蓝圆鲹分离蛋白水溶性蛋白酶的鉴定及性质

Identification and Characterization of Endogenous Proteinase from Protein Isolate of Blue Round Scads(Decapterus maruadsi)

以蓝圆鲹(Decapterus maruadsi)为研究对象,采用酸/碱溶解-等电点沉淀法制备蓝圆鲹肌肉分离蛋白(acid/alkaline aided protein isolate,API/KPI),分析比较不同分离蛋白与肌肉全蛋白(total protein,TP)的自身降解规律,并对其水溶性蛋白酶的酶学性质展开研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)结果显示,TP与KPI均会发生强烈的自身降解现象,API则无明显的自身降解。酶谱与酶活力测定结果表明,KPI保留了TP超过80%的酶活性,而API则仅剩10%的水解活性。酶学性质结果表明,TP的内源酶具有两个最适pH值,分别为3.0与9.0,而KPI的最适pH值为9.0;TP与KPI内源酶的最适温度均为60℃。荧光底物结果表明,TP与KPI的最适荧光底物为Boc-Gln-Arg-Arg-MCA,且对羧基端为Arg的底物均具有较高的水解活性。抑制剂结果显示,TP和KPI水溶性蛋白酶都能被丝氨酸蛋白酶抑制剂强烈抑制,暗示水溶性丝氨酸蛋白酶在碱法等电点制备的分离蛋白凝胶劣化中起关键作用。

Protein isolate was prepared from the skeletal muscle of blue round scads by acid or alkaline aided isoelectric solubilization/precipitation,respectively.The autolysis of total protein from skeletal muscle(TP)and protein isolates(acid or alkaline aided protein isolate,API or KPI),as well as their endogenous proteinase,were comparatively studied.The results showed that endogenous proteinase had a strong autolysis capacity for TP and KPI incubated at 50℃for 120 min,while no significant auto-degradation was detected in API.KPI remained more than 80%proteolytic activity toward casein,while API remained about 10%of total proteinase activity from TP.A similar result was found in myofibrillar based zymography.Endogenous proteinase from TP and KPI had the same optimal temperature of 60℃.TP had two optimal activity at pH 3.0 and 9.0,while KPI exhibited a single activity peak at pH 9.0.Fluorescent substrate specificity analysis showed that the endogenous proteinase in both TP and KPI had highest activity toward Boc-Gln-Arg-Arg-MCA,and released highly hydrolytic active to substrates with Arg residue in the carboxyl side.Serine proteinase inhibitors strongly suppressed the degradation of myofibrillar protein,indicating that the major endogenous proteinase from TP and KPI was serine type proteinase,which might play a key role in modori phenomenon of KPI.