担子菌漆酶的分离纯化及其性质研究

采用硫酸铵盐析、DEAE纤维素柱层析、Phenyl SepharoseTM6 Fast Flow疏水柱层析等方法，得到电泳纯的漆酶同工酶Lac1,纯化倍数为318.4，活力回收率为18.6％。用SDSPAGE测得该酶分子量为60.3kD，而经质谱分析为55.94kD。最适反应温度为65℃，最适反应pH值为2.2～2.8，酶的等电点pI（室温）为4.02，其N末端序列为AIGPVTDL，用硫酸酚法测得其含糖量为49.2％。25℃条件下，以ABTS(2,2'azinobis(3ethylbenzthiazoline6sulphonate)为底物的Km为17.5μmol/L。该酶在45℃，pH3.0～9.5较稳定。Cu^2＋对酶活有明显的促进作用，Fe^2＋完全抑制酶的活性，Mn^2＋和Ag^＋对酶活无明显影响。DTT（Dithiothreitol,二硫苏糖醇）和NaN3完全抑制酶的活性。Koshland试剂对漆酶的活力影响比较大，色氨酸可能是酶活力的必需基团。

Purification and Properties of Laccase from Basidiomycete

Laccase produced by Basidiomycete was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation, DEAE-cellulose and hydrophobic interaction column chromatography. Purification of about 318.4 fold was achieved with an overall yield of 18.6%. Its molecular weight was estimated to be about 60.3 kD by SDS-PAGE, and that of it was 55.94 kD by mass spectrum. The optimum temperature and pH of the enzyme activity were 65 degrees C and 2.2 - 2.8 respectively. The isoelectric point was 4.02 (room temperature). Its N-terminal sequence was AIGPVTDL. The carbohydrate content was 49.2% by the phenol-sulfuric acid method. Michaelis constant of the enzyme for ABTS was 17.5 micromol/L. The enzyme activity was stable under 45 degrees C and in the pH range of 3.0 - 9.5. The activity was enhanced by Cu2+, and was strongly inhibited by Fe2+. While Mn2+ and Ag+ had no effect on laccase activity. Dithiothreitol and sodium azide inhibited completely the activity. Trp was possible essential residue for enzyme activity.