Enzyme/enzymatic redox enzymes

Therefore, for preparative applications of redox enzymes, effective and simple methods for the continuous recycling of the active cofactors have to be available. In addition, such systems must be stable over long time periods and the separation of the product must be simple to render technical processes economically feasible. Until now, this problem has generally been solved by the application of a second enzymatic reaction (enzyme-coupled regeneration, Fig. 2). 

As most enzymes function under compatible ambient conditions, bio-bio cascades had already been successfully developed by the 1970s. By far, most examples have been reported in the field of carbohydrates, using combinations of enzymatic conversions (up to eight enzymes in one-pot), as well as for the in situ cofactor regeneration of enzymatic redox reactions towards amino and hydroxy acids. 

A quantitative model for the electrocatalyitic response of enzymatic amperometric biosensors requires consideration of the diffusion of all the involved species and the kinetics of the redox-enzyme catalytic cyde, as is depicted in Figure 2.27. 

Only a few years ago it was widely accepted that the cofactor regeneration problem represented a serious obstacle with respect to the commercial viability of enzymatic redox processes. Hopefully it is clear from the preceding discussion that there is no longer a cofactor regeneration problem anymore than there is an enzyme problem . The number of readily available enzymes has increased dramatically in the last decade and advances in in vitro evolution have made it possible to routinely optimize the performance of enzymes. The coupling of enzymes in multi-enzyme cascade processes is an attractive way to regenerate cofactors, shift equilibria towards products and remove intermediate products that cause inhibition. Hence, we expect that multi-enzyme cascade processes will become much more common in the future. 

Electroenzymatic reactions are not only important in the development of ampero-metric biosensors. They can also be very valuable for organic synthesis. The enantio- and diasteroselectivity of the redox enzymes can be used effectively for the synthesis of enantiomerically pure compounds, as, for example, in the enantioselective reduction of prochiral carbonyl compounds, or in the enantio-selective, distereoselective, or enantiomer differentiating oxidation of chiral, achiral, or mes< -polyols. The introduction of hydroxy groups into aliphatic and aromatic compounds can be just as interesting. In addition, the regioselectivity of the oxidation of a certain hydroxy function in a polyol by an enzymatic oxidation can be extremely valuable, thus avoiding a sometimes complicated protection-deprotection strategy. 

Nonaqueous enzymatic redox reactions have been limitedby stability owing to solvents and highly reactive substrates (H202). Here we have shown evidence of methods to alleviate these concerns for reactions with CPO. In experimental systems, the in situ production of H202 by GOx was shown to function equally well and more reproducibly than added H202. In situ production is experimentally easier and prevents enzyme deactivation owing to high peroxide levels. GOx was more solvent stable than CPO therefore, the GOx system may be useful for this and other redox systems. 

The metabolically important functions of the Bn-derivatives are directly concerned either with enzymatically controlled organometalhc reactions involving protein-bound adenosylcobamides (such as coenzyme B12, (3)), or methyl-Co -corrinoids (such as methylcobalamin, (4)), or with enzyme-controlled redox reactions. Studies on the underlying biologically relevant organometalhc chemistry of the Bi2-coenzymes in homogeneous (protic) solution, as well as the characterization of the enzymatic processes themselves have attracted considerable interest. ... 

Coupling between a biologically catalyzed reaction and an electrochemical reaction, referred to as bioelectrocatalysis, is the constructional principle for enzyme-based electrochemical biosensors. This means that the flow of electrons from a donor through the enzyme to an acceptor must reach the electrode in order for the corresponding current to be detected. In case a direct electron transfer between the active site of an enzjane and an electrode is not possible, a small molecular redox active species, e.g. hydrophobic ferrocene, meldola blue and menadione as well as hydrophilic ferricyanide, can be used as an electron transfer mediator. This means that the electrons from the active site of the enzyme reduce the mediator molecule, which, in turn, can diffuse to the electrode, where it donates the electrons upon oxidation. When these mediator molecules are employed for coupling of an enzymatic redox reaction to an electrode at a constant potential, the resulting application can be referred to as mediated amperometry or mediated bioelectrocatalysis. 

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