南极磷虾羧肽酶的分离纯化及酶学性质

Purification and enzymatic properties of carboxypeptidase from Euphausia superba

南极磷虾生活在极寒环境,其体内特殊的蛋白酶系统是其维持正常新陈代谢的关键因素。羧肽酶是南极磷虾蛋白酶系统中一类主要且关键的蛋白酶,酶学性质研究对其后续基础研究开展及商业利用都具有指导意义。本研究以南极磷虾为原料,采用缓冲液提取、硫酸铵分级盐析,DEAE-琼脂糖凝胶FF阴离子层析和Sephadex G-100凝胶层析,从南极磷虾匀浆液中分离纯化出一种羧肽酶,SDS-PAGE结果显示其分子量为30 ku。酶学性质研究结果显示:该酶最适温度为30℃;最适pH为8.0。热稳定性较差,即使在4℃下保温超过2 h酶活性会急剧下降。金属离子Ni^2+、Mn^2+、Zn^2+和Mg^2+具有激活作用,且激活作用依次增强;而Ca^2+、Fe3+和Cu^2+起抑制作用,Cu^2+抑制效果最明显。对巯基有修饰作用的抑制剂DTT和β-巯基乙醇对其有抑制作用,推测该酶活性中心存在二硫键;金属蛋白酶抑制剂EDTA和1,10-菲罗啉对该酶活性有明显的抑制作用,说明了其具有金属蛋白酶特性;而丝氨酸蛋白酶抑制剂PMSF对该酶抑制作用并不强烈。以脲酰-L-苯丙氨酸为底物溶液,测得该酶Km为0.0059 mg/mL、Vmax为4.9091 U/min。

Euphausia superba lives in an extremely cold environment and its unique proteinase plays a key role in maintaining its metabolic reactions at low temperature.Its carboxypeptidase,as a main and key proteinase,was investigated in this research and it provided support for its further basic research and commercial application.Carboxypeptidase from E.superba was extracted first with buffer solution and then purified respectively with ammonium sulfate,DEAE-agarose gel FF anion chromatography and Sephadex G-100 gel chromatography.The results of SDS-PAGE showed its molecular weight was about 30 ku.Aspects such as optimal temperature,optimal pH,thermal stability,activations and inhibitions allowed an in-depth understanding of the enzymatic properties of carboxypeptidase from E.superba.Its optimal temperature was 30℃and optimal pH was 8.0.The low thermal stability resulted in a significant diminishing of enzyme vitality after two hours of autolysis,even at 4℃.Ni^2+,Mn^2+,Zn^2+and Mg^2+increased the carboxypeptidase activity and their activations became more significant from left to right.Ca^2+,Fe3+and Cu^2+inhibited its enzymatic activity and Cu^2+was the strongest inhibitory.DTT andβ-mercaptoethanol,inhibitors of thiol modification,showed inhibitory on the carboxypeptidase.It indicated that there were disulfide bonds in its active center.Metalloproteinase inhibitors EDTA and 1,10-phenanthroline had significant inhibitory on its enzymatic activity,which was similar to the characteristics of metalloproteinase,and serine protease inhibitor PMSF didn’t show significant effect on its activity.The kinetic constants of Km and Vmax were revealed at 0.0059 mg/mL and 4.9091 U/min respectively with hippuryl-L-phenylalanine as a substrate.