海参蛋白酶体的提取及其酶学性质研究  

Extraction and Enzymatic Properties of Proteasome from Wall of Sea Cucumbers

作　　者：李秀芬[1,2] 牛海玲[1,2] 张蕊[1,2] 李冉[1,2] 侯红漫[1,2] 张公亮[1,2] 孙黎明[1,2] 

机构地区：[1]大连工业大学食品学院,辽宁大连116034 [2]国家海洋食品工程技术研究中心,辽宁大连116034

出　　处：《中国食品学报》2013年第12期65-70,共6页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31201300;30901124);辽宁省教育厅项目(LJQ2012048)

摘　　要：目的：从海参体壁组织中分离提取蛋白酶体,研究其酶学性质。方法：新鲜海参组织经匀浆、Tris-HCl缓冲液浸提后得到粗提物,再经（NH4）2SO4盐析、二乙氨乙基葡聚糖纤维素和丙烯葡聚糖凝胶S-300分离纯化后得到海参蛋白酶体。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析,其分子质量200 ku。结果：海参蛋白酶体特异性最强的检测底物Boc-GAA-MCA,最适pH 8.0,最适反应温度45℃。40℃时蛋白酶体活性稳定,在1 h的孵育过程中呈现一定的热激活效应。在50~60℃,酶活力随孵育时间的延长迅速下降。K＋、Ca2＋、Mg2＋、Zn2＋均可明显抑制其活性。抗痛素、N-甲苯磺酰-L-赖氨酸氯甲基化酮、N-甲苯磺基-L-苯乙胺酰氯甲基酮及E-64均可显著抑制该蛋白酶体活性。结论：海参体壁蛋白酶体具有典型的蛋白酶体的复合型活性,其中胰蛋白酶样活性较强,之后是糜蛋白酶样活性,而肽基谷氨酰肽水解酶样活性最弱。Proteasome is extracted from body wall of sea cucumbers and its enzymatic properties are studied.Fresh sea cucumber was homogenized,and crude enzyme was extracted from the homogenate by Tris-HCl（50 mmol/L,pH 7.5）.The extraction is then further purified by ammonium sulphate precipitation,DEAE-Sephacel and Sephacryl S-300 chro-matography.The purity of the obtained proteasome was proved by SDS-PAGE,with a molecular weight of 200 ku.Boc-GAA-MCA was found to be the specific substrate of the purified proteasome.Maximal activity appeared at pH 8.0 and at 45 ℃.The purified proteasome demonstrated Stable activity even a thermal activation effect was observed at 30 ~40 ℃,but the activity decreased rapidly at 50~60 ℃.TLCK,TPCK and E-64 could inhibit significantly the activity,which in-dicated a typical proteasome property with serine-protease-like,chymotrypsin-like,and cysteine-proteinase-like activities.

关 键 词：海参 蛋白酶体 纯化 酶学性质 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程]