四川农业大学学报 ›› 2008, Vol. 26 ›› Issue (03): 251-257.doi: 10.16036/j.issn.1000-2650.2008.03.002

• 研究论文 • 上一篇    下一篇

鲢鱼(Hypophthalmichthys molitrix)背肌中组织蛋白酶B2的纯化与酶学性质鉴定

李树红1,2,3, 周小秋1, 马长伟3   

  1. 1. 四川农业大学 动物营养研究所, 四川 雅安 625014;
    2. 四川农业大学 信息与工程技术学院, 四川 雅安 625014;
    3. 中国农业大学 食品科学与营养工程学院, 北京 100083
  • 收稿日期:2008-01-03 出版日期:2008-09-30 发布日期:2017-03-03
  • 基金资助:
    国家自然科学基金资助项目(30371122)

Purification and Characterization of Cathepsin B2 from Dorsal Muscle of Silver Carp(Hypophthalmichthys molitrix)

LI Shu-hong1,2,3, ZHOU Xiao-qiu1, MA Chang-wei3   

  1. 1. Institute of Animal Nutrition, Sichuan Agricultural University, Yaan 625014, Sichuan, China;
    2. College of Information and Engineering Technology, Sichuan Agricultural University, Yaan 625014, Sichuan, China;
    3. College of Food Science and Nutritional Engineering, China Agricultural University. Beijing 100083, China
  • Received:2008-01-03 Online:2008-09-30 Published:2017-03-03

摘要: 对鲢鱼背部白肌中的组织蛋白酶B2,进行了分离纯化及酶学性质的鉴定。目的酶经粗提后,再通过酸处理、硫酸铵分级沉淀、超滤、DEAE-Sephacel、Sephacryl S-100、SP-Sepharose F. F.、Blue-sepharose 6 F. F.、Con A-Sepha-rose等步骤纯化,纯化倍数达到396.5倍。经SDS-PAGE分析,该酶呈现了分子量约为25和20KDa的2条带。组织蛋白酶B2的最适反应温度和pH分别为30~35℃和pH5.5。巯基还原剂DTT、L-Cys、β-Me均显著地激活了该酶的活性。50μmol/L的E-64能够完全抑制其活性。Cl-浓度为100μmol/L以内时,对组织蛋白酶B2起到激活作用,200μmol/L时则表现为抑制作用。25 mmol/L的P2O74-,即可有效抑制该酶的活性。该纯化酶能够水解Z-Arg-Arg-MCA、Z-Phe-Arg-MCA,但是不能够水解L-Arg-MCA。组织蛋白酶B2水解Z-Arg-Arg-MCA、Z-Phe-Arg-MCA的Km值分别为7.15及226μmol/L,表明Z-Arg-Arg-MCA是该酶的最适底物。

关键词: 组织蛋白酶B2, 鲢鱼, 纯化, 鉴定

Abstract: Silver carp cathepsin B2 was extracted from the dorsal muscle.Then it was purified to 396.5 folder by a series of procedures of acidification,ammonium sulfate precipitation,ultral-filtering,DEAE-Sephacel,Sephacryl S-100,SP-Sepharose F. F.,Blue-sepharose 6 F. F. and Con A-Sepharose.The purified enzyme assumed two bands of about 25 and 20KDa on SDS-PAGE.The optimum temperature was 30~35℃,and the optimum pH was pH 5.5.Cathepsin B2 was efficiently activated by Cys,DTT and β-ME,but was completely inhibited by E-64 of 50μmol/L.Cl-within 100μmol/L activated cathepsin B2,but when reaching 200μmol/L,it shoued effect of inhibition.P2O74- of 25mmol/L inhibited the hydrolysing activity of cathepsin B2 significantly.Substrate specificity analysis indicated that it could hydrolyze Z-Phe-Arg-MCA and Z-Arg-Arg-MCA but not L-Arg-MCA;that Z-Arg-Arg-MCA was the optimum substrate judging from the Km values of 7.15 and 226μmol/L.

Key words: cathepsin B2, silver carp(Hypophthalmichthys molitrix), purification, characterization

中图分类号: 

  • TS254