Laymon R A, Adney W S, Mohagheghi A, Himmel M E, Thomas S R
Applied Biological Sciences Branch, Alternative Fuels Division, National Renewable Energy Laboratory, Golden, CO 80401, USA.
Appl Biochem Biotechnol. 1996 Spring;57-58:389-97. doi: 10.1007/978-1-4612-0223-3_35.
The process of converting lignocellulosic biomass to ethanol via fermentation depends on developing economic sources of cellulases. Trichoderma reesei cellobiohydrolase (CBH) I is a key enzyme in the fungal cellulase system; however, specific process application requirements make modification of the enzyme by site-directed mutagenesis (SDM) an attractive goal. To undertake SDM investigations, an efficient, cellulase-free host is required. To test the potential of Escherichia coli as a host, T. reesei CBH I cDNA was expressed in E. coli strain GI 724 as a C-terminal fusion to thermostable thioredoxin protein. Full-length expression of CBH I was subsequently verified by molecular weight, Western blot analysis, and activity on soluble substrates.
通过发酵将木质纤维素生物质转化为乙醇的过程取决于开发经济的纤维素酶来源。里氏木霉纤维二糖水解酶（CBH）I是真菌纤维素酶系统中的一种关键酶；然而，特定的工艺应用要求使得通过定点诱变（SDM）对该酶进行修饰成为一个有吸引力的目标。为了进行SDM研究，需要一个高效的、无纤维素酶的宿主。为了测试大肠杆菌作为宿主的潜力，里氏木霉CBH I cDNA在大肠杆菌菌株GI 724中作为与热稳定硫氧还蛋白的C端融合蛋白进行表达。随后通过分子量、蛋白质免疫印迹分析以及对可溶性底物的活性验证了CBH I的全长表达。
