中国明对虾蛋白酶的分离纯化及其酶学性质研究  

Isolation,Purification and Properties of Protease from Fenneropenaeus chinensis

作　　者：林建城[1] 郑云忠[1] 闵志勇[1] 

机构地区：[1]莆田学院环境与生物工程学院,福建莆田351100

出　　处：《中国海洋大学学报（自然科学版）》2015年第6期57-63,共7页Periodical of Ocean University of China

基　　金：福建省高校服务海西重点建设项目(2008HX02);福建省区域科技重大项目(2009N3002)资助

摘　　要：以中国明对虾（Fenneropenaeus chinensis）肝胰脏为材料,通过硫酸铵沉淀分级分离、Sephadex G-100和DEAE-32柱层析纯化,经PAGE获得蛋白酶,该酶的比活力为96.752 U/mg.SDS-PAGE显示一谱带,测得酶亚基分子量为36.3kDa,凝胶层析法测得酶分子量为100.9 kDa,推断该蛋白酶由3个相同亚基组成,等电聚焦电泳法测得酶的等电点pI为9.65.以酪蛋白为底物,研究蛋白酶催化反应的动力学参数.结果表明：蛋白酶的最适pH、最适温度、Km和Vmax分别为8.0、65℃、4.578 mg/mL和0.273 U/min;酶在pH为5.0～10.0范围内较稳定,在20～45℃之间具有较好的热稳定性,在50℃以上热稳定性迅速下降.Mg2＋对酶活力没有影响,Ca2＋、Ba2＋、Cu2＋、Mn2＋、Co2＋、Zn2＋和Hg2＋对酶有不同程度的抑制作用,其中Hg2＋的抑制作用最强,10 mmol/L Hg2＋可使酶活力完全丧失,而Fe2＋对酶有激活作用.EDTA对酶活力没有影响,推断该蛋白酶为非金属蛋白酶.The protease was purified from the hepatopancreas of Fenneropenaeus chinensis by ammonium sulfate fractionation,then chromatographed on Sephadex G-100 followed by DEAE-cellulose （DE-32） column.The purified enzyme determined to be homogeneous by polyacrylamide gel electrophoresis （PAGE） and SDS-PAGE.The specific activity of the purified enzyme was 96.752 U · mg-1.Enzyme molecular weight was determined to be 100.9 kDa;it contained three subunits of the same mass （36.3 kDa）.The pI value was calculated to be 9.65 by isoelectric focusing.The optimum pH and optimum temperature of the enzyme for the hydrolysis of casein（enzyme substrate） were determined to be pH 8.0 and 65 ℃,respectively.The Km and Vm values were determined to be 4.578 mg/mL and 0.273 U/min by plot of Lineweaver-Burk,respectively.The stability of the enzyme was investigated,and the results showed that the enzyme was stable in a pH range from 5.0 to 10.0 and at temperatures＜45 ℃.The stability of enzyme dropped rapidly at temperatures＞50 ℃.The effects of metal ions on the enzyme were also studied.Mg2＋ had no influence on enzyme activity.Fe2＋ activated the enzyme,while Ca2＋ 、Ba2＋ 、Cu2＋ 、Mn2＋ 、Co2＋ 、Zn2＋ and Hg2-showed various degrees of inhibitory effects on the enzyme.Hg2＋ inhibited the enzyme most strongly,when its concentration reached 10 mmol · L-1,enzyme activity completely ceased.EDTA had no influence on the enzyme indicating the protease was nonmetallic enzyme.

关 键 词：中国明对虾 蛋白酶 分离纯化 酶学性质 

分 类 号：Q556.1[生物学—生物化学]