Laboratory of Biocatalysis and Bioprocessing, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
J Biotechnol. 2010 Oct 1;150(1):108-14. doi: 10.1016/j.jbiotec.2010.07.007. Epub 2010 Jul 16.
A gene encoding an esterase of Bacillus subtilis ECU0554 previously isolated from soil was cloned and overexpressed in Escherichia coli BL21. The recombinant esterase (recBsE) showed the best enantioselectivity (E>100) towards DL-menthyl acetate, in contrast to DL-menthyl esters propionate and butyrate. A high ratio of substrate to catalyst (S/C-ratio, ≥50) was achieved in the kinetic resolution of DL-menthyl acetate by using whole cells of recombinant E. coli BL21. Some key parameters of the biocatalytic process, including amount of cosolvent, catalyst loading and substrate loading, were optimized. Compared with the process catalyzed by wild-type whole cells of B. subtilis ECU0554, the second-generation bioprocess using whole cells of recombinant E. coli BL21 afforded a 40-fold improvement in S/C-ratio and a 75-fold improvement in the volumetric productivity per biocatalyst loading. Moreover, the substrate loading was increased up to 200 g L(-1) (∼1 M), the biocatalyst loading was reduced to 2.5 g L(-1) and the space-time yield was improved from 54 g L(-1) d(-1) to 202 g L(-1) d(-1).
先前从土壤中分离出的枯草芽孢杆菌 ECU0554 的酯酶基因被克隆并在大肠杆菌 BL21 中过表达。与 DL-薄荷基丙酸酯和丁酸酯相比，重组酯酶（recBsE）对 DL-薄荷基乙酸酯表现出最好的对映选择性（E>100）。在使用重组大肠杆菌 BL21 全细胞动力学拆分 DL-薄荷基乙酸酯时，实现了高的底物与催化剂比（S/C-比，≥50）。优化了生物催化过程的一些关键参数，包括共溶剂用量、催化剂用量和底物用量。与枯草芽孢杆菌 ECU0554 野生型全细胞催化过程相比，使用重组大肠杆菌 BL21 全细胞的第二代生物过程的 S/C-比提高了 40 倍，每单位生物催化剂的体积产率提高了 75 倍。此外，底物浓度可提高至 200 g L(-1)（约 1 M），生物催化剂用量减少至 2.5 g L(-1)，时空产率从 54 g L(-1) d(-1)提高到 202 g L(-1) d(-1)。
