龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

Purification and Characterization of a Bromoperoxidase from Gracilaria lemaneiformis

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定,活力单位较低或无;经DEAE cellulose 52离子交换层析,去除了结构多糖及藻胆蛋白,酶催化反应稳定,得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明:该溴过氧化物酶为单体酶,分子量约66 kD,溴化单氯双甲酮时的最适pH值为6.0,在40℃以下和pH 3.0～9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性,而Fe^(2+)、Fe^(3+)、Cu^(2+)、Zn^(2+)和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明,该酶对Br^-、H_2O_2的Km分别为53.5μmol/L和38μmol/L。

A bromoperoxidase from Gracilaria lemaneiformis was purified to homogeneity using a multi-step process of ammonium sulfate precipitation （AS）, dialysis, and DEAE-cellulose 52 anion exchange chromatography. The bromoperoxidase activity was unstable or undetectable in crude extract solution. However, it became stable with electrophoretic purity after this multiple purification process. The anion exchange chromatography purification was a critical step in the purification process, which effectively eliminated the phycobiliprotein and smucilaginous polysaccharides. The purified bromoperoxidase was a monomeric enzyme with the relative molecular masses of 66 kD as determined by denaturing and native gradient gel electrophoresis. The optimal pH for hromoination was 6.0 and bromoperoxidase activity was stable as stored at a broad pH range of 3.0-9.0. Of a range of compounds tested, only vanadium enhanced bromoperoxidase activity. Kinetic studies for the bromination of monochlorodimedone （MCD） showed that the Km values of Br^- and H2O2 are 53.5 μmol/L, 38 μmol/L respectively.