Sei-Eok Yun

Electrochemical evaluation of the reaction rate between methyl viologen mediator and diaphorase enzyme for the electrocatalytic reduction of NAD+ and digital simulation for its voltammetric responses

Abstract The electrocatalytic reduction of NAD + using diaphorase enzyme was studied. Methyl viologen was used as an electron transfer mediator between an electrode and the enzyme. A catalytic wave for the reduction of NAD + when all the species were in the solution was measured with cyclic voltammetry at a gold-amalgam electrode which showed low background currents at negative potentials. Steady-state currents could be obtained under the conditions of slow scan rate, low methyl viologen concentration, and high NAD + concentration as the electrode reaction was converted to an electrochemical-catalytic (EC′) reaction. The bimolecular rate constant for the reaction of the reduced methyl viologen with the oxidized diaphorase was estimated as 7.5×10 3 M −1 s −1 from the slope of the current versus [MV 2+ ] plot. Another slope of the current against the square root of the enzyme concentration also gave a close value of 6.7×10 3 M −1 s −1 . In the calculation of the rate constant, the stoichiometric factor when it is not one-to-one was considered. With the evaluated rate constant, digital simulation using the suggested reaction mechanism was compared with the experimentally obtained voltammograms. Satisfactory agreement indicates that the evaluation methods of the rate constant and the suggested mechanism are appropriate for the mediated enzyme-catalyzed electrochemical reactions.

Optimization of the mediated electrocatalytic reduction of NAD+ by cyclic voltammetry and construction of electrochemically driven enzyme bioreactor

The optimal concentrations of diaphorase, methyl viologen (MV2+) and NAD+ in the mediated electrocatalytic reduction of NAD+ were decided by applying cyclic voltammetry. The steady-state catalytic current was achieved under the conditions of 1.5 U diaphorase ml−1, 0.2 mM MV2+, and 4.8 mM NAD+ at the scan rate of 2 mV s−1, suggesting that the rate of the electrocatalytic reaction is the highest under the former conditions. However, NAD+ was effective at 0.3 mM as it was at 4.8 mM when the electrocatalysis is coupled with an enzymatic synthesis requiring NADH. In effect, the electrochemical procedure under the conditions of 1.5 U diaphorase ml−1, 0.2 mM MV2+, and 0.3 mM NAD+ worked satisfactorily to drive an enzymatic reduction of pyruvate to d-lactate in the presence of d-lactate dehydrogenase.

The formation of a diaphorase enzyme multilayer bound to a self-assembled monolayer for the mediated enzyme-catalyzed reduction of NAD+

Abstract A diaphorase enzyme multilayer was constructed over an aminoethanethiol self-assembled monolayer on a gold electrode for the enzyme-catalyzed reduction of NAD + . Methyl viologen dissolved in a solution was used as a mediator to transfer electrons between an electrode surface and the immobilized enzyme, and the reduced enzyme consequently reduced the NAD + substrates. As the layers of the enzyme were assembled, stepwise enhancements of the catalytic reduction currents were observed in the cyclic voltammetric measurements. It is supposed that more enzyme was accumulated with the multilayer formation than with a monolayer and the rate of reaction between the mediator and the enzyme was increased. The height of the catalytic wave could be controlled by the number of enzyme layers and the multilayered-enzyme electrode is considered to be used for an efficient catalytic NAD + reduction. The concentration of glutaraldehyde used as a crosslinking reagent for the attachment of the enzyme layer affected the current heights and its density around the enzyme seemed to be related to the catalytic reactivity. An optimal temperature maintained during the voltammetric measurements was necessary to obtain a better catalytic reduction wave.

ELECTRIC FIELD EFFECTS WITH ALCOHOL DEHYDROGENASE

Extending previous work with electrostimulation of yeast dehydrogenases in cell suspension, we attempted to influence the alcohol dehydrogenase system in vitro using methylene blue as the acceptor. According to the conformational coupling model and the free radical hypothesis, the possibility of capacitive current treatment was tested by short applications of E = 10, 20, and 30 V/cm at a frequency of 50 Hz. Besides a temperature rise, no significant kinetic data from spectrophotometry or polarography could be measured, nor could inductive coupling using magnetic fluxes of 1, 5, and 10 mT. On the other hand, marked changes of relative electrofusion yield were found after interaction of dehydrogenases with membrane surfaces of barley protoplasts. It was confirmed that in unbuffered solution containing 0.5 M mannitol, the isoelectric point (PI) of enzymes determines the relative fusion yield: Fr 1 for pI > 7. We must conclude that only the position of dehydrogenases at or within cell ...

The role of immobilized rennet on carbon cloth in flavor development during ripening of Gouda cheese

Rennet-free Gouda (RFG) cheese was prepared to investigate the influence of rennet on the non-volatile and volatile profiles of cheese and was characterized by HPLC and GC/MS analyses. Chymosin, a major protease in rennet, was immobilized onto oxidized and chemically modified carbon cloth. The chymosin immobilization efficiency was 60.4%, and the milk-clotting activity used as an index of the stability of the immobilized chymosin decreased by around 20% in 2 weeks. However, the activity was maintained at 70–80% from 2 weeks to 32 weeks and was more stable than that of chymosin solution alone. Non-volatile (organic acids) and volatile profiles of the RFG cheese and rennet-containing normal Gouda cheese were not significantly different during ripening with a few exceptions. Therefore, it can be concluded that cheese flavor is developed by lactic acid fermentation, irrespective of the presence of rennet.