Kinetic study and modeling of mercuric reduction by recombinant microorganisms

The kinetics and modeling of complex biocatalytic processes, which involve several polypeptides and require bioenergy and cofactors, were investigated. The reduction of {dollar}rm Hgsp{lcub}2+{rcub}{dollar} by recombinant Escherichia coli, encoded by the plasmid-borne mer operon, served as model system. It is carried out by the simultaneous action of three proteins, which transport {dollar}rm Hgsp{lcub}2+{rcub}{dollar} across the cellular envelope into the cytoplasm, where mercuric reductase catalyzes the reduction of {dollar}rm Hgsp{lcub}2+{rcub}{dollar} to elemental mercury, using NADPH as electron donor and FAD as cofactor.; The reduction process was analyzed into its two components, the transport system and the enzymatic reaction, and a mathematical model was developed for each of them. The parameters of the models were determined using experimental data. Intact and permeabilized cells were used to measure the {dollar}rm Hgsp{lcub}2+{rcub}{dollar} transport and reduction rate, respectively. The kinetic data confirmed the validity of the model and identified the transport system as the rate-determining step of the overall {dollar}rm Hgsp{lcub}2+{rcub}{dollar} reduction process.; Based on the predictions of the model, optimization of the {dollar}rm Hgsp{lcub}2+{rcub}{dollar} reduction rate was pursued by cloning the mer operon onto plasmids maintained at 3 to 140 copies per cell. The 47-fold overall gene amplification led to a linear 7-fold increase of the reaction rate and a similar increase of the mercuric reductase concentration. In contrast, the transport rate increased only 2.4-fold, paralleling the increase of the transport protein concentration. Evaluation of those results indicated that optimization of the biocatalytic rate requires selective amplification of the transport protein expression to increase the intracellular {dollar}rm Hgsp{lcub}2+{rcub}{dollar} concentration to optimal, subinhibitory levels and allows mercuric reductase to function at higher turnover rates.