JPS63216481A - Carboxypeptidase and production thereof - Google Patents

Abstract

PURPOSE:To obtain the titled enzyme having an activity to successively separate amino acids from C-terminal of a peptide, exhibiting high otpimum action temperature and having high thermal stability, by culturing a specific highly thermophilic bacterium belonging to Thermus genus in a nutrient medium. CONSTITUTION:A highly thermophilic bacterium belonging to Thermus genus and capable of producing carboxypeptidase, e.g. Thermus aquaticus YT-1 (ATCC 25104) is cultured in a nutrient medium (usually at about neutral pH and 40-80 deg.C for 12hr-10 days under aeration and agitation) and the objective enzyme exhibiting optimum action temperature of 75-80 deg.C is separated from the cultured product. The enzyme has a molecular weight of 62,000 measured by gel-filtration method using Sephadex G-200, an amino acid composition shown in the Table and an optimum action pH of 8.0-9.0. The crude enzyme liquid extracted from the bacteria cell retains about 85% of the activity by heating at 80 deg.C for 20hr at pH 7.2 and about 65% of the activity by heating at 90 deg.C for 20hr.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to carboxypeptidase and a method for producing the same, and more particularly, to a carboxypeptidase that exhibits an optimum operating temperature at 75 to 80°C and has excellent heat resistance, and a method for producing the same. .

[Prior art and problems to be solved by the invention] Carboxypeptidases are found in the microbial and animal kingdoms.

Many species have been isolated from the plant kingdom, and their structures and properties have been investigated in detail. Carboxypeptidases are classified into serine carboxypeptidases, metallocarboxypeptidases, and cysteine carboxypeptidases depending on their catalytic activity.

However, the carboxypeptidases reported so far have been obtained from thermophilic organisms and have not yet been purified from thermophilic organisms such as thermophilic bacteria.
Furthermore, there are few reports on metallocarboxypeptidases isolated from bacteria.

[Means for solving problems]

Therefore, the present inventors conducted repeated research to search for thermophilic bacteria that produce carboxypeptidase with excellent heat resistance, and found that highly thermophilic bacteria belonging to the genus Thermus produce the desired enzyme. They discovered this and completed the present invention.

That is, the present invention relates to a carboxypeptidase that has the action of sequentially releasing amino acids from the C-terminus of a peptide, exhibits an optimum operating temperature at 75 to 80°C, and has high thermal stability, and a carboxypeptidase belonging to the genus Thermus. The present invention provides a method for producing carboxypeptidase by culturing highly thermophilic bacteria capable of producing it.

The carboxypeptidase of the present invention can be produced by culturing a highly thermophilic bacterium belonging to the genus Thermus and having the ability to produce the enzyme in a nutrient medium, producing the enzyme in the culture, and collecting the enzyme. .

Examples of microorganisms capable of producing the target carboxypeptidase include Thermus aquaticus (Thermus d) YT-1 (ATCC25
104), and natural or artificial mutant strains thereof can also be used in the present invention as long as they have the ability to produce the enzyme.

The nutrient medium may be any medium as long as the above-mentioned microorganisms can grow sufficiently and produce the desired enzyme. For example, a medium containing polypeptone, yeast extract, whey protein, etc. with inorganic salts such as calcium sulfate is preferably used. used.

Cultivation is usually preferably carried out by an aerated agitation culture method, and an antifoaming agent is added if necessary. Generally, the culture is adjusted to a pH near neutrality and kept at a temperature of 40 to 80°C for 12 hours or more.
This is carried out for about 10 days to ensure sufficient production of carboxypeptidase. This enzyme is mainly accumulated in the bacterial cells.

Carboxypeptidase can be collected from the culture using a combination of known methods as appropriate; one example is, after the completion of the culture, bacterial cells are collected from the culture by centrifugation, etc.
Store frozen if necessary.

After crushing the bacterial cells, the supernatant obtained by centrifugation is dialyzed against an appropriate buffer to obtain a crude enzyme solution extracted from the bacterial cells.

Next, purified carboxypeptidase can be obtained by appropriately combining the crude enzyme solution with ordinary operations used for enzyme purification, such as ammonium sulfate salting out, column chromatography, gel filtration, and electrophoresis.

Next, the properties of the enzyme of the present invention are shown below. Note that the enzyme activity was measured by the following method.

1) Measurement of activity against general synthetic peptides Measurement of enzymatic activity against general synthetic peptides is carried out using the Matheson
””MaCan, J, Bioche+++,,
42+ 95+ 1964), amino acids produced by enzymatic action were reacted with ninhydrin and fixed colorimetrically. Actually, 1mM various peptide solutions dissolved in buffer solution 0.1+j! After adjusting the enzyme reaction temperature to
Distilled water 0 to the 0 reaction stop solution in which the reaction was stopped by adding #
．． After adding 7 liters to make 1 tal, add 0.5 tsl of 0.2M citrate buffer (p)I 5.0) and 1.2 mJ of ninhydrin reagent, stir and mix, and add 7.5 tsl in a boiling water bath. Heating was performed for a minute. Thereafter, the mixture was quickly sufficiently cooled in ice water, diluted with 2.5 mx of 60% ethanol, and the absorbance value at 570 nm was measured using a spectrophotometer at room temperature. Also, depending on the content of the analysis.

The enzyme reaction was performed by changing the proportions of the substrate solution, enzyme solution, distilled water, or buffer solution. As for the unit of enzyme activity, the ninhydrin-positive substance was converted into the amount of leucine, and the amount of enzyme that produced the ninhydrin-positive substance corresponding to 18M leucine was defined as 1 unit (U).

2) Measurement of activity against peptides containing proline residues Measurement of enzyme activity against peptides containing proline residues was carried out by '1aron and Mlyner (Biochem,
Biophys.

Res, Cosmun, 32.658.1968
) After bringing 0.1 ml of 1mM various peptide solutions dissolved in the solution to the enzyme reaction temperature, the enzyme solution 0.
．． A reaction was carried out by adding 1 ml of each. The reaction conditions were usually pH 8.5, 70°C, and 30 minutes. The reaction was stopped by adding 0.1e+β of an O, lM acetic acid solution, and further 0.1 tml of distilled water was added to make the total volume 1 ml.

Then, 2.5 ml of glacial acetic acid and 2.5 ml of ninhydrin reagent.
After stirring and mixing, the mixture was heated in a boiling water bath for 30 minutes. After heating, the sample was sufficiently cooled in ice water, returned to room temperature, and the absorbance value at 480 nm was measured using a spectrophotometer. The unit of enzyme activity is usually pH 8, 5, 7, 5℃
Enzyme solution IIIj! The amount of proline produced per minute was calculated, and the amount of enzyme that produced lpM of proline was defined as 1 unit (U).

Measurements were made using the above two methods, and the specific activity of the enzyme was expressed in units of activity per IIIg of protein in the enzyme solution.

Determined by gel filtration method. The relationship between the elution position and molecular weight of the purified enzyme and standard protein is shown in Figure 1.

As a result, the molecular weight of this enzyme was estimated to be 62,000.

2) Amino acid composition The results of amino acid analysis of this enzyme excluding tryptophan and the calculated number of residues when the molecular weight is 62,000 are the first
Shown in the table. The constituent amino acid composition is Glu(n),
The content of Gly, Set, and Ala is relatively high, and Cys
was not included.

Table 1 Asx 6.2 Thr 4.6 Ser 12.9 Glx 13.6 Pro 5.4 Gly 12.6 Ala 11.7 Half Cys O,0Val
5.8 Table 1 (continued) Amino acid content (mol%) Met
O,71ie
3.2L e u
8.7Tyr 1,
2Phe 2.91, y
s 3.4His
2.6 Arg 4.5T r p
n, d.

Total 100.0 3) Optimum action pH The optimum action pH of this enzyme is ptl 5. O to 10.0
at 70°C using Cbz-Phe-Tyr as a substrate.
, measured under reaction conditions of 30 minutes. Buffers were added to each p at room temperature.
0.05M citrate buffer prepared at pH 5-6
).

0.05M phosphate buffer (pH 6-B), 0.05M
Tris-HCj! Buffer (pH 8-9) and 0.
05MHz B O4'K CI N a O
H buffer (pH 9-10) was used. The results are shown in Figure 2. The optimum working pH was in the range of 8.0 to 9.0. Relative activity decreased on the alkaline side from pH 9.5 and on the acidic side from pH 7.

4) Optimal temperature of action The optimal temperature of action of the purified enzyme is between 30°C and 95°C.
pH 8,50,0 using Cbz-Phe-Tyr as a substrate
The measurement was performed using a 5M Tris-HCIII solution for 30 minutes. The results are shown in Figure 3. This enzyme is 7
It showed an optimal action temperature between 5°C and 80°C, and had relative activity of about 95% at 85°C and about 85% at 95°C, even at extremely high temperatures. The activity at low reaction temperatures is about 75% at 60°C, about 55% at 50°C, and about 20% at 30°C.
Met.

5) Thermostability The thermostability of enzyme activity was measured using a crude enzyme solution extracted from bacterial cells and a purified enzyme. Enzyme heat treatment at pH 7, 20, 05M
Tris-HCI buffer was used at 80°C to 95°C for 20 hours, and the remaining activity after heating was at pH 8.5.
The enzyme activity was measured by reaction at 70° C. for 30 minutes using Cbz-Phe-Tyr as a substrate, and relative activity values are shown based on each unheated enzyme activity.

The protein concentration during heating was 10 μg/I11 for the purified enzyme, and the protein concentration for the bacterial cell extraction crude enzyme solution was set to have the same activity as the purified enzyme when unheated. The results obtained are shown in FIG. Purified enzyme is heated at 80℃ for 20 hours to reduce the concentration to about 75% and 90%.
Approximately 40% of the activity remained even after heating at ℃ for 20 hours. In addition, the crude enzyme solution extracted from bacterial cells exhibits higher thermal stability than the purified enzyme; approximately 85% after heating at 80°C for 20 hours;
Approximately 65% activity remained after heating for 20 hours.

6) Effect of metal ions Table 2 shows the results of measuring the effects of various metal ions on the activity of this enzyme. Various metal ions were added to the enzyme solution at a concentration of 1mM, and after being left at 25°C for 30 minutes, Cbz-Phe-Tyr was added in the presence of metal ions.
as a substrate at pH 5 and 70°C.

The enzymes reacted for 30 minutes were Ca'', Co
”.

It was stable against Mg1, but almost lost its activity against Z n 2 +. Also, Cu", Fe"°.

It was influenced to some extent by Su"°, Mn".

Table 2 Metal ions (1mM) Relative activity (%) Con
trol 100.0CaC1z
97.9GoCI,
106.3CuSoa 76.
3MgC1g 103.0FeSo
, 74.5SiO2g
65. lZnC1g
17.2MnC1,83,5 7) Effect of reagents Table 3 shows the results of measuring the effects of various reagents on the activity of this enzyme. The enzyme solution and various reagents at respective concentrations were mixed, and after being left at 25°C for 30 minutes, the pH was adjusted to 5.
The reaction was carried out at 70°C for 30 minutes.

The activity of this enzyme is 1mM EDTA, 1mM 1-10
It was strongly inhibited by metal chelators such as phenanthroline. In addition, it was significantly inhibited by the SH reagent PCMH, but was hardly affected by 1 m M N-ethylmaleimide, 1 mM monoiodoacetic acid, 1 mM 2-mercaptoethanol, 0.1 mM cysteine, 10 μM amastatin, and 10 μM bestatin. There wasn't. It was significantly inhibited by SDS at 0.05% and guanidine hydrochloride at 0.5M.

Table 3 Control -100,0EDTA
” 1.0
mM 27.81.1O-Phen
anthroline 1. OmM 14.
3PCMB”
1.0-M 63.0N-f! t
hy1maleside 1. Od 9
4.6 Monoiodoacetic acid
1. OmM 96.7 Table 3 (Continued) Reagent Concentration Relative Activity (%) 2-Mercaptoethanol 1. O
mM 95.8Cysteine
O, 1mM 91.9Amastatin
0.01mM 100.0Bes
tatin O,01d 99
．． 7SDS” 0.05χ
47.3PCMB; p-chloromercribenzoic acid.

SDS: Sodium dodecyl sulfate 8)-value and Vmax.

The i value and Vmax of this enzyme are Cbz-Phe-Ty
It was determined by changing the enzyme reaction temperature using r as a substrate. After performing the enzyme reaction at various concentrations, the liberated tyrosine was identified and quantified using a Hitachi amino acid automatic analyzer model 835.

0.056.0.057.0.058 respectively at 90℃
゜0.068mM. On the other hand, Vmax is 0.4 B, 1.64.5.41.9.52 μM/
It was ll1in. Although the value hardly changed depending on the reaction temperature, Vmax differed considerably and showed the highest value at 90°C.

9) Substrate specificity The specificity of this enzyme for various substrates was measured and Cbz-Ph
Relative activity values based on e-Tyr are shown in Table 4. The enzyme reaction was carried out at pH 8.5 for 30 minutes at 70°C. This enzyme shows broad (low) specificity for peptides that contain Pro at the C-terminus, excluding Cbz-Gly-Pro, and has weak activity against peptides that have Pro at the second residue from the C-terminus. Indicated. The activity of hydrophobic amino acids varies depending on the type of amino acid located 2nd residue from the C-terminus.
Comparing the activities of peptides with , Guy showed higher activity. Also, it has Tyr at the C-terminus and the second residue is G.
When lu and Phe peptides were compared, PheO showed higher activity.

Such results indicate that Cbz-Gly-Leu and Cbz-(
yly-Pro-Leu, Cbz-Qly-(rly and Cbz-Qly-Pro-Leu-Qly were also obtained.

Table 4 Peptide Relative activity Cbz-Glu-Phe 7.5C
bz-Glu-Tyr 3.7Cb
z-Gly-Ala 83.2Cb
z-Gly-Gly 18.9Cb
z-Gly-Leu 84.0Cb
z-Gly-Phe 91.0Cb
z-Gly-Pro O,0Cbz
-Gly-Val 57.4Cbz
-Phe-Tyr 100.0Cbz
-Tyr-Glu 30.0Cbz
-Gly-Pro-Leu 22.3C
bz-Gl-Pro-Lau-Gl 123
．． 910) Hydrolytic action of this enzyme on peptides: Cbz-Gly-Pro-Leu-Gly,
Thr-Thr-Met-Pro-Leu-Trp,
Tyr-Leu-Gly-Tyr-Leu-Glu-G
The hydrolysis effect on ln-Leu-Leu-Arg was measured. The oxygen reaction was carried out at 70°C using pH 8, 50, 1M formic acid-ammonia buffer, and the enzyme/substrate (W
/W) were conducted at 1:30, 1:40, and 1 near 0, respectively.

Regarding the effect on Cbz-Gly-Pro-Leu-Gly, after Gly was first released, Leu was released relatively slowly. And Cbz-Q with Pro at the C-terminus
It had no effect on ly-Pro, and the result was the same as the previous analysis of substrate specificity. Thr-Thr-Met-Pr
For o-Leu-Trp, Trp + Leu is rapidly released from the C4° terminus, and the release rate of Leu is different from that of the previous peptide where Pro is located under the same conditions (2nd residue from the C-terminus). The results were shown.

Next, Thr-Thr-Met with Pro at the C-terminus
-Also shows weak activity against Pro.
When released, Met and Thr were released almost simultaneously.

Tyr-Leu-Gly-Tyr-Leu-Glu-G
For ln-Leu-Leu-^rg, the C-terminal Ar
It was hydrolyzed sequentially from g to <lu, and also acted on Gin.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

Example Polypeptone 0.4%, yeast extract 0.2%.

Ca5Oa・2HzOO, 15g/'j! was dissolved in groundwater, adjusted to pH 7.2 with NaOH, and then heated at 120°C for 60°C.
Those that were sterilized for minutes were considered to be cultivated. This medium contains Thermus aquaticus YT-1 (ATCC25104).
was inoculated and cultured with shaking at 75°C for 24 hours.

Next, medium 11 having the same composition as above was placed in a 31-volume jar fermenter, sterilized, and then inoculated with the preculture solution at a rate of 2%, and heated at 250 rpm. OVVM.

A second preculture was carried out at 75° C. for 24 hours at 0.5 kg/−.

Main culture contains 0.8% polypeptone and 0.4% yeast extract.
, CaSO4”2HzOO, 15g/x.

Dissolve 0.4% whey protein in ground water, use a medium that has been pH adjusted and sterilized in the same way as the pre-medium, and use a 4% secondary culture solution.
The cells were inoculated and cultured at 65°C until reaching the stationary phase. In addition, an antifoaming agent (Ad
ecanol LG126) was added.

After culturing, the bacterial cells were collected by centrifugation according to a conventional method.

Next, the bacterial cells were added to 0.05M Tris-HC1 buffer (
p) After adding 1 (10% ■) to 17.2), use a powerful ultrasonic shaker (Brasov Ng, model 200100V).
20 KHz) at maximum output for 10 minutes to disrupt the bacterial cells. The temperature of the sample at this time was 10℃
I did the following. After that, the bacterial fragments were centrifuged (35,
OOOxg for 20 minutes), and the resulting supernatant was thoroughly dialyzed against the same buffer as above to obtain a bacterial cell extraction crude enzyme solution.

Next, the enzyme was purified by the following method.

l) Salting out with aluminum sulfate Bacterial cell extraction Crude enzyme solution 50+*l and ammonium sulfate 28.
Add 0.05g to reach 80% saturation, leave for 30 minutes, and centrifuge (10,OOOXg for 30 minutes).The resulting precipitate is mixed with 0.05M5Tris-HCl.
After dissolving in No. 11 buffer (pH 7.2) and thoroughly dialyzing against the same buffer, the enzyme fraction is obtained as an ammonium sulfate precipitated enzyme fraction.

2) DEAE Sephacel chromatography 80% ammonium sulfate precipitated enzyme fraction 7011 at 0.05M in advance
D equilibrated with Tris-HC1 buffer (pH 7,2)
Adsorb onto an EAE Sephacel column (2,4×24G). The column was washed with the same buffer, then OM ~0.5
M Sodium chloride (NaCj) is eluted using the same buffer 10,100 O under conditions that provide a linear concentration gradient.The elution rate is 40 tsll/hour, and the eluate is
Collect 1 mff at a time.

The amount of protein ft (absorbance value at 280 m) and hydrolysis activity for carbobenzoxy (Cbz)-Phe-Tyr were measured for each fraction, and eluted fractions 24 to 54 containing carboxybebutigase activity were determined. Collect, 0.05M Tris
- After dialysis with HCl buffer (pH 7,2),
DEAE Sephacel chromatography enzyme fraction.

3) Phenyl Sepharose CL-4B chromatography Phenyl Sepharose CL-4B column (1,2X20
(J) was 0.05M Tri containing IMNaCJ in advance.
s-HCj! Equilibration was performed with lil buffer (pH 7,2). 300ml of the previous enzyme solution was IM NaC with the same buffer solution.
1 and then adsorbed onto a column. After that, IM~0.
0.0 under the condition of linear concentration gradient of 05M NaCl
5M Tris-HC1 @ buffer solution (pH 7,2) 70
0 m#, 0.05M to OM NaCl 200 ml of the same buffer, 100 ml of the same buffer without NaCl, 100 tal of 0.05M Tris-HCI buffer (pH 9,0), 100 tal of distilled water
m1゜Further elution is performed sequentially with 100ml of ethanol, elution speed is 20s+4! / hour, and the eluate was 1% 5
■ Amount of protein in both fractions (280
The absorbance value of M) and the hydrolysis activity against Cbz-Phe-Tyr were measured, and the elution fractions 11kL38 to 160 containing carboxybebutigase activity were collected and 0.05M
After repeated dialysis with Tris-HCIII solution (pH 7,2), the enzyme fraction was subjected to phenyl Sepharose CL-4B chromatography.

4) After concentrating 610 liters of the enzyme solution on Sephadex G-200 gel chromatography using a collodion bag, 0.05M Tris-HCIg solution (pH 7.2
) and finally concentrated to 3 all. The concentrated sample was prepared using Sephadex G, which had been equilibrated with the same buffer solution in advance.
-200 column (2x70cm) and elute with the same buffer, elution rate is 5a+j! / hour, and the eluate is fractionated in 2.5+mi portions for each fraction.The amount of protein (absorbance value at 280m+) and Cbz-Phe
The hydrolysis activity against -Tyr is measured, and elution fractions -48 to -74 containing carboxybebutigase activity are collected and used as Sephadex G-200 chromatography enzyme fractions.

5) Enzyme solution 65nj obtained by purification by gel filtration after re-chromatography using DEAE Sephacel! 0.05M Tris-HCI! Buffer (pn 7.2
) equilibrated with a DEAE Sephacel column (1,7X
20cm). After washing with the same buffer,
OMtag/hour, eluate is 1 fraction 5 tall
Take out portions one by one. Each hundred percent protein eluted! (28Q+
The absorbance value of m) and the hydrolysis activity for Cbz-Phe-Tyr are measured. Enzyme activity is the first DE/l
As was observed in the purification process using Sephacel, the activity was fractionated in a shoulder-like manner in front of the main peak of activity, so the eluted fraction containing carboxypeptidase activity was divided into two fractions and collected. Both fractions 33 to 48 from which the activity was eluted first were subjected to DEAE Sephacel rechromatography and designated as enzyme fraction (2). Each enzyme fraction is 0.05
M Tris-HCI! Dialysis is performed with a buffer solution (pH 7,2). The specific activity obtained here is 325.5 for enzyme fraction I.
U/■, the enzyme fraction ■ was 712.2 U/■. 6) DEAE Sephacel re-chromatography for disc electrophoresis gel extraction, semiphoresis was performed at pH 8.0 using a 7.5% polyacrylamide gel. ν4, concentrated enzyme fraction I, and ■ are loaded with a protein amount of 220 μg per column and electrophoresed for about 2 hours at a constant current of 3 mA per column. After electrophoresis, enzyme fraction 1. 1 of n
The book was stained, and the other gels were sliced every second and each half was injected with 1 vglt of 0.05M Tris-HC121.
% buffer solution (pi(7,2)) and crush it to extract the enzyme.The absorbance value of the stained gel of enzyme fraction I, ■ is 550.The densitometer pattern of the fin and As a result of measuring the hydrolysis activity for Cbz-Phe-Tyr, the pattern of carboxypeptidase activity was the same when enzyme fraction 1.II obtained by DEAE Sephacel rechromatography was purified by disk electrophoresis gel extraction.Disc electrophoresis The above carboxypeptidase activity showed a main activity peak at a relative mobility of approximately 0.35, and weak activity was also observed throughout the region with lower mobility. The main activity peak with a relative mobility of 0.35 was purified by disk electrophoresis using the enzyme fraction (2) that had a high specific activity in the chromatography purification process, and the carboxypeptidase active fractions were collected and subjected to disk electrophoresis. This was used as the migrating enzyme fraction.

7) Re-gel extraction by disk electrophoresis Concentrate the main active fraction solution by disk electrophoresis of the enzyme solution ■ with a collodion bag, perform gel extraction again by disk electrophoresis in the same manner as above, and extract Cbz-Phe-Tyr. Both fractions containing hydrolytic activity against 0.0:M Tris −
After dialysis with HCl buffer (pH 7,2), a purified enzyme solution is obtained.

The results of the above purification steps are summarized in Table 5.

When the purified enzyme was compared with the crude enzyme solution extracted from bacterial cells using Cbz-Phe-Tyr as a substrate, the specific activity was purified 412.5 times, and the yield of activity was 21.6%.

〔Effect of the invention〕

The carboxypeptidase of the present invention has an optimal action temperature of 75
~80°C, and has excellent thermal stability at high temperatures. Because it has high heat resistance and true carboxidase action that sequentially releases amino acids from the C-terminus of peptides,
This enzyme is expected to have a wide range of applications, including research on the structure and function of enzymes, 9 analytical reagents, and 1 food industry applications.

Furthermore, this enzyme can be efficiently produced using thermophilic bacteria.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between elution position and molecular weight of the enzyme of the present invention and standard protein, in which A is chymotrypsin (molecular weight 25,000), B is ovalbumin (molecular weight 43,000), and C is Bovine serum albumin (molecular weight 67.000), D is aldolase (molecular weight 11
60.000), E represents the enzyme of the present invention, the second
The figure is a graph showing the relationship between pi and residual activity of the enzyme.
Sodium citrate for pH 5-6, sodium phosphate for pH 6-8, Tris-HCl for pH 8-9, pH 9-1
For O, each sodium borate buffer (0.05M) was used. FIG. 3 is a graph showing the relationship between temperature and the relative activity of the enzyme, FIG. 4 is a graph showing the relationship between temperature and the residual activity of the enzyme, and FIG. 5 is a graph showing the lv value and Vmaχ of the enzyme of the present invention. 30°C (black circle), 50°C (square). The results are shown when the ink was incubated at 70°C (white circles) or 90°C (triangles) at pH 8.5 for 30 minutes. Residual activity (%) Figure 4 Temperature (°C) l/S mM X 10-”

Claims (3)

[Claims] (1) A carboxypeptidase that has the action of sequentially releasing amino acids from the C-terminus of a peptide, exhibits an optimum operating temperature at 75 to 80°C, and has high thermal stability. (2) It belongs to the genus Thermus and has the ability to sequentially release amino acids from the C-terminus of peptides, exhibits an optimal temperature of action between 75 and 80°C, and has the ability to produce carboxypeptidase with high thermostability. 1. A method for producing carboxypeptidase, which comprises culturing thermophilic bacteria in a nutrient medium, producing the carboxypeptidase in the culture, and collecting the carboxypeptidase. (3) The method according to claim 1, wherein the highly thermophilic bacterium having the ability to produce carboxypeptidase belonging to the genus Thermus is Thermus aquaticus YT-1 (ATCC 25104).