暗纹东方鲀胃蛋白酶的分离纯化及部分性质研究  

Purification and Property of Stomach Proteinase from Fugu obscurus

作　　者：张继平[1] 郭照良[1] 唐冬生[1] 陈建酬[1] 贺顺莲[1] 

机构地区：[1]佛山科学技术学院动物科学系,广东佛山528231

出　　处：《湖南农业大学学报（自然科学版）》2004年第6期526-529,共4页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：广东省自然科学基金项目(04011645);佛山科学技术学院重点项目(2002032)

摘　　要：为了探明 pH 值、温度及矿物质离子对暗纹东方鲀胃蛋白酶活性的影响效果,用暗纹东方鲀的胃为材料,经柠檬酸缓冲液抽提,硫酸铵分级沉淀,CM-纤维素离子交换柱、Sephedex G75 层析柱纯化,获得比活为 7.13μmol/(min·mg)的胃蛋白酶.酶经聚丙烯酰胺电泳,表现为单一蛋白带,经 SDS-PAGE 测得酶的亚基相对分子质量为18 600.酶水解酪蛋白的最适温度为50 ℃,在pH2.5, ℃下酶催化酪蛋白水解的Km值为0.715 mmol/L;pH5.0, 3737 ℃下酶催化 N-卞氧羰酰 L-赖氨酸对-硝基酚酯(CBZ-Lys.pNP)的 Km 值为 0.112 mmol/L.几种矿物质离子对酶活力的影响表明,Ca2+,Mn2+,Mg2+对酶有激活作用,Zn2+,Fe2+对酶有抑制作用.To investigate the effects of pH value,temperature and some metalions on its activity,stomach proteinase was purified from Fugu obscurus by following procedures:citric acid buffer (pH 5.0) extraction,ammonium sulfate precipitation,ion-exchange chromatography on CM-cellulose,then by gel filtration through Sephadex G-75.The final proteinase was homogeneous on PAGE and SDS-PAGE.The relative molecular mass of the enzyme subunit was 18 600 by SDS-PAGE.The specific activity of the enzyme was 7.13 μmol/(min·mg).The optimum temperature is 50 ℃ for the hydrolysis of casein. Mchaelis-Menten contant (Km) is 0.715 mmol/L for hydrolysis of casein at pH 2.5 and 37 ℃ while the Km is 0.112 mmol/L for hydrolysis CBZ-Lys.pNP at pH 5.0 and 37 ℃.The effects of some metal ions on the activity of enzyme showed that the Ca2+,Mn2+,Mg2+ can activate the enzyme,Zn2+,Fe2+ can inhibite the enzyme.

关 键 词：暗纹东方鲀 胃蛋白酶 纯化 性质 

分 类 号：S965.225[农业科学—水产养殖] Q356.1[农业科学—水产科学]