摘要
首次发现野生革耳菌 (Panusrudis)在 39℃液体振荡培养条件下 ,不需诱导剂的诱导 ,能持续产生漆酶 .优化后培养基的碳氮源分别为 2 %蔗糖和 2 6mM酵母抽提物 ,最高酶活达 1 .6× 1 0 4U/L ,属于目前已知的组成型漆酶高产菌株 .发酵液经硫酸铵沉淀 ,DEAE Sepharose ,SephacrylS 2 0 0 ,MonoQ柱层析分离纯化后 ,可得电泳纯漆酶蛋白 .该酶为单体蛋白 ,分子量为 58kDa .以愈创木酚为底物 ,该酶的最适作用pH为4 5,最适反应温度为 50℃ ,Km值为 0 .2 1mM ,并表现出较好的热稳定性 。
The laccase of Panus rudis was produced constitutively in shaking flask culture without induction. The optimum carbon source and nitrogen source were 2% sucrose and 26mM yeast extract respectively. The maximum enzyme activity obtained was 1.6×10 4U/L. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE Sephrose, Sephacryl S 200 and Mono Q chromatography. The enzyme is a monomer with the molecular mass of 58kDa on SDS PAGE. With guaiacol as the substrate, the enzyme optimal pH is 4.5, the optimal temperature is 50°C, and the Km is 0.21mM. Panus rudis laccase show good thermostability and is valuable for further studies on laccase catalytic mechanisms and industry applications.
基金
中国科学院"百人计划"
安徽省自然科学基金资助项目 (982 12 4 11)
安徽省教育厅自然基金重点资助项目 (2 0 0 0J10 13)
