Purification and enzymatic properties of carboxypeptidase from Euphausia superba

Abstract:

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℃. Ni²⁺, Mn²⁺, Zn²⁺ and Mg²⁺ increased the carboxypeptidase activity and their activations became more significant from left to right. Ca²⁺, Fe³⁺ and Cu²⁺ inhibited its enzymatic activity and Cu²⁺ was the strongest inhibitory. DTT and β -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 K_m and V_max were revealed at 0.005 9 mg/mL and 4.909 1 U/min respectively with hippuryl-L-phenylalanine as a substrate.