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

摘要:

副溶血弧菌C20发酵液经离心，超滤浓缩，盐析，DEAE-Sepharose Fast Flow和Sephardex G-100分离纯化得到电泳纯的褐藻胶裂解酶，其分子量为40 kD。酶的最适温度为30 ℃，在此温度下保温3.5 h，相对酶活仍保持在90%；酶的最适pH为7.2，在pH 7.0~7.6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1.38 mg/mL，Vmax为0.052 mg/(mLmin)。

Abstract:

Alginate lyase was successfully purified from fermented broth of V. 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. Mg2+, Na+, NH4+, Ca2+, methanol and Tween-80 with certain concentration had activation of the enzyme while ethanol, acetone, DTT, urea, SDS, Zn2+, Cu2+, Fe2+ and Al3+ showed inhibition. This enzyme specifically degraded polyguluronate with Km of 1.38 mg/mL and Vmax of 0.052 mg/(ml•min).