RESUMEN
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 %.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Thermotoga maritima/enzimología , Aldehído Reductasa , Aldo-Ceto Reductasas , Estabilidad de Enzimas , Cinética , Estereoisomerismo , Especificidad por Sustrato , TemperaturaRESUMEN
The gene encoding a novel short-chain alcohol dehydrogenase in the thermophilic bacterium, Carboxydothermus hydrogenoformans, was identified and overexpressed in Escherichia coli. The enzyme was thermally stable and displayed the highest activity at 70 °C and pH 6.0. It preferred NAD(H) over NADP(H) as a cofactor and exhibited broad substrate specificity towards aliphatic ketones, cycloalkanones, aromatic ketones, and ketoesters. Furthermore, ethyl benzoylformate was asymmetrically reduced by the purified enzyme, using an additional coupled NADH regeneration system, with 95 % conversion and in an enantiomeric excess of (99.9 %). The results of this study may lead to the discovery of a novel method for asymmetric reduction of alcohols, which is an important tool in organic synthesis.
Asunto(s)
Alcohol Deshidrogenasa/aislamiento & purificación , Alcohol Deshidrogenasa/metabolismo , Bacterias Grampositivas/enzimología , Bacterias Grampositivas/metabolismo , Alcohol Deshidrogenasa/genética , Clonación Molecular , Coenzimas/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Bacterias Grampositivas/genética , Concentración de Iones de Hidrógeno , NAD/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
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.