白腐真菌漆酶的纯化及性质

Purification and characterziaton of laccase from white-rot fungi

液体发酵培养白腐真菌F9,粗酶液经盐析、透析浓缩、葡聚糖G-100柱层析、DEAE-纤维素离子交换层析四步分离纯化,得电泳纯漆酶。经SDS-PAGE法测定酶的相对分子质量约为6×104,酶活回收率达46.47%,纯度提高了18.86倍。F9漆酶最适反应温度为40℃,最适反应pH为4.8,在35℃以下、pH 4.8～5.4的范围内稳定性较强。其催化愈创木酚的Km为4.61 mmol/L,vm为6.27 mmol/(L.min)。K+对其有激活作用,而Fe2+、Fe3+对其有明显抑制作用。

The laccase of white-rot fungi F9 was purified through salting out,dialyzing and condensing,Sephadex G-100 column chromatography and DEAE-cellulose ion-exchange chromatography with a final purification of 18.86-fold and an overall yield of 46.47%.The purified enzyme was identified with a molecular mass of 6×104 by SDS-PAGE method.The optimum pH and the temperature were 4.8 and 40 ℃ respectively.When the temperature was below 35 ℃ and the pH was 4.8-5.4,the laccase exhibited maximal stability.Km and vm for oxidizing guaiacol were 4.61 mmol/L and 6.27 mmol/（L·min）,respectively.The laccase was activated by K＋ and inhibited by Fe2＋,Fe3＋.