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

ObjectiveThe 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.MethodCrude 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.ResultMolecular 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.ConclusionCarboxypeptidase 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.