南极磷虾体内胰蛋白酶的纯化及性质研究

Purification and characterization of serine proteinase from Euphausia superba

以南极磷虾(Euphausia superb)为研究对象,通过硫酸铵分级沉淀、Phenyl-Sepharose疏水层析、DEAE-Sepharose FF离子交换层析等方法,从南极磷虾体内分离纯化出胰蛋白酶。其纯化倍数为5.44倍,比活力为38.3 U/mg,得率为26%。SDS-PAGE电泳结果显示,该酶的分子质量为28 ku。蛋白酶最适温度为37℃、最适pH为7.5,Mg2+、Ca2+、Mn2+对南极磷虾蛋白酶具有激活性,Zn2+、Cu2+、Fe3+具有酶活抑制性,其中Cu2+的抑制性最强。酶的动力学实验结果表明,以BApNA为底物测得Km为0.073 mmol/L,Vmax为1.44×10-2mmol/L·s,kcat为0.6S-1,kcat/Km为8.22×103,PMSF作为蛋白酶抑制剂,对南极磷虾蛋白酶作用机制为不可逆抑制。

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 Km value was 0. 073 mmol/L, Vmax value was 1.44 × 10^-2 mmol/L · s,kcat value was 0.6 S-1 and k cat/Km value was 8.22 × 10^3. PMSF was a protease inhibitor, and its mechanism of action was irreversible inhibition.