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

Cloning, heterologous expression and characterization of a ferulic acid esterase gene from thermophilic Clostridium thermocellum

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 °C，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。

Abstract:

[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 °C. 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.