Purification and characterization of serine proteinase from Euphausia superba

Abstract:

A serine protease from Euphausia superba was purified by a series of procedures, including ammonium sulfate precipitation, column chromatographies on DEAE-Sepharose and Phenyl-Sepharose.The purification multiple of the protease was 5.44 times, and the yield of the protease was 26%, with specific activity of 38.3 U/mg.As shown in the result of SDS-PAGE electrophoresis, the molecular weight of this protease is 28 ku.The optimum temperature of protease was 37℃ and the most suitable pH was 7.5.Mg²⁺, Ca²⁺and Mn²⁺were activated to protease from Euphausia superba.However, Zn²⁺, Cu²⁺and Fe³⁺ were inhibited to the enzyme activity, and the inhibition ability of Cu²⁺ was the strongest.The enzyme kinetics experiments were performed by using BApNA as substrate.The results showed that the K_m value was 0.073 mmol/L, V_max value was 1.44×10^-2 mmol/L·s，k_cat value was 0.6 S^-1 and k_cat/K_m value was 8.22×10³.PMSF was a protease inhibitor, and its mechanism of action was irreversible inhibition.