Enantioselective hydrolysis of dl-menthyl benzoate by cell-free extract of newly isolated Acinetobacter sp. ECU2040.

Production of l-menthol by bioprocesses attracts increasing attention nowadays. Herein, we attempted to develop a bioresolution process for production of l-menthol through enantioselective hydrolysis of dl-menthyl benzoate using a newly isolated bacterium from soil samples. Among 129 active soil isolates screened rapidly by thin-layer chromatography, an outstanding bacterial strain numbered ECU2040, which was subsequently identified as Acinetobacter species, was finally selected as our target enzyme producer due to its highest activity and the best enantioselectivity toward l-substrate as confirmed by chiral gas chromatography. The catalytic performance of the cell-free extract from Acinetobacter sp. ECU2040 was preliminarily examined, indicating that its optimal pH and temperature for the reaction were 7.5 and 37 °C, respectively. Under the optimal conditions, the enzymatic reaction was performed on a 1-L scale, affording l-menthol in 48 % yield and 71 % ee.

An efficient bioprocess for enzymatic production of L-menthol with high ratio of substrate to catalyst using whole cells of recombinant E. coli.

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).