摘要
采用硫酸铵盐析、DEAE纤维素柱层析、PhenylSepharoseTM6FastFlow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 31 8.4,活力回收率为 1 8.6%。用SDS PAGE测得该酶分子量为 60 .3kD ,而经质谱分析为 5 5 .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 4.0 2 ,其N末端序列为AIGPVTDL ,用硫酸 酚法测得其含糖量为 49.2 %。 2 5℃条件下 ,以ABTS( 2 ,2’ azinobis ( 3 ethylbenzthiazoline 6 sulphonate)为底物的Km为 1 7.5 μmol L。该酶在 45℃ ,pH3.0～ 9.5较稳定。Cu2 + 对酶活有明显的促进作用 ,Fe2 + 完全抑制酶的活性 ,Mn2 + 和Ag+ 对酶活无明显影响。DTT(Dithiothreitol,二硫苏糖醇 )和NaN3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。
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 60.3kD by SDS-PAGE,and that of it was 55.94kD by mass spectrum.The optimum temperature and pH of the enzyme activity were 65℃ 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 17.5μmol/L.The enzyme activity was stable under 45℃ and in the pH range of 3.0～9.5.The activity was enhanced by Cu 2+,and was strongly inhibited by Fe 2+.While Mn 2+and Ag+ had no effect on laccase activity.Dithiothreitol and sodium azide inhibited completely the activity.Trp was possible essential residue for enzyme activity.
出处
《微生物学报》
CAS
CSCD
北大核心
2003年第1期73-78,共6页
Acta Microbiologica Sinica
基金
国家"863"项目资助 ( 2 0 0 1AA2 1 4 1 61 )
关键词
担子菌
漆酶
分离纯化
性质
Basidiomycete,Laccase,Purification,Properties
