JPS63219377A - Low-temperature protease and isolation thereof - Google Patents
Low-temperature protease and isolation thereofInfo
- Publication number
- JPS63219377A JPS63219377A JP5387487A JP5387487A JPS63219377A JP S63219377 A JPS63219377 A JP S63219377A JP 5387487 A JP5387487 A JP 5387487A JP 5387487 A JP5387487 A JP 5387487A JP S63219377 A JPS63219377 A JP S63219377A
- Authority
- JP
- Japan
- Prior art keywords
- protease
- low
- activity
- krill
- temperature range
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 31
- 239000004365 Protease Substances 0.000 title claims abstract description 30
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 27
- 238000002955 isolation Methods 0.000 title 1
- 230000000694 effects Effects 0.000 claims abstract description 25
- 241000239366 Euphausiacea Species 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 101710118538 Protease Proteins 0.000 claims abstract description 5
- 210000002808 connective tissue Anatomy 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 102000018389 Exopeptidases Human genes 0.000 claims abstract description 4
- 108010091443 Exopeptidases Proteins 0.000 claims abstract description 4
- 238000002523 gelfiltration Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 102000035195 Peptidases Human genes 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 2
- 239000007853 buffer solution Substances 0.000 abstract 2
- 239000007788 liquid Substances 0.000 abstract 2
- 239000000463 material Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- -1 acryl Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002196 fr. b Anatomy 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は低温水域（海域）、特に南氷洋に生息するオキ
アミより得られる低温プロテアーゼ及びその単離方法に
関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a low-temperature protease obtained from krill that inhabits low-temperature waters (sea areas), particularly the Antarctic Ocean, and a method for isolating the same.
（従来の技術）
本発明者らはオキアミが捕獲後、常温（南氷洋気温）に
放置すると急速に自己消化し、蛋白質溶解を生ずること
に着目し、オキアミ中にプロテアーゼ、キチナーゼ等の
溶解酵素が存在することが予想された。(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. It was expected that it would.
（発明が解決しようとする問題点）
本発明の目的はオキアミに存在すると予想される酵素及
びその単離方法を提供することにある。(Problems to be Solved by the Invention) An object of the present invention is to provide an enzyme expected to exist in krill and a method for isolating the same.
（問題点を解決するための手段）
本発明はオキアミより抽出されたプロテアーゼであって
、分子量約５万及び約３．３万のエキソプロテアーゼ及
びエンドプロテアーゼ活性を有する２種類のプロテアー
ゼを含み、約５〜２０℃の低温領域に至適温度領域を有
し、ＮａＣｌ耐性を有す全低温プロテアーゼ及びその単
離方法に係る６本発明において目的のプロテアーゼは、
例えばオキアミを原料として、これに適量の生理リン酸
緩衝液を加え、ホモゲナイズし、粗シ濾過により結合組
織を除去後、遠心分離し、その上澄液をゲル濾過して濃
縮することにより得られる。(Means for Solving the Problems) The present invention is a protease extracted from krill, which contains 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 and about 33,000. The protease of interest in the present invention relates to a total low-temperature protease having an optimum temperature range in the low-temperature range of 5 to 20°C and is resistant to NaCl, and a method for isolating the same.
For example, it can be obtained by using krill as a raw material, adding an appropriate amount of physiological phosphate buffer to it, homogenizing it, removing connective tissue through coarse filtration, centrifuging, and concentrating the supernatant through gel filtration. .
本発明のプロテアーゼは具体的には例えば次の様にして
得られる。Specifically, the protease of the present invention can be obtained, for example, as follows.
未加熱（生鮮状態）のまま、捕獲後急速凍結されたオキ
アミを凍結のまま細切して、２〜５倍量の生理リン酸緩
衝液（Ｐ Ｂ Ｓ ）を加えてワーリングプレングーを
用いてホモゲナイズし、四重が一ゼでシ濾過して結合組
織を除去後、１０．ＯＯＯｘｇｔ’ｌＯ〜３０分遠心分
離して上澄液を採り、この上澄液を５ｅｐｌ＋ａｃｒｙ
ｌ Ｓ−４００（スウェーデン、サルマシャ社！１１り
を用いて、デルｌ過分離を行−うことにより、プロテア
ーゼ活性１０倍以上に濃縮された低温プロテアーゼ酵素
液が得られる。Unheated (fresh) krill was captured and quickly frozen, cut into small pieces while still frozen, and mixed with 2 to 5 times the amount of physiological phosphate buffer (PBS) using a Waring pre-gun. After homogenization and filtration with quadruple filter to remove connective tissue, 10. OOOxgt'lO ~ Centrifuge for 30 minutes, collect the supernatant, and add 5 epl + acryl
A low-temperature protease enzyme solution with a protease activity concentrated by more than 10 times can be obtained by performing a Del.l superseparation using a S-400 (Salmasha, Sweden! 11).
本発明のプロテアーゼの特性は次の通りである。The properties of the protease of the present invention are as follows.
（１）酵素活性の検出
濃縮プロテアーゼ標品（Ｐ Ｂ Ｓ溶液）を牛血清アル
ブミンを基質として反応させ、トリクロル酢酸（Ｔ Ｃ
Ａ ）沈澱を行い、上清の酸可溶性画分の増加によりプ
ロテアーゼ活性の検出を試みたが、拳法では酵素活性は
認められず、ポリアクリルアミドゲル電気泳動（ＰＡＧ
Ｅ）にかけ牛血清アルブミンのバンドの消失により酵素
活性を調べた。その結果牛血清アルブミンバンドが完全
に消失し、低号子領域に複数のバンドが出現したことか
ら、オキアミ酵素液中にプロテアーゼ活性が確認された
。(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 of the supernatant, but no enzyme activity was observed with Kenpo, and polyacrylamide gel 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.
本プロテアーゼは切断部位特異性が比較的高いエンドプ
ロテアーゼであり、アミノ酸１０個以上のペプチド断片
を与えることを示している。このために、先のＴＣＡ法
では本酵素の活性が見出されなかった原因である。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）デルｌ過による酵素の精製
オキアミホモジネートを５ｅｐｈａｃｒｙｌ Ｓ −２
００゜Ｓ −３００，Ｓ −４００テＰ Ｂ Ｓ 全４
１衝１．！−Ｌ”ｌイ（分画した結果、Ｓ−４００７！
１１最適であり、１回のデルｌ過分離で９０％以上の活
性分画を果たすことが出来でおり、この活性分画には２
つの分画区が得られ、両分Ａ区より）は分子量約５万、
画分Ｂからは分子量約３．３万のものが得られた。(2) Purification of enzyme by filtration of krill homogenate to 5ephacryl S-2
00゜S -300, S -400TEP B S Total 4
1 strike 1. ! -L”l (As a result of fractionation, S-4007!
11 is optimal, and more than 90% of active fractions can be achieved in one del l overseparation, and this active fraction contains 2
Two fractions were obtained, both fractions A) had a molecular weight of approximately 50,000,
Fraction B had a molecular weight of about 33,000.
（３）酵素の基質特異性
本酵素はアルギニンに最も高い基質特異性を示し、リジ
ン及びロイシンに対しても活性を示したが、チロシン及
びフェニルアラニンに対しては活性を示さなかった。こ
の基質特異性はトリプシンと類似しており、Ａ、Ｂ両画
分はトリプシンと同様アルギニンに対して最も高い活性
を示した。リジンに対してはトリプシンは分解初期速度
はアルギニンの５０％以上であったのに対し、Ａ ＋
Ｂ 両ｌｉｔ分ともかなり低い分解速度を与えた。また
トリプシンはロイシンに対して分解活性を示さなかった
が、Ａ、 Ｂ１１１ｉｌｌｉ分ともに弱いロイシン分解
活性を示した。(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 tyrosine and phenylalanine. This substrate specificity is similar to that of trypsin, and both fractions A and B showed the highest activity toward arginine, similar to trypsin. For lysine, the initial degradation rate of trypsin was more than 50% that of arginine, whereas for A +
B. Both lit portions gave fairly low decomposition rates. Also, trypsin did not show any degrading activity for leucine, but both A and B111illi showed weak leucine degrading activity.
（４）至適ｐＨ
種々のｐＨのリン酸緩衝液中で８画分を牛血清アルブミ
ン（ＢＳＡ）と反応させ、ＰＡＧＥ法で活性を測定する
と、ｐＨ６，５〜８．０では高い活性を示した。(4) Optimal pH When 8 fractions were 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）至適温度
本発明のプロテアーゼは、種々の温度で牛血清アルブミ
ンと反応させ分析した結果、第１表に示されるようにＡ
、Ｂ両画分共に５℃及び１０℃でも活性を示し、２０’
Ｃで最大活性を示した。しかも３７℃以上のより高い温
度で活性が低下したことより、低温プロテアーゼであり
、他に例のない特異的なプロテアーゼであることが明ら
かである。(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 20'
The maximum activity was shown at C. Furthermore, 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.
即ち本プロテアーゼは、低温領域特に５〜２０℃にかけ
て特異的に至適温度領域を有することを特徴とする低温
プロテアーゼである。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.
第 １ 表
（６）熱安定性
本発明のプロテアーゼはＡ、Ｂ両画分ともに５０〜７０
℃で処理すると酵素活性の部分的低下が起こり、８０℃
以上では完全に失活した。Table 1 (6) Thermostability The protease of the present invention has a thermostability of 50 to 70 for both fractions A and B.
A partial decrease in enzyme activity occurs when treated at 80°C.
Above that, it was completely deactivated.
（７）ＮａＣ１耐性
本発明のプロテアーゼは第２表に示すように１０％Ｎａ
１ｌ存在下でも酵素活性の低下は認められなかった。(7) NaCl resistance The protease of the present invention is resistant to 10% NaCl as shown in Table 2.
No decrease in enzyme activity was observed even in the presence of 1 liter.
第２表
（発明の効果）
本発明のプロテアーゼは低温において優れた酵素活性を
有し、！ＪたＮａＣ１耐性にも優れている。Table 2 (Effects of the Invention) The protease of the present invention has excellent enzymatic activity at low temperatures! It also has excellent resistance to NaCl.
従って低温及び／又は高い塩濃度での使用が可能である
。Therefore, use at low temperatures and/or high salt concentrations is possible.
（以 上）(that's all)
Claims (2)
分子量約５万及び約３．３万のエキソプロテアーゼ及び
エンドプロテアーゼ活性を有する２種類のプロテアーゼ
を含み、約５〜２０℃の低温領域に至適温度領域を有し
、ＮａＣｌ耐性を有する低温プロテアーゼ。(1) A protease extracted from krill,
A low-temperature protease containing two types of proteases having exoprotease and endoprotease activities with molecular weights of about 50,000 and about 33,000, having an optimum temperature range in the low-temperature range of about 5 to 20°C, and having NaCl resistance.
緩衝液を加え、ホモゲナイズし、粗濾過により結合組織
を除去後、遠心分離し、その上澄液をゲル濾過して濃縮
することを特徴とする分子量約５万及び約３．３万のエ
キソプロテアーゼ及びエンドプロテアーゼ活性を有する
２種類のプロテアーゼを含み、約５〜２０℃の低温領域
に至適温度領域を有し、ＮａＣ１耐性を有する低温プロ
テアーゼの単離方法。(2) Using krill as a raw material, add an appropriate amount of physiological phosphate buffer to it, homogenize it, remove connective tissue by rough filtration, centrifuge, and concentrate the supernatant by gel filtration. Contains two types of proteases with molecular weights of approximately 50,000 and 33,000, having exoprotease and endoprotease activities, has an optimum temperature range in the low temperature range of approximately 5 to 20°C, and has NaCl resistance. Methods for isolating proteases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5387487A JPS63219377A (en) | 1987-03-09 | 1987-03-09 | Low-temperature protease and isolation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5387487A JPS63219377A (en) | 1987-03-09 | 1987-03-09 | Low-temperature protease and isolation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63219377A true JPS63219377A (en) | 1988-09-13 |
Family
ID=12954893
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5387487A Pending JPS63219377A (en) | 1987-03-09 | 1987-03-09 | Low-temperature protease and isolation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63219377A (en) |
-
1987
- 1987-03-09 JP JP5387487A patent/JPS63219377A/en active Pending
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