罗非鱼肠蛋白酶的分离纯化及其性质  

Purification and characterization of protease from intestine of tilapia

作　　者：吴燕燕[1] 李来好[1] 郝志明 杨贤庆[1] 

机构地区：[1]中国水产科学研究院南海水产研究所,广东广州510300 [2]国家加工食品质量监督检测中心,广东广州510330

出　　处：《水产学报》2010年第3期357-366,共10页Journal of Fisheries of China

基　　金：农业部“九四八”项目(2006-G40);中央级公益性专项资金项目(2007ZD05);广东省重点项目(2005B33201002)

摘　　要：以罗非鱼肠为原料,采用超声波辅助提取技术、电泳技术和层析技术等对罗非鱼肠道蛋白酶进行研究,结果表明:将鱼肠匀浆经超声波提取的粗酶液,用30%～70%的硫酸铵盐进行盐析、HitrapTM Q FF阴离子交换柱纯化及Sephadex G-100凝胶柱分离纯化,得到罗非鱼肠蛋白酶纯品,其比活为335U/mg,得率为32.8%;SDS-PAGE电泳为单一蛋白酶带,分子量为28ku。该酶最适pH为8.0～8.5,在pH7.0～9.0的条件下稳定;最适温度为37～42℃,热稳定性好;该酶的Km值和Vmax值分别为0.605g/L和9.407μg/min。金属离子Ag+、Pb2+对蛋白酶有完全抑制作用,Na+、K+对该酶无抑制作用。丝氨酸蛋白酶抑制剂能完全抑制该酶活性,胃蛋白酶抑制剂和脲素对该酶有一定抑制作用,EDTA没有明显抑制作用,DTT能激活该酶活性,该酶为丝氨酸蛋白酶。Tilapia intestines as enzyme raw materials are studied systematically by ultrasonic extraction technology of auxiliary, electrophoresis, chromatography techniques and so on for the first time. The results showed that： after organic mashing and ultrasound-assisted extracting, 30% -70% of ammonium sulfate fractions, QFF anion chromatography and Sephedax G-100 gel chromatography, the purified protease from tilapia intestines was obtained. The purified protease specific activity is 335 U/mg and yield is 32.8%. The result of SDS-PAGE showed a single band and the subunit molecular weight of tilapia intestines protease was 28 ku. The protease optimum pH value and temperature are 8.0 -8.5 and 37 -42℃ respectively. It is stable at pH range from 7.0 to 9.0 and has good thermal stability. Enzyme Km value and Vmax value are 0.605 g/L and 9.407 ug/min. Ag＋ and Pb2＋ can inhibit the enzymatic activity completely while Na＋ and K＋ without inhibition. PMSF can strongly inhibit the activity of the enzyme. Pepstatin A and urea partly inhibit the activity of the enzyme. EDTA has no effect on the enzyme while DTT can activate the enzyme activity. The protease from tilapia intestines is a kind of serinc protease. Further work should be carded out on the sequences of the enzyme and its applications.

关 键 词：罗非鱼肠 蛋白酶 纯化 性质 

分 类 号：S254.1[农业科学—农业机械化工程] S917[农业科学—农业工程]