南蓝鳕鱼糜中L型组织蛋白酶的研究  

Studies on cathepsin L in the surimi of southern blue whiting

作　　者：胡亚芹[1] 姚燕佳[1] 陈建初[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院,浙江杭州310029

出　　处：《水产学报》2010年第3期336-341,共6页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2007AA091802);浙江省钱江人才计划项目(2009R10056);浙江省教育厅项目(N20090182);中央高校基本科研业务费专项资金;浙江大学2008年度优秀青年教师资助计划(紫金计划)项目;国家"十一五"科技支撑计划(2008BAD91B00)

摘　　要：从南蓝鳕鱼糜中提取肌动球蛋白,对肌动球蛋白进行凝胶层析,测定各收集组分中L型组织蛋白酶的活性。结果表明,组织蛋白酶L依然存留于肌动球蛋白样品中,多次漂洗以及稀释-沉淀处理亦不能有效将其除去。凝胶层析图谱分析表明,该酶可能是肌动球蛋白非结合型酶。对凝胶层析所得粗酶液进行酶学性质分析,结果得知该酶最适温度为45℃,正好落在鱼糜凝胶劣化发生范围之内。专一性底物及酶激活剂、抑制剂影响研究表明,该酶为内含巯基的半胱氨酸型组织蛋白酶。该酶最适pH为5.5,在近中性pH范围内依然有较高的残留活性,表明该酶具有潜在凝胶劣化能力。According to the conventional method, actomyosin was extracted from the surimi of southern blue whiting （ Micromesistius australias）. After Sepharose-6B gel filtration of the actomyosin obtained, cathepsin L activity was monitored in each of the fractions collected. The results showed that the activity values in the actomyosin samples were much higher than that in the washing water, suggesting that cathepsin L could not be removed completely during conventional leaching and still remained in actomyosin sample. Further purification of actomyosin by dilution-precipitation could not remove cathepsin L effectively, indicating that cathepsin L might be dissolved together with actomyosin in the presence of high ion-strength solutions. The profile of the gel filtration showed hat the main peak of cathepsin L was obviously separated from that of actomyosin, suggesting that cathepsin L was non-binding with actomyosin. Fractions that showing the main peak of cathepsin L were pooled to be called Lmi~, partially purified cathepsin L. Lmix could strongly hydrolyze Z-Phe-Arg-MCA, a specific substrate for cathepsin L, rather than Z-Arg-Arg-MCA, spcefic substrate for cathepsin B, nor Arg-MCA, substrate for cathepsin H, as well as Boc-Gln-Ala-Arg-MCA, substrate for trypsin and trypsin-like proteases. The above results indicate that Lmix was a crude cathepsin L. Cathepsin L specific inhibitors, such as E-64, specific inhibitor for cathepsins, and leupeptin, inhibitor for both cathepsins and trypsin-like proteases, could suppress the activity of Lmix completely. Activators for cathepsin L, such as DTT, EDTA ＋ DTT, could effectively improve the activity of Lmix. As a result, the effect of activators and inhibitors confirmed the partially purified crude enzyme, Lmix, tO be a thiol-type cysteine protease. The temperature dependence and pH dependence of the activity of Lmix were also investigated. Zmix had optimum temperature of 45 ~C which was right in the temperature range of modori phenomena. Zmix had optimum pH of 5.5, but ke

关 键 词：南蓝鳕 L型组织蛋白酶(组织蛋白酶L) 漂洗 酶抑制剂 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程] S917[轻工技术与工程—食品科学与工程]