副溶血弧菌C20褐藻胶裂解酶的纯化及酶学特性研究

Purification and Enzymatic Characteristics of Alginate Lyase from V.parahaemolyticus C20

副溶血弧菌C20发酵液经离心,超滤浓缩,盐析,DEAE-SepharoseFastFlow和SephardexG-100分离纯化得到电泳纯的褐藻胶裂解酶,其分子量为40kD。酶的最适温度为30℃,在此温度下保温3.5h,相对酶活仍保持在90%;酶的最适pH为7.2,在pH7.0～7.6范围内稳定;Mg2+、Na+、NH4+、Ca2+、甲醇和适量浓度的Tween-80对酶有激活作用;Zn2+、Cu2+、Fe2+、Al3+、乙醇、丙三醇、DTT、尿素和SDS等对酶有较强的抑制作用;此褐藻胶裂解酶对聚古罗糖醛酸有很强的底物专一性,其Km值为1.38mg/mL,Vmax为0.052mg/(mL-min)。

Alginate lyase was successfully purified from fermented broth of 14. parahaemolyticus C20 by ultra-filtration, ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography. The purified alginate lyase with molecular mass of 40 kD had optimal temperature of 30℃ and maintained 90% of its activity after 3.5-hour incubation at 30℃. The enzyme with optimal pH of 7.2 was stable within the pH value range of 7.0 to 7.6. Mg^2＋, Na^＋, NH^4＋, Ca^2＋, methanol and Tween-80 with certain concentration had activation of the enzyme while ethanol, acetone, DTT, urea, SDS, Zn^2＋, Cu^2＋, Fe^2＋ and Al^3＋ showed inhibition. This enzyme specifically degraded polyguluronate with K,,of 1.38 mg/mL and Vmax of 0.052 mg/（ml.min）.