Overcoming the thermodynamic limitation in asymmetric hydrogen transfer reactions catalyzed by whole cells

Abstract

Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness.

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

“…For efficient utilization of biocatalysts the consumed amount of cofactors has to be regenerated sufficiently during the biotransformation process (Wandrey, 2004). Different strategies for cofactor regeneration were developed for oxidoreductase catalyzed whole cell biotransformation processes making use of glucose metabolism or cofactor regeneration by applying a second enzymatic reaction (Eckstein et al, 2004;Faber, 2000;Goldberg et al, 2006Goldberg et al, , 2007aWichmann and Vasic-Racki, 2005). For the enzyme-mediated cofactor regeneration two different approaches are established: the enzyme-coupled and the substrate-coupled cofactor regeneration.…”

Cloning and Overexpression of an NADH-Dependent Alcohol Dehydrogenase Gene fromCandida marisInvolved in (R)-Selective Reduction of 5-Acetylfuro[2,3-c]pyridine

“…Movement of the equilibrium point of the reaction can be overcome by removal of the acetone produced in the reaction mixture. Several procedures for this are possible, including continuous evaporation of it in vacuo, 33,34) but these operations are not easy on an industrial scale.…”

A highly efficient ADH‐coupled NADH‐recycling system for the asymmetric bioreduction of carbon‐carbon double bonds using enoate reductases

“…For ketone reduction employing 2-propanol as hydrogen donor a stripping process has been reported to remove the formed most volatile compound (acetone) to overcome the thermodynamic limitation and thereby enhancing the conversion (Goldberg et al, 2006). In order to get a deeper insight into the position of the equilibrium of the herein reported system, the Gibbs free energy (DG) for the redox reaction methyl-vinyl ketone þ 2-propanol !…”