JPH04126079A - Carboxypeptidase b-like enzyme originated from microorganism - Google Patents

Abstract

PURPOSE:To obtain a carboxypeptidase B-like enzyme suitable for the production on industrial scale by culturing a microbial strain belonging to genus Chaetomium. CONSTITUTION:A microbial strain belonging to genus Chaetomium and capable of producing carboxypeptidase, e.g. Chaetomium sp. F-33 (FERM P-11711) is cultured and the objective carboxypeptidase B-like enzyme accumulated in the bacterial cell is collected therefrom. The enzyme hydrolyzes and isolates the terminal arginine or lysine of a peptide having arginine or lysine as carboxy terminal. It has an optimum pH of about 8.5 (in tris-HCI buffer solution), an optimum temperature of 55 deg.C, a stable pH range of 7-10 (25 deg.C, 9hr), a thermal stability to retain 100% activity at 4 deg.C and pH 7.5 after 1 year and 90% activity at 55 deg.C after 5hr and a molecular weight of about 450,000.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel carboxypeptidase B-like enzyme of microbial origin.

(Prior art) Carboxypeptidase B (hereinafter referred to as CPa5e B) is already known (J.E., Folk, The.
Enzyraes (3rd ed,) 357-79
(1971) ), this C'Pa5e B cleaves arginine or lysine at the C-terminus of peptides, and is therefore widely used as a biochemical reagent for analyzing the primary structure of proteins. Furthermore, since CPa5e B has an inactivating effect on, for example, kinin, it is expected to be used as a pharmaceutical.

CPa5e B exists in the pancreas of humans, orchids, pigs, rats, sharks, starfish, etc., and products made from porcine pancreas extract are commercially available. However, pancreatic extract contains other enzymes,
For example, since it contains kallikrein and trypsin, both enzymes and CPa5e can be used on an industrial scale.
Separating it from B would require a very large expense.

Furthermore, animal organs have the disadvantage that they are not suitable as raw materials for enzyme production in terms of resources for industrial scale production.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to provide a CPa5e B-like enzyme derived from a microorganism to replace CPa5e 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 Ch belonging to Chaetomium was collected from the soil of Tomitabayashi City, Osaka Prefecture.
It has been found that aetosium sp. F-33 produces an enzyme having the desired properties of carboxypeptidase. After purifying this enzyme and examining its properties in detail, we discovered that it is a new type of cPase B-like enzyme! This led to the completion of the present invention.

The CPa5e B-like enzyme of the present invention has the following properties.

(1) Action: The 8-terminal arginine or lysine of a peptide whose carboxy terminus is arginine or lysine is released by hydrolysis.

(2) Substrate specificity Acts on nigericyl-glycyl-tyrosyl arginine, bradykinin, and neurotensin fragment 1-8, and releases C-terminal arginine by hydrolysis.

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

(4) Optimum temperature = 55°C (5) pH stability: Stable at pH 7 to 10 when kept at 25°C for 9 hours.

(6) Thermal stability: When maintained at 4°C and pH 7.5, it shows 100% activity for at least 1 year.

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

(The molecular weight is approximately 450,000 when measured by Nigel filtration method.

(8) Isoelectric point: 5.49 The CPa5e B-like enzyme of the present invention is Chaetomium
Chaetomium sp., especially Chaetomium sp, strain F-33. This strain is a new strain isolated by the inventors from soil collected from Tomitabayashi City, Osaka Prefecture. The mycological properties of this strain are shown below.

(A) Growth status on culture medium ■ Broth agar medium Growth on broth agar medium does not occur at all at 5°C. 30
℃, the colony diameter reaches 4.7-5.71 mm in 7 days. Its appearance is white and cotton-like. After 14 days of culture, the colony diameter reaches 7-8 CI11. After 21 days of culture, the surface becomes dense and moist and woolly, but no coloration is observed and no pericarp is formed.

Growth is poor at 37°C and does not reach maturity.

■Tsapek agar medium Growth on Tsapek agar medium does not occur at all at 5°C. At 30°C, the colony diameter reaches 4-5 cm in 7 days. The colony is white and cotton-like, and is wetter than the colony on the broth agar medium. After 14 days of culture, the colonies reach a diameter of 7 to 80I11 and have a partially black border color. After 30 days of culture, the color becomes almost entirely black. No ascus formation is observed.

■Oatmeal agar medium Growth on oatmeal agar does not occur at 5°C.

At 30°C, the diameter of the colony reaches 5 to 6C11 in 7 days, and the colony becomes woolly in appearance. In 14 days, the diameter of the colony reaches 7-8C11, and the underside becomes dark olive green to greenish-yellow.

■Potato glucose agar medium Growth on potato glucose agar medium does not occur at 5'C. At 23°C, the diameter of colonies is 4-5 in 9 days.
It reaches C1+ and has a white cotton-like appearance. Colonies reach a diameter of 6.5-7.5 cm in 14 days.

Leaving the center and circumference, the inside of the middle part has a black border color,
The back side of the center is blackish brown. As a result, the entire product becomes wet and the colored portions have an oily taste. Many asci are observed, but ascospores are rare. Black edged part↓
Formation of cysts in two children is observed.

(B) Physiological and ecological properties ■Optimal growth conditions pH 5-7 Temperature 23°C-27°C ■Growth range pi (4-8 Temperature 15°C-37°C ■Other notable characteristics Potato glucose By culturing in a medium, a carboxypeptidase B-like enzyme is produced within the bacterial cells.

(C) Microscopic findings ■ Percipitum: Transparent when young, and becomes black-rimmed when mature. Oval to bulb shaped, size 200 to 240 x 1
It is 50-200 uta.

■ Crown hair: Dark olive-brown to almost black with septa and straight hair. It branches at right angles, forming a complex network. Ends at a plump tip without tapering. Width at base: 7-7.5μ.

■Filthy hairs: similar to apical hairs, but narrower in width. Width at base 5~
Next to 6μ.

■Ascospores: Club-shaped, 8 spores■Ascospores: Colorless at first, later becoming dark olive-brown. Wide oval to circular shape. 6-ma x 4-6 μm.

Based on the above mycological properties, “A Monograph of
Ketomium (A monograph of the
genuschaetomium) J (1970)
[Seth (HJ, 5eth) 'J, 1 Ketomiashie (
Chaetomiaceae) J (1963)
(Ames (1, M, An+es)) and "Illustrated Encyclopedia of Fungi (Part 2) J (1978) [Keisuke Tsubaki et al.], and when searching this, it was determined that this strain belongs to Chaetomium. i to Chaetomium subinosum (
However, the difference is that the ascospores of Chaetomium spinosum are rabbit-shaped (described in the above-mentioned literature), whereas the ascospores of Chaetomium spinosum are broadly oval to circular. In addition, ketomium
Spinosum does not grow at 37°C and turns grayish-yellowish on oatmeal agar medium (described in the above-mentioned literature), whereas the underside of this strain turns dark olive green to greenish-yellow;
It is different in that it grows poorly at 7°C, but can grow.

Therefore, the present inventors identified this strain as Chaetomium (
Chaetomium) sp, was named F-33. This strain was transferred to the National Institute of Microbiology, Agency of Industrial Science and Technology,
No. 1717 (FERM P-11717).

In this specification, abbreviations for amino acids, peptides, protecting groups, etc. shall follow the symbols commonly used in the field.

Notes to Article 1: The medium for culturing the production strain to produce the CPa5e B-like enzyme of the present invention does not need to be special (a normal medium can be used. As a carbon source, glucose, sucrose, glycerol, Starch etc. are used.

Meat extract, yeast extract, malt extract, polypeptone, casamino acids, various amino acids, general amino acids,
Soy protein, fish meal, etc. are used. As the salts, NaCl, KCl, potassium tartrate, sodium nitrate, potassium phosphate, magnesium sulfate, calcium chloride, etc. are used. Others include bran, rice bran, rapeseed meal,
Molasses, cornstarch liquor, potato extract, and vitamins are also useful.

In particular, zero carboxypeptidase B-like enzyme (hereinafter referred to as CP
Potato extract/shakerose medium is suitable as a medium for culturing (including seed culture and production culture) of producing bacteria (a5e B-like enzyme) (described in detail in Examples). .

Culture is carried out at pl (5 to 7, preferably around pl (6, 25
C. to 37.degree. C., preferably 30.degree. C., with shaking or stirring under aeration. The culture time is, for example, 40 to 100 hours when using a Sakaro flask, and 45 to 50 hours when using a fermentor.

group! ■Collection of CPa5e B-like enzymes mainly exist within the bacterial body, so
After culturing, the bacteria are collected by filtration or centrifugation, and the enzymes are extracted by crushing the cells. For example, a method of grinding the bacterial cells using a mortar, a ball mill, etc. in the presence of glass peas, sea sand, etc. (commercially available devices include Dyno Mill A, a freeze-thaw method available from Bachofen, and an enzyme treatment method). The method of disrupting the bacterial cells is not particularly limited, but a method with a high recovery rate is preferred.After disrupting the bacterial cells, a buffer solution is added and then filtered or centrifuged to obtain a crude enzyme solution.

To obtain a purified enzyme from this crude enzyme solution, methods commonly used for enzyme purification may 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, a purified enzyme can be obtained by subjecting a crude enzyme solution to ammonium sulfate fractionation, followed by ethanol fractionation, chromatography, and gel filtration. As for the above chromatography treatment, for example, first, an ion exchange column (
Q-Sepharose, DEAE Toyovar, etc.) and then a hydrophobic column (Butyl Toyopal, etc.). Examples of the Kel filtration carrier include Toyo Pearl H-55S.

30tMBz-Gly-Arg O, 1rxi! and 10
Add 0.05 yri of the enzyme solution to a 0.85 m mixture of 0 mM Tris-HCl (pH 8.5) and start the reaction at 55°C. After reacting for 15 minutes, this was quickly cooled with water, and then 1r of 200ml citrate buffer (pH 5,0) was added.
Add IIl and 1 part of ninhydrin reagent sequentially and stir quickly to stop the reaction. The resulting reaction solution is heated in boiling water for 5 minutes and then cooled on ice. Add to this 60% (v/
v) After adding 2 d of ethanol and mixing thoroughly, 57
Measure the absorption at 0na+.

One unit (U) of the present enzyme is defined as the amount of enzyme capable of decomposing 1 mol of substrate (liberating 1 mol of Arg) per minute at 55°C.

The ninhydrin reagent used in this measurement is ninhydrin 15
g with methyl cellosolve 300 d? The tested liquid A and 0. OIM KCN 5d with methyl cellosolve 25
It is prepared by mixing BWl in which 0111 is dissolved at a ratio of 1:5.

Of the following 11 properties of enzymes, substrate specificity, inhibition, molecular weight,
The isoelectric point and Km value were measured using purified specimens obtained in Examples described later. For optimum pH and stable pi (range, optimum temperature and thermal stability, a partially purified sample obtained as follows was used. First, ammonium sulfate was added to the bacterial cell extract at 80% saturation. The precipitate was
It was dialyzed against 100mM) ') HCl (pH 7.5). After adsorbing this dialysate to Q-Sepharose equilibrated with the same buffer, the NaCl concentration of the eluate was adjusted to 0 to
A partially purified sample was obtained by elution linearly increasing to 1M.

(2) Action The arginine or lysine at the carboxy terminus of a peptide whose carboxy terminus is arginine or lysine is released by hydrolysis. It works best on peptides with arginine at the end.

■Substrate specificity The degradability of the synthetic peptides shown in Table 1 was investigated. First, 5%
100 d l-lis-hydrochloric acid (pi(
Each substrate (synthetic peptide) was added to 8,5) at a ratio of 11 parts, and reacted at 55°C for a predetermined time. The substrate decomposition rate was calculated by quantifying the free amino acids produced by the ninhydrin method (according to the activity measurement method). The results are shown in Table 1.

(Left below) From Table 1, it can be seen that the CPa5eB-like enzyme of the present invention: the C-terminus is A
It can be seen that the rg peptide is degraded best.

Similar to Arg, peptides with the basic amino acid Lys at the C-terminus are also degraded relatively well, but the rate is slower than that of A.
It was about 179 for rg. In this respect, the enzyme is
CPa5e B, e.g. The EnzyIle,
3rd ed.

3, 57-79 (1971).
(This enzyme has a peptide with a terminal or ^rg and a peptide with a terminal L
ys peptide at a similar rate).

In the case of peptides in which the second amino acid residue from the C-terminus is Phe (aromatic amino acid), similar resolution is observed, but with Gly, Ala, lie, Leu
It can be seen that in the case of aliphatic amino acids such as, the resolution differs depending on the type of C-terminal amino acid.

Next, we investigated the degradation of various natural peptides by this enzyme.

1 phosphate buffer solution (pH 7) containing 1 Ill'l of substrate.
, 5), the enzyme was allowed to react at 55°C for a predetermined period of time. The released amino acids were measured using an amino acid analyzer (Hitachi High Speed Amino Acid Analyzer Model 835). The results are shown in Table 2.

Similar to the results in Table 2, high decomposition activity was shown when the second amino acid residue from the C-terminus was an aromatic amino acid such as Tyr or Phe. In contrast, Ala has 173 to 1
The activity decreased to /4, and no decomposition occurred at all with Leu. Furthermore, the CPa5e B-like enzyme degraded relatively well even when the second amino acid residue from the C-terminus was Pro. This is the above TheEnzyr@e
CPa5e B described in
This is a big difference from the fact that it hardly degrades peptides.

Thus, the CPa5e B-like enzyme of the present invention
Although it is similar to conventional CPa5e B in that it effectively degrades peptides having basic amino acids such as , Lys, etc. at the C-terminus, it is clear that it differs in the following points.

(i) Peptides having Arg at the C-terminus are degraded nearly 10 times faster than peptides having Lys at the C-terminus.

(ii) Peptides in which the second amino acid from the C-terminus is Pro are also degraded relatively well.

(iii) When the second amino acid from the C-terminus is an aromatic amino acid, decomposition is relatively successful.

■To i! lpH and Stable pH Using this enzyme, an enzymatic reaction was carried out according to the activity measurement method under conditions of pl (pH 6 to 10). The relative activity of the enzyme at each pH is shown in Figure 1. ,・ is Tris-hydrochloric acid, △ is KHzPO4-NazHPO4, and is boric acid KCI-NazCO: +, and ○ is Naz
The results using each buffer of HPO4-citric acid are shown. From FIG. 1, it can be seen that the optimum pH is about 8.5 at 55°C.

This enzyme was added to a Britton-Robins solution at a specified pH.
on buffer at a ratio of 2.76 x 10-'U/ml and maintained at 25°C for 9 hours, after which the residual activity was measured. The results are shown in FIG. It can be seen from FIG. 2 that the stable pH range is from pH 7.0 to 10.0.

■ Range of suitable temperature for action Using this enzyme and using Bz-Gly-Arg as a substrate,
Enzyme reactions were carried out under temperature conditions in the range of 70°C according to the activity assay method. The relative activities are shown in FIG. From FIG. 3, it can be seen that the optimum temperature for this enzyme is about 55°C.

■Thermostability When this enzyme was kept at pH 7, 5, and 4℃, it did not lose its activity for at least one year. Even after keeping it at pH 7, 5, and 55℃ for 4 hours, 90% of the residual activity remained. Demonstrative.

This enzyme was added at a concentration of 2.76X10-'U/s+1 at pH 7.
, 5, the temperature shown in Figure 4 (55°C to 100°C)
The remaining activity was determined by holding for 15 minutes. The results are shown in FIG. From Fig. 4, this enzyme is 1 at pH 7.5.
It can be seen that it is stable up to 55°C when held for 5 minutes and is completely inactivated at 80°C or higher.

■Inhibition 10 hours containing the compounds shown in Table 3 at a ratio of 0.7++M
The enzyme (3,8X
10-qU/ml) for 10 minutes, the residual activity was measured. The results are shown in Table 3.

(Leaving space below) Table 3 Compound residual activity (%) Iodoacetic acid iodoacetic acid N-ethyl malei TPCK" MSFb LCKC ZPCK'DTT" EDTA '0-Phenence mouth PCMB' Ringg:p RiU11! As shown in Table 3, this enzyme is similar to CPa5e B.
It was strongly inhibited by metal chelation rules such as DTA and o-phenanthroline. From this, it is clear that this enzyme is a metal enzyme. However, this enzyme is DTT,
Since it is also inhibited by PCMB, -3S binding,
It is believed that the -5H group is also necessary for the expression of activity. Therefore, it is considered to be a different enzyme from CPa5eB, which is the same metal enzyme.

Next, we investigated the effects of various ions on the activity of this enzyme. First, 100mM containing 1mM EDTA
Add the enzyme solution to Tris-HCl (pH 7.5) at 4°C.
Dialyzed for 2 hours, then again in 100mM Tris-HCl (
EDTA was removed by dialysis against pi(7,5). The compounds shown in Table 4 were added thereto at a concentration such that the desired ion was 1 mM, and the activity of the enzyme was measured.

(Margin below) Table 4 NaCI CoC1□ eSOa eCIz PbAc.

Zn5O.

gSOa nC1z CaC1t aC1z uSOa HgC1°CdC1! As shown in Table 4, no activation was observed for ionic species within the range tested, but rather inhibition. In particular, when CoCl2 was added, activation was significantly reduced. This is a feature that is significantly different from the conventional CPa5e B.

■Molecular weight The molecular weight determined by gel filtration using Toyovar HW55S is approximately 450,000.

■Fast system using isoelectric point fast gel rEp3-9 (
The isoelectric point of the enzyme was measured using pI Calibresion Kit pH 2.5 to 6.5 as a standard. As a result, the isoelectric point of this enzyme was 5.49.

■KIII value Bz-Gl for Bz-Gly-Arg
1001M Tris-HCl (pH
8,5), and the K+s value was calculated. As a result, the Km value was 0.59mM.

As a result of a comparison between the existing enzyme lacking 1IIIλL and CPa5e B that has been reported so far, the following differences were revealed.

■Both enzymes are basic amino acids Arg or Ly.
Arg or L at the terminal of a peptide having s at the C-terminus
ys is specifically released by hydrolysis.

The CPa5e B-like enzyme of the present invention works more favorably on substrates containing Arg, and its degradation rate is about 10 times faster for substrates containing Arg than for substrates containing Lys. In contrast, the conventional CPa5eB
acts to the same extent on substrates with Arg at the C-terminus and substrates with Lys at the C-terminus.

This enzyme acts on peptides whose second amino acid residue from the C-terminus is Phe, an aromatic amino acid, and peptides whose second amino acid residue from the C-terminus is Pro, an aliphatic amino acid, and degrades them relatively well. In contrast, the conventional CPa5
e B has low degradability in these.

■Both enzymes are metalloenzymes, but in the case of this enzyme Co
No activation by C1z or Zn5O is observed.

It is also inhibited by DTT or PCMB.

On the other hand, the conventional CPa5e B was recognized to be activated by CoC1 or ZnSO4, and was activated by DTT.
or less susceptible to inhibition by PCMB.

In this way, the enzyme of the present invention has a C-terminal Arg
Alternatively, the property of specifically releasing Lys is the same as that of conventional CPa5e B. however,
It is a new enzyme with completely different substrate specificity and sensitivity to inhibitors. Therefore, this enzyme was named CPa5e B-like enzyme.

(Margin below) (Example) The invention will be explained below with reference to examples.

(A) Cultivation of bacterial cells Add 500 g of water to 200 g of peeled and cut potatoes into 1 cm cubes! The mixture was then heated and boiled to perform extraction for 1 hour. After cooling, pass through a, and finally ↓, 1.
Distilled water was added to make the solution 000d. 2% (-/v) sucrose was added to the potato extract thus obtained to prepare a potato extract/sucrose medium.

This potato extract/sucrose medium was sterilized and Ch
aetomium sp, F-33 strain was inoculated, and 30
Seed culture was carried out at ℃ & trowel for 3 days. Separately, the above potato extract/sucrose medium is 3.5! was placed in a 52-volume fermenter and sterilized at 120°C for 15 minutes.

Add 300 d of the above-mentioned seed culture to this, and heat at 30°C.
1 stirring number 1100 rpm, aeration amount 1! /Il]n conditions. After 45 hours of culture, when the enzyme activity reaches its maximum, bacteria were collected by suction filtration using exposed filter paper.
70 g of bacterial cells were obtained.

(B) Isolation and Purification of Enzyme To 70 g of the bacterial cells, approximately the same amount of sea sand was added and the bacterial cells were crushed in a mortar. To this, 1100Il1 Tris-HCl (
pH 7,5) Add 200if and leave it for a while.
Suction filtration using exposed filter paper was performed to obtain a filtrate (crude enzyme extract) of 200 females (total activity 7.73×10-'U, specific activity 0.0232). The above extraction operation was performed under water cooling.

Using this crude enzyme extract, purification was performed as described below. First, the crude enzyme extract was subjected to ammonium sulfate fractionation at 40 to 60% saturation, and the enzyme was recovered as a precipitate.

This precipitate was dissolved in 100mM Tris-HCl (pH 7.5).
), dialysis was carried out overnight at 4°C, followed by ethanol fractionation (40-60% Si/ν), and the enzyme was again recovered as a precipitate (total activity 4.18 x 10-'U).
, specific activity? , 64; yield 54%).

This precipitate was dissolved in 1100II Tris-HCl (pH 6.0).
After dissolving in the same buffer, it was dialyzed against the same buffer. dialysis fluid,
After adsorption onto Q-Sepharose (1,6 x 13 cm; manufactured by Pharmacia) that had been equilibrated with the same buffer, 100 mM Tris-HCl (pH 6, o
; 0. The enzyme was eluted by washing the column extensively with IM NaC1 (containing IM NaCl) and increasing the NaClfi degree linearly from 0.1M to 0.4M. This enzyme was eluted when the NaCIfi degree of the eluent was around 0.3M (total activity 3.39XlO-'U, specific activity 40.8; yield 4
4%).

Next, the active fraction was dissolved in 100mM Tris-HCl (pH 7).
, 5), DEAE Toyo Pearl 650M (I
It was adsorbed onto a 9 cm (manufactured by Tosoh) column. 0.1M
100mM Tris-HCl (pH 7) containing NaCl
, 5), then 0.0~0.15M Na
Elution of 2 Id per fraction was performed using a linear concentration gradient of C1. This CPa5e B-like enzyme is NaCl
The total activity was 2.60 as it was eluted from a concentration of around 0.12M.
X10-'U, specific activity 144; yield 34%).

The eluted active fraction was treated with 11001II Tris-HCl (p
After dialysis against H7,5,3M NaCl), butyl h
After adsorption on Yopar 650 (IX9cm; manufactured by Tosoh Corporation) and washing with the same buffer, the enzyme was eluted by linearly decreasing the NaCl concentration from 3M to OM. This CPa5e B-like enzyme was eluted around 1 gate of NaC 11 degrees (total activity 1.78×10-'U, specific activity 278, yield 23%).

The eluted active fraction was dissolved in 100+aM Tris-HCl (
The internal solution was thoroughly dialyzed against Q-Sepharose (1×
9 cm; manufactured by Pharmacia). This is 0
．． After washing with the same buffer containing 23M NaCl, Na
C! Enzyme elution was performed using an eluent whose concentration was linearly increased from 0.23M to 0.4M. Enzyme activity is NaCl
It was completely eluted when the concentration was about 0.4 (total activity 0).
．． 618XIO-'U; specific activity 170. Yield 8.0
%).

Furthermore, the eluted active fraction was dialyzed against 100mM Tris-HCl (pH 9,0), and the dialyzed solution was equilibrated with the same buffer.
M (0.5X5CI11, manufactured by Tosoh Corporation), and
．． It was thoroughly washed with the same buffer containing 18M NaCl. Enzyme elution was performed using a linear concentration gradient from 0.18M to 0.25M NaCl. Enzyme activity is about 0 NaClJ degree
．． Total activity eluted around 2M 0.274 Xl0-
'U, specific activity 106; yield 3.5%).

Finally, the active fraction 12- was filtered by ultrafiltration (Amicon 8 (50
(type) to concentrate to 2 txR. This concentrated enzyme solution was mixed with 100mM Tris-HCl (pH 7.5).
) was applied to a Toyo Pearl HW 55S column (2, CX90cm, manufactured by Tosoh) at a flow rate of 45
．． 2 d/fraction was collected at 6 d/h (
Total activity 0.027 xLO-'U H specific activity 34.2
, yield 0.35%).

Through the above series of operations, the yield was 0.35% and the specific activity was 1.
470 purified enzymes were obtained. The purity of this purified sample is p
As a result of investigation using H7,5 Fast Gel (manufactured by Pharmacia), it became clear that it was single.

When the properties of the purified enzyme and partially purified enzyme were investigated, the properties described in the "Properties of Enzyme" section were confirmed.

(Effects of the Invention) According to the present invention, a novel carboxypeptidase B-like enzyme is thus obtained. This enzyme is easy to produce because it is of microbial origin. Since it has different substrate specificity from known carboxypeptidases, it is possible to take advantage of this difference in properties and use it for various purposes, such as as a reagent for determining the primary structure of proteins.

4. Figure 1 is a graph showing the optimum pH of the enzyme of the present invention; Figure 2 is a graph showing the stable pH range of the enzyme; Figure 3 is a graph showing the optimum temperature of the enzyme; and FIG. 4 is a graph showing the stable range of the enzyme.

that's all

Claims (1)

[Claims] 1. A carboxypeptidase B-like enzyme having the following physicochemical properties. (1) Effect: arginine or lysine at the carboxy terminus of a peptide whose carboxy terminus is arginine or lysine,
Released by hydrolysis. (2) Substrate specificity: glycyl-glycyl-tyrosyl-arginine, bradykinin, neurotensin fragment 1-8
and releases C-terminal arginine by hydrolysis. (3) Optimum pH: about 8.5 (in Tris-HCl buffer). (4) Optimum temperature: 55°C (5) pH stability: When kept at 25°C for 9 hours,
Stable at pH 7-10. (6) Thermal stability: When kept at 4°C and pH 7.5, it shows 100% activity for at least 1 year. It shows 90% residual activity when heated at 55°C for 5 hours. (7) Molecular weight: The molecular weight when measured by 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.