Mediator-coupled enzyme systems

As previously mentioned, the ideal biocatalysts for the regeneration of NAD(P) are NOXs require exclusively the molecular oxygen provided by the atmosphere to perform the re-oxidation of nicotinamide cofactors. 

Nevertheless, various novel approaches have been suggested during the last years where the in situ oxidation of the NAD(P)H cofactors is promoted by the concerted action of an enzymatic activity and a suitable redox mediator. 

Actually, the use of dyes, quinones, or metal ions as redox mediators for the preparative recycling of NAD(P) cofactors was first investigated by Lee and Whitesides [71] 30 years ago. Diaphorase was shown to increase the reaction rate between NADH and a mediator, but the rate-limiting step was the re-oxidation of the mediator by O2, which resulted in overall reaction rates that were significantly lower than those achieved with conventional enzymatic systems. 

More recently, it was shown that such Umitations can be overcome either by the choice of a more suitable redox mediator or by the concomitant exploitation of different types of enzymatic activities, such as laccases. 

Subsequentiy, new developments have been achieved by combining the use of mediators with the exploitation of the laccase enzymes (EC 1.10.3.2), which allow a fast re-oxidation by utilizing oxygen as a terminal electron acceptor. A proof-of-prindple demonstration for such systems was given by the Arends group in 2009 

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Biomechanical straw pulps treated with enzyme mediator systems have been studied using mid-lR spectroscopy [108]. IR spectra indicated that a manganese-based enzyme system in combination with hydrogen peroxide generated the glucose oxidase couple, which removed aromatic ring structures. 

D-Glucose oxidase has been immobilized for use in a number of analytical systems. Immobilization of the enzyme onto nonporous glassy carbon electrodes by carbodi-imide-mediated coupling to superficial oxides generated by anodic oxidation has afforded an immobilized enzyme electrode with which hydrogen peroxide released enzymically from D-glucose may be measured amperometri-cally. Properties of the enzyme immobilized on amino-organo-sylochrome have been studied. 

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