韦伯灵芝漆酶的分离纯化及其性质

采用硫酸铵分级盐析、透析、聚乙二醇浓缩和高效液相色谱柱层析从韦伯灵芝TZC-1 发酵液中分离纯化得到电泳纯漆酶，其纯化倍数为37.1 倍，酶活性回收率为21.3%。活性-PAGE(聚丙烯酰胺凝胶电泳)结果表明该漆酶由一种同工酶组成，纯化漆酶经SDS-PAGE 检测显示为单一条带，其分子质量约为40kD。该漆酶催化底物ABTS 的最适反应温度为50～60℃，最适反应pH 值为4.6，在60℃以下和pH3.0～5.0 范围内保持稳定，以ABTS为底物的表观Km 值为13.8μmol/L。Fe2+ 完全抑制酶活，Al3+ 和Mn2+ 对该漆酶也有明显抑制作用，Hg+、Cu2+ 和Mg2+ 对该酶有明显激活作用，而Zn2+、Ba2+ 及K+ 对该酶活性影响不大。

Purification and Characterization of Laccase from Ganoderma weberianum

The laccase from Ganoderma weberianum TZC-1 was purified using fractional ammonium sulfate precipitation, dialysis, polyethylene glycol condensation and high performance liquid column chromatography. Compared to the crude extract, its purity exhibited a 37.1-fold improvement and the recovery rate of enzyme activity was 21.3%. Meanwhile, SDS-PAGE analysis revealed a single band with molecular weight of 40 kD. Moreover, the optimal reaction conditions of this laccase towards ABTS as substrate included 50－60 ℃ of reaction temperature and pH 4.6. The Km value was 13.8 μmol/L under these optimal reaction conditions. Furthermore, the activity of the enzyme exhibited excellent stability below 60 ℃ and pH 3.0－ 5.5, but could be inhibited by Fe2+, Al3+ and Mn2+ and enhanced by Hg+, Cu2+ and Mg2+. No obvious effect on laccase activity was observed due to Zn2+, Ba2+ and K+.