嗜热细菌Clostridium thermocellum阿魏酸酯酶基因的克隆、异源表达及酶学特性分析

摘要

[目的]阐明嗜热细菌Clostridium thermocellum XynZ蛋白的阿魏酸酯酶催化域的酶学特性,为其在生物质能源及其它发酵工业中的应用奠定基础.[方法]分别构建了C.thermocellum XynZ的阿魏酸酯酶催化域(FAE)及该阿魏酸酯酶催化域和碳水化合物结合域(FAE-CBM6)编码基因的原核表达载体,并在大肠杆菌菌株BL21(DE3)中异源表达,在此基础上分析比较了温度、pH、底物、金属离子及CBM6结合域对阿魏酸酯酶活性的影响.[结果]重组FAE酶及FAE-CBM6酶发挥催化活性的适宜pH值为5.0-9.0,适宜温度为50-70℃,它们对不同金属离子的响应有差异.[结论]在同一反应条件下,FAE-CBM6酶的酶活均比FAE高,说明CBM6结合域的存在对于阿魏酸酯酶活性有促进作用.%[Objective] To elucidate the characteristics of a ferulic acid esterase catalytic domain within a XynZ protein from thermophilic Clostridium thermocellum to provide a basis for its application in bioenergy and other fermentation industries.[Methods] A prokaryotic expression vector for the ferulic acid esterase catalytic domain (FAE) within XynZ protein as well as for this catalytic domain adding an extra carbohydrate binding module 6 (FAE-CBM6) was constructed and expressed in E.coli BL21(DE3) cells,respectively.Then,the effects of temperature,pH,substrates,metal ions and the CBM6 module on the catalytic activity of ferulic acid esterase were estimated.[Results] The favorable pH for optimal catalytic activity of recombinant FAE or FAE-CBM6 protein ranged from 5.0 to 9.0,and the favorable temperatures being 50-70 ℃.Furthermore,different metal ion had a positive or negative effect on catalytic activity of these two recombinant proteins.[Conclusion] Under the same reaction condition,FAE-CBM6 usually produced a higher catalytic activity than FAE,indicating that the CBM6 module plays a key role in improving the ferulic acid esterase activity.