JPS6054037B2 - Manufacturing method of elastase - Google Patents
Manufacturing method of elastaseInfo
- Publication number
- JPS6054037B2 JPS6054037B2 JP23164584A JP23164584A JPS6054037B2 JP S6054037 B2 JPS6054037 B2 JP S6054037B2 JP 23164584 A JP23164584 A JP 23164584A JP 23164584 A JP23164584 A JP 23164584A JP S6054037 B2 JPS6054037 B2 JP S6054037B2
- Authority
- JP
- Japan
- Prior art keywords
- elastase
- pancreas
- extract
- autolysis
- water
- 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.)
- Expired
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- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 本発明はエラスターゼの製造方法に関する。[Detailed description of the invention] The present invention relates to a method for producing elastase.
エラスターゼは、啼乳動物膵臓中に存在している所謂セ
リンプロテアーゼの一種であるが、不溶性硬タンパク質
であるエラスチンを分解する、きわめて特異なプロテア
ーゼである。近年、エラスチンの分解現象と、加令、動
脈硬化症、あるいは肺気腫、肺炎、血管炎、等との関係
が注目されており(金攻全らConnectiveTi
ssue6(1)1(1974))、将来エラスターゼ
の医薬品としての利用が高まるものと考えられる。この
ような観点から、エラスターゼを効率よく工業的規模で
製造する方法の開発が望まれている。従来エラスターゼ
の製造方法に関して多くの報告がなされているが、その
ほとんどは研究室レベルでの製造方法である。Elastase is a type of so-called serine protease present in the pancreas of mammals, and is an extremely unique protease that degrades elastin, which is an insoluble hard protein. In recent years, attention has been focused on the relationship between elastin degradation and aging, arteriosclerosis, emphysema, pneumonia, vasculitis, etc.
ssue6(1)1 (1974)), it is thought that the use of elastase as a pharmaceutical will increase in the future. From this point of view, it is desired to develop a method for efficiently producing elastase on an industrial scale. Many reports have been made regarding methods for producing elastase, but most of them are produced at the laboratory level.
現在までに報告されているエラスターゼの製造方法とし
て最も代表的なものにLewisらの方法(J、Bio
lchemZ四 705(1956))がある。この方
法は、膵臓及びパンクレアチンの稀酢酸抽出物に硫安を
45%飽和に加えて、生じた沈澱をO、IM炭酸緩衝液
に溶解し、次いで脱塩により生じるオイグロブリン画分
をO、IM炭酸塩緩衝液に溶解し、硫安を35%飽和に
加えてエラスターゼの沈澱を得る。The most representative elastase production method reported to date is the method of Lewis et al. (J, Bio
lchemZ4 705 (1956)). This method involves adding ammonium sulfate to a dilute acetic acid extract of pancreas and pancreatin to 45% saturation, dissolving the resulting precipitate in O, IM carbonate buffer, and then desalting the euglobulin fraction resulting from O, IM. Precipitate the elastase by dissolving it in carbonate buffer and adding ammonium sulfate to 35% saturation.
しかる後、再びO、IM炭酸緩衝液に溶解して結晶化さ
せるものである。特公昭50−21557にかかる方法
はレwisらの方法の45%硫安飽和で得られる沈澱を
pH5〜10g)水溶液に溶解し、5〜50Cに1時間
以上放置した後に再度塩析することを特徴とするもので
、基本的にはレwisらの方法と同様である。Thereafter, it is dissolved again in O, IM carbonate buffer and crystallized. The method disclosed in Japanese Patent Publication No. 50-21557 is characterized in that the precipitate obtained by saturation with ammonium sulfate at 45% according to the method of Rewis et al. is dissolved in an aqueous solution (pH 5 to 10 g), left at 5 to 50 C for 1 hour or more, and then salted out again. This is basically the same method as that of Rewis et al.
特開昭52−110892にかかる方法は膵臓抽出液を
直接陽イオン交換体に作用させエラスターゼを吸着させ
た後、溶出し分画することを特徴とするものである。The method disclosed in JP-A-52-110892 is characterized in that a pancreatic extract is directly applied to a cation exchanger to adsorb elastase, and then eluted and fractionated.
また特開昭52−61287では、膵臓に啼乳動物の−
指腸あるいは、その抽出液を加えてエラスターゼ前駆
物質を活性化することを特徴とするエラ’スターゼの製
造方法を示している。Furthermore, in Japanese Patent Application Laid-open No. 52-61287, the pancreas contains -
This shows a method for producing elastase, which is characterized by adding denum or an extract thereof to activate an elastase precursor.
上記の方法は、いずれも膵臓抽出液を得るための前処理
が全くなかつたり、あるいはきわめて不十分であるため
、膵臓抽出液を収率よく、しかも清澄に得ることがきわ
めて困難である。In all of the above-mentioned methods, pretreatment for obtaining a pancreatic extract is completely absent or extremely insufficient, so it is extremely difficult to obtain a clear pancreatic extract with a good yield.
それに加えて、抽出液のタンパクその他生体成分の含量
が多く、その後の精製過程に於て操作が煩雑となり、高
純度のエラスターゼを収率よく工業的な規模で製造する
方法としては十分とは言い難い。In addition, the content of proteins and other biological components in the extract is high, making the subsequent purification process complicated, making it difficult to use as a method for producing high-purity elastase on an industrial scale in good yield. hard.
これらの点に特に注意して研究した結果、高純度のエラ
スターゼを得る本発明を完成させるに至つた。As a result of research paying particular attention to these points, the present invention for obtaining highly pure elastase has been completed.
本発明は噛乳類の膵臓を20〜6Cf′C好ましくは4
0〜50℃に少なくとも1時間以上自己消化させたのち
、消化枦液に10〜30%の水可溶性有機溶剤存在下に
アルカリ土類金属を1%以上に加え、生じた沈澱を除去
してエラスターゼ抽出液を得ることを特徴とする。The present invention uses the pancreas of chewing mammals with 20 to 6 Cf'C, preferably 4
After autolysis at 0 to 50°C for at least 1 hour, 1% or more of an alkaline earth metal is added to the digestive fluid in the presence of a 10 to 30% water-soluble organic solvent, the precipitate formed is removed, and elastase is added. It is characterized by obtaining an extract.
必要によりこのエラスターゼ抽出液に対して、通常行な
われるとおり透析した後、硫安を25〜45%飽和好ま
しくは、35%飽和に加えて生じた沈澱PH8〜11.
5の水に溶解した後、脱塩しPHを中和することにより
エラスターゼが複合体を結晶として析出させ、或いは上
記の脱塩の後弱塩基性陰イオン交換体に通じ、未吸着画
分を集めた後に中和することにより、純エラスターゼ結
晶を析出させることも出来る。If necessary, this elastase extract is dialyzed as usual, and then ammonium sulfate is added to saturation of 25 to 45%, preferably 35%, and the resulting precipitate has a pH of 8 to 11.
After dissolving in water in step 5, elastase precipitates the complex as a crystal by desalting and neutralizing the pH, or after the above desalting, passes through a weakly basic anion exchanger to remove the unadsorbed fraction. Pure elastase crystals can also be precipitated by neutralizing after collection.
本発明にかかるエラスターゼは牛、豚、羊等、啼乳動物
の膵臓に含有されているが、特に豚膵臓に多量に存在し
ているため、工業的製造のための原料として豚膵臓を用
いるのが有利である。Elastase according to the present invention is contained in the pancreas of dairy animals such as cows, pigs, and sheep, but since it is present in particularly large amounts in pig pancreas, it is difficult to use pig pancreas as a raw material for industrial production. is advantageous.
精製エラスターゼは低温下ではPH4〜10.5の範一
囲できわめて安定であるが、20℃以上では不安定であ
る事が知られている。しかし膵臓自己消化時に於けるエ
ラスターゼの安定性はきわめて高く、プタ膵臓細切物を
20〜6(代)で少なくとも1時間以上例えば50′C
3時間の自己消化を行なつた場合に於いても、公知の方
法(特開昭52−61287)と比較したかぎり、ほと
んどエラスターゼの分解が認められない事を発見した。
この条件で得られる自己消化液はきわめて粘性が低く、
従つて枦過が速やかで且つ大量の清澄枦液を得ることが
出来た。Purified elastase is extremely stable at low temperatures in the pH range of 4 to 10.5, but is known to be unstable at temperatures above 20°C. However, the stability of elastase during pancreatic autolysis is extremely high.
It was discovered that even when autolysis was carried out for 3 hours, almost no elastase degradation was observed as compared with the known method (Japanese Patent Application Laid-Open No. 52-61287).
The autolytic fluid obtained under these conditions has extremely low viscosity;
Therefore, the filtration was quick and a large amount of clear liquid was able to be obtained.
更に比較的高温度下での自己消化により、結合組織タン
パクその他の不要タンパクの多くが低分子化を受け、そ
の後のエラスターゼ製造工程に都合の良い消化枦液を得
ることが出来た。Furthermore, by autolysis at relatively high temperatures, many of the connective tissue proteins and other unnecessary proteins were reduced to low molecular weight molecules, making it possible to obtain a digested fluid suitable for the subsequent elastase production process.
自己消化清澄戸液に、アルカリ土類金属を1%以上にな
る様に加えた後、エタノール、メタノール、アセトン等
、水可溶性有機溶剤を10〜30%となる様に加えてタ
ンパク質沈澱を除去し、エラスターゼ含有溶液を得る。After adding alkaline earth metals to the autolysis solution to a concentration of 1% or more, protein precipitates are removed by adding water-soluble organic solvents such as ethanol, methanol, acetone, etc. to a concentration of 10 to 30%. , to obtain an elastase-containing solution.
ここで使用しうるアルカリ土類金属塩としては例えば塩
化カルシウム、塩化マグネシウム等の水溶性塩が有効で
ある。本発明による上記方法とLewisらの方法との
エラスターゼ精製度を下表に参考として示した。As alkaline earth metal salts that can be used here, for example, water-soluble salts such as calcium chloride and magnesium chloride are effective. The elastase purification degrees of the above method according to the present invention and the method of Lewis et al. are shown in the table below for reference.
精製度はレWisらの方法によつて得られたエラスター
ゼ含有溶液の粗抽出液の精製度を1.0としてその倍率
を示している。但し本発明方法により得られるエラスタ
ーゼのエラスチン分解能の測定は、不溶性エラスチンの
分解により生じる可溶性エラスチン分解物の量をチロシ
ン価として算出し、1分間に1μyのチロシンの遊離す
る活性を1単位とするRObbinsらの,方法(Ar
ch.sBiOchem,.BiOphys,.皿、3
96、(1975))の改良法により測定した。The degree of purification is expressed as a magnification, assuming that the degree of purification of the crude extract of the elastase-containing solution obtained by the method of Le Wis et al. is 1.0. However, the elastin decomposition ability of elastase obtained by the method of the present invention is measured by calculating the amount of soluble elastin decomposed product produced by the decomposition of insoluble elastin as the tyrosine value, and using RObbins, where 1 unit is the activity of releasing 1 μy of tyrosine per minute. et al.'s method (Ar
ch. sBiOchem,. BiOphys,. plate, 3
96, (1975)).
タンパク質はLOwry法(JIBiOlIchem●
M良・265・ (1951))に従い、卵白アルブミ
ンを標準として定量した。Proteins are prepared using the LOWry method (JIBiOlIchem●
Quantification was performed using ovalbumin as a standard according to M. Ryo, 265, (1951)).
以下に本発明を実施例をもつて更に詳細に説明する。The present invention will be explained in more detail below with reference to Examples.
豚膵臓細切物1k9を強く攪拌しながら50℃に3時間
自己消化にかけた後、3eの水を加え、枦過により、3
.5eの清澄液を得た。After autolyzing 1k9 pieces of shredded pork pancreas at 50°C for 3 hours with strong stirring, 3e of water was added and filtrated.
.. A clear solution of 5e was obtained.
清澄液に80fの酢酸カルシウムを加えたのち、アセト
ン500m1を加えた。生じる沈澱を遠心分離により除
きエラスターゼ含有の遠心上清3.8eを得た。該上清
を透析して塩及び有機溶剤を除き、透析内液に固形硫酸
アンモニウムを35%飽和になるように溶解し、生じた
沈澱を遠心分離により集め、100m1の水に懸濁し、
アンモニア水でPHを10に調節する事により沈澱を溶
解した。After adding 80 f of calcium acetate to the clarified liquid, 500 ml of acetone was added. The resulting precipitate was removed by centrifugation to obtain elastase-containing centrifugal supernatant 3.8e. The supernatant was dialyzed to remove salts and organic solvents, solid ammonium sulfate was dissolved in the dialysis solution to a saturation of 35%, and the resulting precipitate was collected by centrifugation and suspended in 100 ml of water.
The precipitate was dissolved by adjusting the pH to 10 with aqueous ammonia.
溶解のPHを8〜11.5に保ちながら透析して脱塩し
た後、酢酸で溶液のPHを7にする事によりエラスター
ゼ複合体を晶出させた。After desalting by dialysis while maintaining the pH of dissolution at 8 to 11.5, the elastase complex was crystallized by adjusting the pH of the solution to 7 with acetic acid.
Claims (1)
0〜30%の水可溶性有機溶剤存在下にアルカリ土類金
属塩を1%以上となる様に加え、生じた沈殿を除去して
エラスターゼ抽出液を得ることを特徴とするエラスター
ゼの製法。 2 膵臓の自己消化は40〜50℃に少なくとも1時間
以上行なう特許請求の範囲第1項の方法。[Claims] 1. After autolyzing the mammalian pancreas, 1.
A method for producing elastase, which comprises adding an alkaline earth metal salt to a concentration of 1% or more in the presence of a 0 to 30% water-soluble organic solvent, and removing the resulting precipitate to obtain an elastase extract. 2. The method according to claim 1, wherein autolysis of the pancreas is carried out at 40 to 50°C for at least 1 hour.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP23164584A JPS6054037B2 (en) | 1984-11-02 | 1984-11-02 | Manufacturing method of elastase |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP23164584A JPS6054037B2 (en) | 1984-11-02 | 1984-11-02 | Manufacturing method of elastase |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP11712179A Division JPS5642588A (en) | 1979-09-11 | 1979-09-11 | Production of elastase |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60133887A JPS60133887A (en) | 1985-07-17 |
JPS6054037B2 true JPS6054037B2 (en) | 1985-11-28 |
Family
ID=16926742
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP23164584A Expired JPS6054037B2 (en) | 1984-11-02 | 1984-11-02 | Manufacturing method of elastase |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6054037B2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61194132U (en) * | 1985-05-23 | 1986-12-03 | ||
JPS61197429U (en) * | 1985-05-29 | 1986-12-09 | ||
JPS62198440U (en) * | 1986-06-05 | 1987-12-17 | ||
JPH01175242U (en) * | 1988-06-01 | 1989-12-13 |
-
1984
- 1984-11-02 JP JP23164584A patent/JPS6054037B2/en not_active Expired
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61194132U (en) * | 1985-05-23 | 1986-12-03 | ||
JPS61197429U (en) * | 1985-05-29 | 1986-12-09 | ||
JPS62198440U (en) * | 1986-06-05 | 1987-12-17 | ||
JPH01175242U (en) * | 1988-06-01 | 1989-12-13 |
Also Published As
Publication number | Publication date |
---|---|
JPS60133887A (en) | 1985-07-17 |
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