Electrochemical enzyme regeneration

Hydrogen peroxide can be avoided if excess electrons are transferred to the anode. However, direct electron transfer between enzymes and solid electrodes is usually very slow because the etixyorahc active sites am often deeply buried within the protein shell and. therefore inaccessible far the electrode (the lunnehttg probability of electrons is a function of distance), in order to accelerate die electron transfer, low molecular weight redox active substances can be used to shuttle the electrons between the enzyme and the electrode. This indirect electrochemical enzyme regeneration is represented, schematically m Figure 16.2-24. 

Figure 3. Principle of an indirect electrochemical cofactor or enzyme regeneration system for an enzymatic reduction process.

Figure 13. Continuous formation of (S )-4-phenyl-2-butanol from 4-phenyl-2-butanone using the electrochemical enzyme membrane reactor under indirect electrochemical NADH regeneration with a high-molecular-weight rhodium catalyst [26,29,30,65].

The following data for the production of chiral y-lactones from me o-diols using the indirect electrochemical NAD" regeneration procedure can be given In a volume of 100 mL using 3.2 mg (1 x 10 mol) of PDMe, 70mg (1 x 10 mol)ofNAD, and 12.5mg(16U)of HLADH, after 20 h 99.5% of the meso-diol (0.5 to 1.0 g = 3.5 to 7.0 mmol) were converted to the enentiomerically pure lactone. Thus, the space-time yield can be calculated to be 6 to 12 g/L -day . The productivity is limited by the small amount of the enzyme. 

In this study, polymerized NADP and polymerized viologen derivatives were also prepared in a similar manner. We investigated their applications to conjugated redox reaction coupled with NAD(P)-linked enzyme reaction and with electrochemical coenzyme regeneration reaction. 

On the other hand, the biocatalytic epoxidation of styrene and derivatives can be achieved with excellent stereoselectivity using SMOs of various origins (Scheme 13.9). Although isolated SMO has been successfully applied in combination with enzymatic NADH regeneration [92,97] or reductive electrochemical cofactor regeneration [98-101], the process based on E. coli whole cells expressing those SMOs has been proven superior in terms of productivity due to the limited stability of cell-free enzymes, and consequently it has been applied in the majority of reported studies. 

It is difficult to incorporate dehydrogenases that are coupled with NAD(P) into amperometric enzyme sensors owing to the irreversible electrochemical reaction of NAD. We have developed an amperometric dehydrogenase sensor for ethanol in which NAD is electrochemically regenerated within a membrane matrix. 

An immobilized-enzyme continuous-flow reactor incorporating a continuous direct electrochemical regeneration of NAD + has been proposed. To retain the low molecular weight cofactor NADH/NAD+ within the reaction system, special hollow fibers (Dow ultrafilter UFb/HFU-1) with a molecular weight cut-off of 200 has been used [32],... 

For enzymatic reductions with NAD(P)H-dependent enzymes, the electrochemical regeneration of NAD(P)H always has to be performed by indirect electrochemical methods. Direct electrochemical reduction, which requires high overpotentials, in all cases leads to varying amounts of enzymatically inactive NAD-dimers generated due to the one-electron transfer reaction. One rather complex attempt to circumvent this problem is the combination of the NAD+ reduction by electrogenerated and regenerated potassium amalgam with the electrochemical reoxidation of the enzymatically inactive species, mainly NAD dimers, back to NAD+ [51]. If one-electron... 

Indirect Electrochemical NAD(P)H Regeneration Without a Regeneration Enzyme... 

To be able to regenerate NADP(H) by an indirect electrochemical procedure without the application of a second regeneration enzyme system, the redox catalyst must fulfill four conditions ... 

In MET, a low-molecular-weight, redox-active species, referred to as a mediator, is introduced to shuttle electrons between the enzyme active site and the electrode.In this case, the enzyme catalyzes the oxidation or reduction of the redox mediator. The reverse transformation (regeneration) of the mediator occurs on the electrode surface. The major characteristics of mediator-assisted electron transfer are that (i) the mediator acts as a cosubstrate for the enzymatic reaction and (ii) the electrochemical transformation of the mediator on the electrode has to be reversible. In these systems, the catalytic process involves enzymatic transformations of both the first substrate (fuel or oxidant) and the second substrate (mediator). The mediator is regenerated at the electrode surface, preferably at low overvoltage. The enzymatic reaction and the electrode reaction can be considered as separate yet coupled. 

The half-wave potential for the electrochemical oxidation of NADH to NAD is ca. -bO.6 V vj. SCE at pH 7. The formal potential for the NADH/NAD couple, however, is only —0.56 V. The overpotential therefore is about 1.2 V. As NAD acts as coenzyme in many enzyme-catalyzed oxidations of practical importance, it would be of interest to regenerate NAD electrochemically. For this purpose it is necessary to find a mediator system which is able to lower the overpotential. Mediator systems accepting two electrons or a hydride atom are most effective. Therefore, dopaquinone electro-generated from dopamine 2" and quinone diimines derived from diaminobenzenes applied successfully. 

The numerical values for ki. .. k4 vary with RG. For instance, for RG = 10, the following values provide the analytical function Jfei = 0.40472, k2 = 1.60185, k3 = 0.58819, and k4 = -2.37294 [12]. The analytical approximations for hindered diffusion provide a way to determine d from experimental approach curves. For this purpose, one can use an irreversible reaction at the UME (often 02 reduction). In such a case, Fig. 37.2, curve 1 is obtained irrespective of the nature of the sample. Besides the mediator flux from the solution bulk, there might be a heterogeneous reaction at the sample surface during which the UME-generated species O is recycled to the mediator R. The regeneration process of the mediator might be (i) an electrochemical reaction (if the sample is an electrode itself) [9], (ii) an oxidation of the sample surface (if the sample is an insulator or semiconductor) [14], or (iii) the consumption of O as an electron acceptor in a reaction catalyzed by enzymes or other catalysts immobilized at the sample surface [15]. All these processes will increase (t above the values in curve 1 of Fig. 37.2. How much iT increases, depends on the kinetics of the reaction at the sample. If the reaction of the sample occurs with a rate that is controlled by the diffusion of O towards the sample, Fig. 37.2, curve 2 is recorded. If the sample is an electrode itself, such a curve is experimentally obtained if the sample potential... 

Ketoreductases (KREDs) are dependent on nicotinamide cofactors NADH or NADPH. Due to the reaction mechanism, these rather costly cofactors are needed in stoichiometric amounts, disclosing an economic problem that has to be dealt with when using these enzymes. Many different possibilities for cofactor recycling have been established with three major approaches finding application in research and industry (Fig. 13). Further regeneration systems, such as electrochemical methods, are not discussed within this review [22-24, 37, 106-108],... 

---