假海源菌ZJCN121岩藻多糖酶的纯化及其性质

Purification and characterization of fucoidanase from Pseudidiomarina ZJCN121

液态发酵培养假海源菌ZJCN121,粗酶液经过丙酮沉淀和Sephadex G-100柱层析分离纯化得到回收率为13.5%,纯化倍数为21.8,比活为20.59 IU·mg-1的电泳纯岩藻多糖酶。采用SDS-PAGE电泳和EIF-PAGE电泳,分别测得酶的相对分子质量为60.2 kDa、等电点为4.3。该酶的最适反应温度为50℃,最适pH为6.5。其催化Fucus vesiculosus岩藻多糖的Km为5.87 mg·mL-1,Vmax为6.11 g·L-1·min-1。Ca2+、Ba2+对酶稍有激活作用,Fe2+、Cu2+则强烈抑制酶活,Mn2+、K+、Zn2+也在一定程度上抑制酶活性,Mg2+对酶的作用效果不明显。

Pseudidiomarina ZJCN121 was cultured by liquid state fermentation. Crude enzyme liquid was separated and purified through acetone precipitation and Sephadex G-100 chromatography with a final fucoidanase purification of 21.8-fold and a specific activity of 20.59 IU/mg and an overall yield of 13.5%. The purified enzyme was identified with molecular mass of 60.2 kDa and isoelectric point of pH 4.3 by SDS-PAGE and EIF-PAGE, respectively. The optimum temperature was 50℃ and the optimum pH 6.5. Km and Vmax for Fucus vesiculosus fucoidan were 5.87 mg.mL-1 and 6.11 g.Ll.min-1, respectively. The fucoidanase was lightly activated by Ca2＋ and Ba2＋, and strongly inhibited by Fe2＋ and Cu2＋. Its activity also was inhibited by Mn2＋, K＋and Zn2＋, while uneffected by Mg2＋.