Characterization of an aldo-keto reductase from Thermotoga maritima with high thermostability and a broad substrate spectrum.

A novel aldo-keto reductase gene, Tm1743, from Thermotoga maritima was overexpressed in Escherichia coli. The enzyme displayed the highest activity at 90 °C and at pH 9. It retained 63 % of its activity after 15 h at 85 °C. The enzyme also could tolerate (up to 10 % v/v) acetonitrile, ethanol and 2-propanol with slightly increased activities. Methanol, DMSO and acetone decreased activity slightly. Furthermore, Tm1743 exhibited broad substrate specificity towards various keto esters, ketones and aldehydes, with relative activities ranging from 2 to 460 % compared to the control. Its optimum substrate, 2,2,2-trifluoroacetophenone, was asymmetrically reduced in a coupled NADPH-regeneration system with an enantioselectivity of 99.8 % and a conversion of 98 %.

Development of a whole-cell biocatalyst with NADPH regeneration system for biosulfoxidation.

A formate dehydrogenase gene (fdh) originated from Candida boidinii was co-expressed in E. coli BL21 (DE3) with the cyclohexanone monooxygenase gene (chmo) cloned from Acinetobacter calcoaceticus NCIMB 9871. The co-expression system was then used as a whole-cell biocatalyst to synthesize chiral phenyl methyl sulfoxide (PMSO) from thioanisole (PMS) and the reaction conditions were investigated. When the initial concentration of PMS was 20 mM, the specific productivity of PMSO in this system was 2.07 µmol g(-1) cw min(-1) (cw: wet cell weight) and the ee value for the R-sulfoxide was 99 %. In contrast, when chmo was the only gene expressed in E. coli, the specific productivity of PMSO was 0.053 µmol g(-1) cw min(-1) with no exact enantioselectivity. Further determination of NADPH concentration in the whole-cell catalysts suggested that co-expression of fdh with chmo significantly improved NADPH supply. Thus, this whole-cell biocatalyst system is highly advantageous for the synthesis of optically pure R-sulfoxide.