文章摘要
吴燕燕,李来好,郝志明,杨贤庆.罗非鱼肠蛋白酶的分离纯化及其性质[J].水产学报,2010,34(3):357~366
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
投稿时间:2009-10-09  修订日期:2009-12-01
DOI:10.3724/SP.J.1231.2010.06643
中文关键词: 罗非鱼肠  蛋白酶  纯化  性质
英文关键词: tilapia intestine  protease  purification  characterization
基金项目: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)
作者单位E-mail
吴燕燕 中国水产科学研究院南海水产研究所 wuyy1028@yahoo.com.cn 
李来好 中国水产科学研究院南海水产研究所  
郝志明 国家食品质量监督检验中心  
杨贤庆 中国水产科学研究院南海水产研究所  
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中文摘要:
      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335 U/mg,得率为32.8%;SDS-PAGE电泳为单一蛋白酶带,分子量为28 ku。该酶最适pH为 8.0~8.5,在pH 7.0~9.0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0.605 g/L和 9.407 μg/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。
英文摘要:
      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 μg/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 serine protease. Further work should be carried out on the sequences of the enzyme and its applications.
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