毛云芝菌固体发酵产漆酶的分离纯化及酶学性质

Purification and Properties of Laccase Produced by Coriolus hirsutus in Solid State Fermentation

摘要:

作者对毛云芝菌利用桑叶和麸皮固体发酵所产漆酶进行了分离纯化,并研究了纯化后漆酶的酶学性质。将毛云芝菌固体发酵漆酶粗提液通过离心、过滤、大孔树脂脱色、硫酸铵分级盐析、DEAE-Sepharose fast flow层析、Sephadex G-100凝胶过滤层析,结果得到电泳纯的漆酶,纯化倍数15.53,酶活回收率51.21%。用SDS-PAGE测得该酶相对分子质量62 600;该酶反应的最适温度为50℃,在柠檬酸-柠檬酸钠缓冲液中最适pH值为4.0,在醋酸缓冲液中最适pH值3.0;氧化ABTS的Km为0.093 mmol/L,对邻苯二酚的Km为3.71 mmol/L。

Abstract:

In this paper,purification and properties of laccase produced by Coriolus hirsutus in solid state fermentation were studied.Crude extract of laccase from Coriolus hirsutus was collected after solid state fermentation.The laccase was purified to electrophoretic homogeneity by the serials of steps of centrifugation,filtration,ammonium sulfate precipitation,ion-exchange chromatography on DEAE-Sepharose fast flow and gel filtration on sephadex G-100.Purification of 15.53 fold was achieved with an overall yield of 51.21%.Its molecular weight was estimated to be about 62 600 by SDS-PAGE.Studies on the properties of the laccase showed that the optimum temperature was 50 ℃ and the optimum pH were 4.0 in citrate buffer and 3.0 in sodium acetate buffer.The laccase catalyzed the oxidation of ABTS and catechol with a michaelis constant of 0.093 mmol/L and 3.71 mmol/L,respectively.