Tris-hydrochloric acid (pH 7.5), and the resulting solution was subjected to Q-Sepharose (1 × 9 cm; Pharmacia) previously equilibrated with the same buffer. Adsorbed. After washing with the same buffer containing 0.23 M NaCl, the enzyme was eluted using an eluent having a linearly increased NaCl concentration from 0.23 M to 0.4 M. The enzyme activity was completely eluted when the NaCl concentration was about 0.4M (total activity).
0.618 × 10 ⁻⁴ U; specific activity 170, yield 8.0%).

Further, the eluted active fraction was subjected to 100 mM Tris-hydrochloric acid (p
After dialysis against H9.0), the inner dialysis solution is adsorbed to DEAE Toyopearl 650M (0.5 × 5 cm, manufactured by Tosoh Corporation) equilibrated with the same buffer, and the same buffer containing 0.18M NaCl is sufficient. Was washed. The enzyme was eluted with a linear concentration gradient of 0.18M to 0.25M NaCl. The enzyme activity was eluted when the NaCl concentration was about 0.2 M (total activity: 0.274 × 10 ⁻⁴ U; specific activity: 106; yield: 3.5
%).

Finally, ultrafiltration of 12 ml of the active fraction (Amicon 8050)
And concentrated to 2 ml. The concentrated enzyme solution is applied to a Toyopearl HW55S column (2.6 × 90 cm, manufactured by Tosoh Corporation) which has been equilibrated with 100 mM Tris-hydrochloric acid (pH 7.5), and 2 ml per fraction is collected at a flow rate of 45.6 ml / hour. (Total activity 0.027 × 10 ⁻⁴ U; specific activity 34.2; yield 0.35%).

By the above series of operations, the specific activity was 1470 with a yield of 0.35%.
Was obtained. As a result of examining the purity of this purified sample using pH 7.5 fast gel (Pharmacia),
It turned out to be single.

When the properties of the purified enzyme and the partially purified enzyme were examined, the properties described in the section “Properties of the enzyme” were confirmed.

(Effect of the Invention) According to the present invention, such a novel carboxypeptidase B-like enzyme is obtained. This enzyme is easy to produce because it is of microbial origin. Furthermore, since the specificity of the substrate is different from that of known carboxypeptidases, it is possible to use such a difference in properties for various uses such as a reagent for determining the primary structure of a protein.

[Brief description of the drawings]

FIG. 1 is a graph showing the optimal pH of the enzyme of the present invention; FIG. 2 is a graph showing the stable pH range of the enzyme; FIG. 3 is a graph showing the optimal temperature of the enzyme; 5 is a graph showing the effect of temperature on stability.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Sawao Murao 2-12, Horikado Midoricho, Sakai-shi, Osaka (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A carboxypeptidase B-like enzyme having the following physicochemical properties: (1) Action: Arginine or lysine at the C-terminal of a peptide having arginine or lysine is released by hydrolysis. (2) Substrate specificity: glycyl-glycyl-tyrosyl-arginine, bradykinin, neurotensin fragment 1-8
To release arginine at the C-terminus by hydrolysis. (3) Optimum pH: about 8.5 (in Tris-HCl buffer). (4) Optimum temperature: 55 ° C (5) pH stability: pH 7 ~
Stable at 10. (6) Thermal stability: When kept at 4 ° C. and pH 7.5, it shows 100% activity for at least one year. It shows 90% residual activity when heated at 55 ° C. for 5 hours. (7) Molecular weight: The molecular weight measured by the gel filtration method is about 450,000. (8) Isoelectric point: 5.49 2. The enzyme according to claim 1, which is derived from the genus Chaetomium.