灵芝EIM-40漆酶的分离纯化及酶学性质

采用冻干浓缩、（NH4）2S04盐析、HiTrapphenyl（FF）疏水层析和QSepharose FastFlow离子交换层析对灵芝EIM-40发酵液进行分离纯化，获得纯化漆酶，纯化倍数为14．6，回收率为5．3％。SDS-PAGE银染的结果为单一条带，相对分子质量约为6．53×104。以愈创木酚和2，2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）为催化底物进行酶学性质研究，最适pH分别为4．8和4．5，最适温度分别为55和50℃，2种底物在pH4．0。5．0范围内，温度低于50℃时，酶的稳定性都很好。以愈创木酚为底物，Km=645．0umol／L；以ABTS为底物，Km=22．2txmol／L。Cu2＋对该酶起激活作用，Fe2＋、Ca2＋、Ba2＋则完全抑制酶的活性。

Purification and properties of laccase from Ganoderma EIM-40

After the crude laccase from Ganoderma sp. EIM40 was purified using lyophilization, fraction- al ammonium sulfate precipitation, HiTrap phenyl （FF） hydrophobic chromatography and Q Sepharose Fast Flow Ion-exchange chromatography, a final yield of 5.3% and a specific activity of 14.6-fold were achieved. SDS-PAGE analysis by silver staining revealed a single band with molecular weight of 6.53 × 104. The optimal reaction conditions of the laccase toward Guaiacol and ABTS as substrate in- clude 55 ℃ and 50 ℃ of reaction temperature and pH d. 8 and pH 4.5. When the temperature was below 50 ℃ and the pH was in the range of 4. 0 to 5.0, the laccase exhibited excellent stability in the two sub- strates. The Km for oxidizing guaiacol and ABTS were 645.0 and 22.2 umol/L, respectively. Laccase could be enhanced by Cu2＋ and inhibited by Fe2＋ , Ca2＋ , and Ba2＋.