Direct enzymatic reduction

FIGURE 2.11 Schematic diagram of the separation-free immunosensor principle for pesticides (H2O2 detected at + 50 mV vs Ag/AgCl via direct enzymatic reduction of HRP) (adapted from [98]). 

Anaerobic bio-reduction of azo dye is a nonspecific and presumably extracellular process and comprises of three different mechanisms by researchers (Fig. 1), including the direct enzymatic reduction, indirect/mediated reduction, and chemical reduction. A direct enzymatic reaction or a mediated/indirect reaction is catalyzed by biologically regenerated enzyme cofactors or other electron carriers. Moreover, azo dye chemical reduction can result from purely chemical reactions with biogenic bulk reductants like sulfide. These azo dye reduction mechanisms have been shown to be greatly accelerated by the addition of many redox-mediating compounds, such as anthraquinone-sulfonate (AQS) and anthraquinone-disulfonate (AQDS) [13-15],... 

As discussed earlier, Azo biological decolorization are mainly reduced in a direct reduction or mediated/indirect reduction with nonspecial azo reductase or reduced enzyme cofactors (Figs. 1 and 3). According to the direct enzymatic reduction mechanism, nonspecial azo reductase can catalyze the transfer of reducing equivalents originating from the oxidation of original electron donor in the azo dyes. In... 

The acceleration mechanism of redox mediators are presumed by van der Zee [15]. Redox mediators as reductase or coenzymes catalyze reactions by lowering the activation energy of the total reaction. Redox mediators, for example, artificial redox mediators such as AQDS, can accelerate both direct enzymatic reduction and mediated/indirect biological azo dye reduction (Fig. 3). In the case of direct enzymatic azo dye reduction, the accelerating effect of redox mediator will be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the azo dye. Possibly, both reactions will be catalyzed by the same nonspecific periplasmic enzymes. In the case of azo dye reduction by reduced enzyme cofactors, the accelerating effect of redox mediator will either be due to an electron shuttle between the reduced enzyme cofactor and redox mediator or be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the coenzymes. In the latter case, the addition of redox mediator simply increases the pool of electron carriers. 

Afanas ev, I.B., Ostrakhovitch, E.A., Mikhal chik, E.V Korkina, L.G. (2001). Direct enzymatic reduction of lucigenin decreases lucigenin-ampMed chemiluminescence produced by superoxide ion luminescence. Journal of Biological and Chemical Luminescence, 16, 305-307. 

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... 

The active form of the catalyst must transfer the electrons or the hydride ion to NAD(P)+, but not directly to the substrate. Otherwise, this non-enzymatic reduction will lead to low chemoselectivity and/or low enantioselectivity. 

The same authors proposed a complex system for the electrochemically driven enzymatic reduction of carbon dioxide to form methanol. In this case, methyl viologen or the cofactor PQQ were used as mediators for the electroenzymatic reduction of carbon dioxide to formic acid catalyzed by formate dehydrogenase followed by the electrochemically driven enzymatic reduction of formate to methanol catalyzed by a PQQ-dependent alcohol dehydrogenase. With methyl viologen as mediator, the reaction goes through the intermediate formation of formaldehyde while with PQQ, methanol is formed directly [77],... 

Fig. 3.105. HPLC chromatograms for enzymatic reduction, (a) Xylidine ponceau-2R (1 = 2,4-xyli-dine, 2 = 2,6-xylidine, 3 = 2,4,5-trimethylaniline). (b) Direct black-38 (1 = benzidine, 2 = 4-aminophenyl). (c) Direct brown-1 (1 = benzidine, 2 = 4-aminobiphenyl). Conditions mobile phase, acetonitrile (A) and water (B) flow rate, 0.7 ml/min 25°C injection volume, 10 p gradient elution 0 min, A 23 per cent, B 77 per cent 0-21 min, A 34 per cent and B 66 per cent 21-30 min, A 60 per cent and B 40 per cent 30-34 min, A 70 per cent and B 30 per cent 34-37 min, A 90 per cent and B 10 per cent and 37—40 min, A 23 per cent and B 77 per cent. Detection at 280 nm. Reprinted with permission from M. Bhaskar et al. [159].

Electro-generated and regenerated bis(bipyridine)rhodium(I) complexes were able to catalyze the selective non-enzymatically coupled electrochemical generation of NADH from NAD . The direct cathodic reduction even at very negative working potentials leads to the formation of large amounts of enzymatically inactive NAD dimers, while the indirect electrochemical reduction via the rhodium complex acting as... 

Formation of the identical sugars of the D-series, 6-deoxymannose (rhamnose) and 6-deoxytalose, seems to proceed by a different pathway. According to Winkler and Markowitz (13), GDP-6-deoxy-D-mannose is first converted to GDP-6-deoxy-D-lyxo-4-hexulose. This 4-keto intermediate is the direct precursor for the unspecific enzymatic reduction leading to GDP-6-deoxy-D-mannose and GDP-6-deoxy-D-talose. For a pyridine-nucleotide requiring enzyme, the transformation seems to be unusual because of its lack of stereospecificity. However, closer examination and evaluation of properties of the different 4-keto-intermediate reductases must await availability of more highly purified enzyme preparations. 

No 3-carboxy-substituted TBCs, derived from L-tryptophan by the Pic-tet-Spengler route, have yet been isolated from mammalian tissues. The same is also true for the dicarboxylic acid 23a derived from the condensation of L-tryptophan with pyruvic acid (36). The 1-carboxy-substituted TBCs 37 and 38, on the other hand, occur in mammalian systems (70,71) and are metabolically decarboxylated (65,S5). Whether a direct enzymatic decarboxylation of racemic material, occurring with the (S) and (R) enantiomers at a different rate, could account for the formation of unequal amounts of the enantiomers of TBC has not been investigated so far. The pyruvic acid route to optically active TBC (Fig. 12) leading from TBC 38a to TBC 29a via DBC 34 is at tifie moment the preferred pathway (85,86,89), although the enzymes involved in the asymmetric reduction leading to TBC 29a and the hydroxylated metabolites TBCs 30a and 33a have been neither isolated nor characterized. 

An actual contribution of humic substances to metal oxide reduction in natural systems has not been demonstrated, and there are processes such as adsorption or decomposition that could limit their effectiveness. Kostka et al. (2002a) observed that AQDS additions elicited a larger increase in Fe(III) reduction by S. oneidensis growing on ferrihydrite than smectite clay minerals. This suggests that the influence of humic substances may depend on soil or sediment mineralogy. Nevertheless, there is ample evidence to suggest that a portion of the anaerobic metabolism that was previously attributed to direct enzymatic Fe(III) and Mn(IV) reduction was actually none-nzymatic reduction by microbially reduced humic substances. 

A second method for separating enzymatic and nonenzymatic Fe(III) reduction by H2S is to block S04 reduction with molybdate (Mo04 ). The technique has been used effectively to demonstrate the importance of enzymatic reduction in marine and freshwater sediments (Section 8.08.6.4.4). As with all inhibitor techniques, there is the possibility that molybdate additions directly or indirectly affect processes other than S04 reduction. For example, it could overestimate biotic Fe(III) reduction if the enzymatic process was stimulated by a cessation of competition with H2S for Fe(III) substrates, or underestimate if S04 reduction was not completely blocked. Despite these potential limitations, the molybdate method produces patterns that are consistent with other types of geochemical data, and it is therefore widely used. 

In addition, technetium may be fixed by bacteri-aUy mediated reduction and precipitation. Several types of Fe(III)- and sulfate-reducing bacteria have been shown to reduce technetium, either directly (enzymatically) or indirectly through reaction with microbially produced Fe(II), native sulfur, or sulfide (Lyalikova and Khizhnyak, 1996 Lloyd and Macaskie, 1996 Lloyd et al, 2002). 

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