JPS63219378A - Activated low-temperature protease - Google Patents

Abstract

PURPOSE:To make optimum temperature protease 5-20 deg.C and to raise NaCl resistance, by adding a surface active agent or an alcohol to protease extracted from krill and preparing low-temperature protease. CONSTITUTION:Krills as a raw material are mixed with a proper amount of physiological phosphoric acid buffer solution, homogenized and coarsely filtered to remove connective tissue. The resultant material is centrifuged, the supernatant liquid is subjected to gel filtration and concentrated to give protease. The protease is low-temperature protease which contains two kinds of proteases having about 50,000 and about 33,000mol.wt., exoprotease and endoprotease activity, about 5-20 deg.C optimum temperature range and NaCl resistance. Addition of a surface active agent or an alcohol further improves enzymatic activity.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to activated low temperature proteases.

(Prior art) The present inventors focused on the fact that krill rapidly self-digests when left at room temperature (Southern Ocean temperature) after capture, resulting in protein dissolution. We predicted that this would happen, and succeeded in isolating the enzyme from krill, and found that the enzyme is a low-temperature protease with an optimum temperature range in the low-temperature region.

The low-temperature protease obtained from krill has excellent enzymatic activity, but it is more desirable to have even better # elementary activity.

(Problems to be Solved by the Invention) An object of the present invention is to provide a further activated low-temperature protease.

(Means for Solving the Problems) The present invention relates to an activated low-temperature protease obtained by adding a surfactant or an alcohol to a low-temperature protease extracted from krill.

The low-temperature protease used in the present invention includes two types of proteases having exoprotease and endoprotease activities with a molecular weight of about 50,000 and about 33,000, and has a molecular weight of about 50,000 to 33,000.
It is a low-temperature protease that has an optimum temperature range in the low-temperature range of 20°C and is resistant to NaCl.

The above-mentioned protease used in the present invention is made from krill, for example, by adding an appropriate amount of physiological phosphate buffer to it, homodenizing it, removing connective tissue by coarse filtration, centrifuging, and draining the supernatant. N,! Obtained by filtration and concentration.

Specifically, the above protease can be obtained, for example, as follows.

Unheated (fresh) krill that was caught and quickly frozen was cut into small pieces while still frozen, and 2 to 5 times the volume of physiological phosphate buffer (PBS) was added to the krill using a Waring Brengu. Homodenize! 7. After removing the connective tissue by crushing the quadruple with one zebra, 10. Centrifuge for 10 to 30 minutes at OOOxg, collect the supernatant, and add 5ephacryl to this supernatant.
By performing Delcy superseparation using S-400 (manufactured by Sarmasha, Sweden), a low-temperature protease enzyme solution with a protease activity concentrated by 10 times or more is obtained.

The properties of the obtained protease are as follows.

(1) Detection of enzyme activity A concentrated protease preparation (PBS solution) was reacted with bovine serum albumin as a substrate, and trichloroacetic acid (TC
A) Precipitation was performed and an attempt was made to detect protease activity by increasing the acid-soluble fraction in the supernatant, but no enzymatic activity was observed, and polyacrylamide delta electrophoresis (PAG) was performed.
E) The enzyme activity was examined by the disappearance of the bovine serum albumin band. As a result, the bovine serum albumin band completely disappeared and multiple bands appeared in the low molecular weight region, confirming protease activity in the krill enzyme solution.

This protease is an endoprotease with relatively high cleavage site specificity, and has been shown to yield peptide fragments of 10 or more amino acids. This is the reason why the activity of this enzyme was not found in the previous TCA method.

(2) Purification of enzyme by Delsi filtration Krill homomononate 5ephacryl S-2
As a result of fractionation using PBS as a buffer with 00゜S-300 and S-400, it was found that S-400 was the most suitable, and more than 90% of the activity could be fractionated with one Delua overseparation. Two fractions were obtained in this active fraction, with fraction A having a molecular weight of approximately 50,000, and fraction B having a molecular weight of approximately 3.
30,000 were obtained.

(3) Substrate specificity of the enzyme This enzyme showed the highest substrate specificity for arginine, and also showed activity for lysine and leucine, but did not show activity for 1,000 rosin and phenylalanine. This substrate specificity was MII with trypsin, and both fractions A and B showed the highest activity against arginine, similar to trypsin. For lysine, the initial decomposition rate of trypsin was more than 50% that of arginine;
Both fractions gave fairly low degradation rates. Also, trypsin did not show degrading activity against leucine, but A.
Both fractions B showed weak leucine degrading activity.

(4) Optimal pH When the B fraction was reacted with bovine serum albumin (BSA) in phosphate buffers of various pH and the activity was measured by PAGE method, high activity was shown at pH 6.5 to 8.0. Ta.

(5) Optimum temperature The protease of the present invention was reacted with bovine serum albumin at various temperatures and analyzed, as shown in Table 1.
Both fractions B showed activity at 5°C and 10'C, and 2
It showed maximum activity at 0°C. However, since the activity decreased at higher temperatures of 37° C. or higher, it is clear that it is a low-temperature protease and is a unique and unique protease.

That is, the present protease is a low-temperature protease characterized by having an optimal temperature range specifically in the low-temperature range, particularly from 5 to 20''C.

Table 1 (6) Thermostability The protease of the present invention has a thermostability of 50 to 70 for both fractions A and B.
Treatment with 'C caused a partial decrease in enzyme activity, with 80
It was completely inactivated at temperatures above °C.

(7) NaC1jt The protease of the present invention is treated with 10% NaCl as shown in Table 2.
No decrease in enzyme activity was observed even in the presence of C1.

Table 2 When a surfactant or an alcohol is added to a low-temperature protease having the above characteristics, its enzymatic activity is further improved. As the surfactant, various cationic, anionic, /ionic and amphoteric surfactants can be used, and particularly preferred ones include sodium dodecyl sulfate (SDS), sodium octyl sulfate, and sodium tetradecyl sulfate. Examples include alkyl tetratum sulfates, dodecylpyridinium chloride, etc. Alcohols include methanol, ethanol, propatool, butanol, octatool, decanol, dodecanol, myristyl alcohol, cetyl alcohol, stearyl alcohol, eifyl alcohol, mercaptometh/ol, mercaptoethanol (MSH
Examples include L mercaptopropanol and mercaptobutanol. The amount of surfactant or alcohol added can be selected from a wide range, but usually the substrate concentration is about 1 to 30% by weight, preferably about 1 to 15% by weight,
More preferably, it is about 3 to 10% by weight. Addition can be carried out according to various known methods.

(Example) Examples will be described below.

Example 1 Unheated (fresh state) krill that was caught and quickly frozen was cut into small pieces while still frozen, and 3 times the amount of physiological phosphate buffer (PBS) was added to the krill, which was then processed using a Waring Brengu. After homodenizing and filtering through four layers to remove connective tissue, 10. Centrifuge for 10 to 30 minutes at OOOxg, collect the supernatant, and add this supernatant to Sepl+acrylS.
-400 was used to perform Delgi permeation separation to obtain a low temperature protease enzyme solution with a protease activity concentrated 10 times or more.

Using the obtained enzyme solution, comb serum albumin (BSA)
When enzyme activity was measured by the method described below using as a substrate, SDS or MSH was added to the measurement.

(A) PAGE method polyacrylamide del electrophoresis method (B) BAPNA method p produced using Na-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as a synthesis substrate
- Measure the amount of nitroaniline A 40%.

The results are shown in Table 3. For comparison, the case where no surfactant or alcohol was added and the case where trypsin was used as the enzyme were described. Note that the concentration indicates the final concentration of the substrate (the same applies below).

As shown in Table 3 of Table t143, the protease of the present invention is MSH
When SDS is not added, only the synthetic substrate is degraded, but in the presence of a high concentration of MSH, BSA degrading activity is exhibited and the degrading activity of the synthetic substrate also increases. In the presence of SDS, the synthetic substrate decomposition activity was lost, but the BSA decomposition activity was promoted, and the activity increased as the concentration of MSH added increased. In the case of trypsin, it can be seen that the enzyme activity is actually inhibited in the presence of a high concentration of MSH.

Example 2 The enzyme activity of fraction A and fraction B of the protease of the present invention in the presence of SDS was investigated. The results are shown in Table 4. For comparison, the case where SDS was not added and the case where trypsin and proteinase K were used were also described.

From Table 4, Table 4R, both fractions A and B showed strong BSA degrading activity at an SDS concentration of 1% or more, and showed high SDS It properties similar to that of proteinase, but trypsin had a high SDS
It lost activity at DS concentration.

Many enzymes are denatured and inactivated in the presence of SDS. Until now, proteinase K is the only known protease that shows activity in SDS, and it has been used in the field of genetic engineering for the purpose of removing proteins when preparing nucleic acids from bacteria and animal cells. Similar to proteinase, the protease of the invention showed no decrease in enzyme activity even in the presence of 8% SDS. Therefore, this enzyme can be used as a reagent for genetic engineering research in the same way as proteinase. Additionally, SDS is used as a surfactant in detergents, toothpaste, etc.

Since the addition of protease to detergents can greatly contribute to improving detergency, various applications are expected.

(Above) Applicant Hayashikane Sangyo Co., Ltd. Agent Patent Attorney 1) Iwao Mura Procedural Amendment September 10, 1988 Patent Application No. 53875 2, Title of Invention Activated Low Temperature Protease 3 ,Relationship with the case of a person who makes a correction Patent applicant: Hayashikane Sangyo Co., Ltd. 4, Agent: Maru Building, 1-2-8 Kunezaki, Kita-ku, Osaka 530 Telephone: 06 (365) 01F 0 (8153) Patent attorney 1 ) Iwao Mura 5, date of amendment order 6, section 7 of "Detailed Description of the Invention" in the specification subject to amendment, content of amendment (1) "Obtained" on page 3, line 7 of the specification [ can get. Furthermore, the protease of the present invention can also be obtained by genetic manipulation of microorganisms. ] Correct.

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Claims (2)

[Claims] (1) Activated low-temperature protease obtained by adding a surfactant or alcohol to low-temperature protease extracted from krill. (2) Low-temperature protease extracted from krill contains two types of proteases with molecular weights of approximately 50,000 and 33,000, having exoprotease and endoprotease activities, and has an optimal temperature in the low temperature range of approximately 5 to 20 degrees Celsius. has an area,
The activated low-temperature protease according to claim 1, which is a low-temperature protease having NaCl tolerance.