南极磷虾羧肽酶A的分离纯化及其酶学性质分析

目的]建立南极磷虾羧肽酶A的分离纯化方法,并研究分析其酶学性质,为南极磷虾羧肽酶A的开发及应用打下基础.方法]南极磷虾粗酶液经硫酸铵分级沉淀、HiTrap DEAE FF阴离子交换柱层析和SEC 70凝胶过滤柱层析,纯化得到的羧肽酶A,以SDS-PAGE分析其分子量;通过分析酶系反应温度、pH、金属离子、抑制剂对南极磷虾羧肽酶A活力的影响,明确其酶学性质和酶促动力学.结果]南极磷虾羧肽酶A分子量为75.0 kD,其最适温度30℃,最适pH 8.0.Mg2+、Zn2+、Mn2+和Ni2+对南极磷虾羧肽酶A有显著的激活作用(P<0.05,下同),且金属离子浓度越高,激活效果越明显;Ca2+、Fe3+、Cu2+和Hg2+对南极磷虾羧肽酶A存在不同程度的抑制作用,以Hg2+的抑制作用最强,Fe3+的抑制作用最弱.金属蛋白酶抑制剂乙二胺四乙酸(EDTA)、3-苯基丙酸(3-phenylpropionic acid)和1,10-菲罗啉(1,10-phen-athroline)对南极磷虾羧肽酶A活力有显著的抑制作用,且抑制剂浓度越高,抑制效果越明显.结论]南极磷虾羧肽酶A具有金属蛋白酶特性,可开发成降解酶制剂在食品工业及医药行业中推广应用.

Separation and purification of carboxypeptidase A from Euphausia superba and its enzymatic properties

Objective]The separation and purification method for carboxypeptidase A from Euphausia superba was established and its enzymatic properties were studied in order to provide technical support for the exploitation and utiliza-tion of carboxypeptidase A from E. superba.Method]Crude enzyme liquid of E. superba was purified by ammonium sul-fate precipitation,column chromatographieson HiTrap DEAE FF ion exchange chromatography and SEC 70 gel chroma-tography. After purification ,carboxypeptidase A was obtained. Its molecular weight was determined by SDS-PAGE electrophoresis analysis. The enzyme properties and enzymatic kinetics of carboxypeptidase A were studied through in-vestigating the effects of temperature,pH,metal ions and inhibitors on activity of carboxypeptidase A.Result]Molecular weight of this protease was 75 kD,the optimal temperature for it was 30℃,and suitable pH was 8.0. Mg2+,Zn2+,Mn2+and Ni2+significantly activated carboxypeptidase A from E. superba(P<0.05,the same below). As the concentration of metal ions increased ,the effects were more obvious. While Ca2+,Fe3+,Cu2+,Hg2+ inhibited the activity of carboxypeptidase A to various extents. The inhibition effects of Hg2+was the strongest ,but that of Fe3+was the weakest. Inhibitors me-talloproteinases ethylenediaminetetraacetic acid(EDTA),3-phenylpropionic acid and 1,10-phenathroline had significant inhibition effects on carboxypeptidase A from E. superba. As the concentrations were higher,the inhibition effects were enhanced.Conclusion]Carboxypeptidase A from E. superba contains features of metalloproteinase,therefore it can be developed into enzyme degradation product for utilization in food industry and pharmaceuticals industry.