Apoenzyme electrodes

An apoenzyme electrode for Cu2+ has been developed by the coupling of immobilized apo-tyrosinase with an oxygen probe (Mattiasson et al., 1979). The detection limit of the sensor was 50 ppm. However, the reusability of such an apoenzyme membrane appears to be questionable because the enzyme activity accumulates during the operation of the sensor whereas the measuring principle requires kinetic control so as to obtain a linear dependence of the sensitivity on the activity of the holoenzyme. 

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-... 

As has been shown, this kind of reaction does take place on the mercury electrode in the presence of cystine. The i-E curves measured on an amalgamated electrode in the presence of peroxidase apoenzyme in the solution also have anodic and cathodic maxima, similar to those obtained in the peroxidase solution. Thus, in the potential region investigated, the S—S groups of the protein globule of peroxidase are electrochemically active. Heme iron in the active center does not take part in the observed redox process. 

Biosensors, known to monitor various analytes both selectively and sensitively, have also been reported for heavy metal detection. Several electrode configurations using whole cells, enzymes or apoenzymes have been designed 4-6). The main advantage of such biosensors is that samples often require little pretreatment and the bioavailable concentration of the toxic heavy metal is measured, rather than the total concentration. However, a limited selectivity and quite low sensitivity characterize these sensors described in the literature. 

The degree of enzyme purity will ultimately affect fuel cell performance, particularly when enzyme preparations are used to form immobilized films on electrode surfaces in DET reactions. Contaminating proteins that do not provide electron transfer effectively foul the electrode. When enzyme immobilization techniques are specific to the enzyme, then enzyme purity may not be as much as an issue, but rarely the immobilization technique is absolutely specific to the cathodic or anodic enzyme. For example, an attractive immobilization strategy is to link a particular enzyme to an electrode via its cofactor (e.g., flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), etc.) [59]. The cofactor is linked to the electrode material first and then the apoenzyme is allowed to naturally bind to the cofactor all other proteins in the enzyme preparation that cannot bind the cofactor remain unbound and can be removed. Enzymes used in fuel cells are not so unique, and proteins in the immobilizing preparation may use the same cofactor but not the same fuel during fuel cell analysis or operation. 

Another method of immobilizing enzyme and mediator is the attachmait of a mediator-containing monolayer to an electrode surface. If this monolayer is also functionalized with an enzyme cofactor meant to bind the enzyme, a catalytic electrode results. For example, gold electrode surfaces ean be eovered with a PQQ monolayer. The monolayer retains the redox properties of PQQ and ean be functionalized further. If FAD eofaetors are then covalently bound, GOx apoenzyme can bind the FAD centers. A covalent molecular chain results and electrons may hop from FAD to PQQ to the electrode surface, thus mediating from the enzyme [87]. PQQ monolayers can also link to covalently bound NAD" ", for mediation to NAD" -dependent dehydrogenase enzymes [88,89]. Functionalizing with monolayers of cytochrome c has also been demonstrated [90]. 

Direct interachon between enzyme and electrode is a particular challenge for many redox enzymes as the redox cofactor is buried deep within the protein structure. The cofactor of GOx, for example, is buried deep within the protein stmcture. One intereshng soluhon to this hmitahon is to anchor the cofactor flavin adenine dinucleohde (FAD) directly to the electrode surface. When the apoenzyme (enzyme without cofactor) is subsequently added, the protein reforms around the anchored FAD and tethers the enzyme in close... 

FIGURE 11.4 Electrical wiring of redox enzymes, (a) Optimal configuration for the electrical contacting of a redox enzyme with the electrode, (b) Reconstitution of an apoenzyme on a relay-cofactor monolayer for the alignment and electrical wiring of a redox enzyme. The structures of the redox relay molecule PQQ and cofactor amino-FAD are shown in the inset. (Reproduced with permission from Ref. [53]. Copyright 2006, Elsevier.)... 

A cofactor (or coenzyme) may be immobilized on an insoluble support at the same time as an enzyme, provided that it forms an undissociable unity with the apoenzyme. For example, during the production of a glucose electrode, FAD (flavin adenine dinucleotide) is bound to glucose oxidase and does not require supplementary immobilization. 

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