Biocatalytic with oxidases

An additional condition may be imposed, even when a cofactor-independent enzyme is used, if a mediator molecule is involved in the electron transfer process, as is often the case with oxidases. Laccases, for example, may employ small-molecule diffusible mediator compounds in their redox cycle to shuttle electrons between the redox center of the enzyme and the substrate or electrode (Scheme 3.1) [1, 2]. Similarly, certain dehydrogenases utiHze pyrroloquinoline quinone. In biocatalytic systems, mediators based on metal complexes are often used. 

An alternative two-step biocatalytic route, first developed at Glaxo in the 1970s, utilized a D-amino acid oxidase and an amidase to provide 7-ACA under physiological conditions (Scheme 1.12). This process has since been established in several companies, with minor modifications. In fact, 7-ACA was manufactured by GSK at Ulverston (Cumbria, UK) using both the chemical and biocatalytic processes in parallel for a period of 2 years during which time the environmental benefits of the biocatalytic process were assessed (see Section 1.6). 

Examples of surface-immobilized mediators are electropolymerized azines for electro-oxidation of The extreme form of this approach is formation of biocatalytic monolayer, comprising a surface-bound mediator species that is itself bound to a single enzyme molecule. Katz et al. report a complete cell based on novel architecture at both electrodes (Figure 7). On the anode side, the FAD center of glucose oxidase is removed from the enzyme shell and covalently attached to a pyrroloquinoline quinone (PQQ) mediator species previously immobilized on a gold surface. The GOx apoenzyme (enzyme with active center removed) is reintroduced in solution and selectively binds to FAD, resulting in a PQQ-... 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

W Adam, W Boland, J Hartmann-Schreier, H-U Humpf, M Lazarus, A Saffert, CR Saha-Moller, P Schreier. a Hydroxylation of carboxylic acids with molecular oxygen catalyzed by the a oxidase of peas (Pisum sativum) a novel biocatalytic synthesis of enantiomerically pure (R)-2-hydroxy acids. J Am Chem Soc 120 11044— 11048, 1998. 

Tissue and Bacteria Electrodes The limited stability of isolated enzymes, and the fact that some enzymes are expensive or even not available in the pure state, has prompted the use of cellular materials (plant tissues, bacterial cells, etc.) as a source of enzymatic activity (48). For example, the banana tissue (which is rich with polyphenol oxidase) can be incorporated by mixing within the carbon paste matrix to yield a fast-responding and sensitive dopamine sensor (Fig. 6.14). These biocatalytic electrodes function in a manner similar to that for conventional enzyme electrodes (i.e., enzymes present in the tissue or cell produce or consume a detectable species). 

Figure 6. Electrical wiring of glucose oxidase with ferroeene units tethered to lysine residues of the protein backbone (cf. Figure 5B). (A) Cyelic voltammograms of a bare Au electrode in the presence of modified GOx (10 mg mL ) and glucose at (a) 0, (b) 0.8 and (c) 5 mM. Experiments were performed in 0.085 M phosphate bulfer, pH 7.0, under argon. (B) Glucose concentration dependence of the current at 0.26 V vs. SCE developed by the biocatalytic system (using a larger electrode). Adapted from Ref. [83] with permission.

The reconstitution method was suggested as a means to introduce the photoisomerizable unit into the vicinity of the biocatalytic redox-center, thereby generating a light-switchable bioelectrocatalyst that operates between fiilly switched ON and OFF states. Apo-glucose oxidase, apo-GOx, was reconstituted with the nitrospiropyran-FAD cofactor unit, (20), Fig. 3-30. 

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