【作者】 吴燕燕； 王萍； 李来好； 杨贤庆； 刁石强；

【Author】 WU Yan-yan~1,WANG Ping~(1,2),LI Lai-hao~1,YANG Xian-qing~1,Diao Shi-Qiang~1 (1.South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510300,China;2.Shanghai Ocean University,Shanghai,201306,China)

【机构】 中国水产科学研究院南海水产研究所； 上海海洋大学；

【摘要】 以凡纳滨对虾虾头为原料,采用层析及电泳技术等对凡纳滨对虾虾头蛋白酶进行研究,结果表明:将虾头匀浆在pH7.0条件下浸提1h,冷冻离心得到粗酶液,采用硫酸铵盐盐析、Hitrap Q HP阴离子交换层析柱及Sephadex G-100凝胶柱分离纯化,得到蛋白酶纯品。针对其生化特性的研究表明:SDS-PAGE电泳为单一蛋白酶带,分子量为16.2kDa;此酶最适反应的pH条件为10,在pH 7.0～10.0的条件下稳定;最适温度为40℃,热稳定性好。金属离子Ca²⁺、Mg²⁺、Cu²⁺、Mn²⁺对蛋白酶有一定的激活作用,Zn²⁺、Hg²⁺对蛋白酶有完全抑制作用。3种有机试剂中,丙酮对蛋白酶有较强的激活作用,乙醇在低浓度时有明显的激活作用,但丙三醇对此酶的抑制效果明显。丝氨酸蛋白酶抑制剂STI、TLCK、PMSF对该酶有强烈的抑制作用,胃蛋白酶抑制剂、TPCK、EDTA对此酶抑制效果不明显,表明这是一种丝氨酸蛋白酶。各种氨基酸修饰剂对此酶的修饰作用表明此酶非巯基酶,色氨酸、组氨酸、丝氨酸是构成该酶活性中心的必需基团,精氨酸与该酶酶活有一定的关系,氨基并非该酶活性中心的必需基团。更多还原

【Abstract】 The protease from the heads of Litopenaeus vannamei were studied using the biotechnology of chromatography and electrophoresis,and the results showed that:After extraction in buffer at pH7.0 lasted 1h,centrifugaljzation,salting using ammonium sulfate,Hitrap Q HP anion exchange chromatography and Sephadex G-100 gel column,the pure protease were obtained from the raw material.The biochemical characteristics of this enzyme were also studied. SDS-PAGE electrophoresis demonstrated a single protease with a molecular weight of 16.2kDa; the optimum pH and temperature are 10.0 and 40℃,It is stable at pH range from 7.0 to 9.0 and has good thermal stability.Ca²⁺,Mg²⁺,Cu²⁺,Mn²⁺ can active the proteases certainly when Zn²⁺, Hg²⁺ can strongly inhibit the activity of the enzyme.Acetone has strong activation for the protease,and the same with ethanol at low concentrations,but glycerol can inhibit the enzyme obviously.The enzyme is identified as a serine protease for the serine protease inhibitor STI, TLCK and PMSF strongly inhibited the enzyme,and the effect of pepsin inhibitors,TPCK and EDTA is the least.The modification of Various amino acid modifiers for this enzyme determined that it is non-sulfhydryl enzyme and tryptophan,histidine,serine is necessaried by the enzyme active center groups,arginine has a certain relationship with the enzyme activity,the amino groups are not the necessary groups.更多还原