Cytochrome laccase electrode

Figure 7. Influence of the lactate concentration on the H2Q converting capacity of the laccase/cytochrome b2 electrode/phosphate buffer pH 6.5, E = +400 mV vs SCE.

The first reports on a reversible DET between redox proteins and electrodes were published in 1977 showing that cytochrome c is reversibly oxidized and reduced at tin-doped indium oxide [30] and gold in the presence of 4,4 -bipyridyl [31]. Only shortly after these publications appeared, papers were published describing the DET between electrode and enzyme for laccase and peroxidase [32,33]. It was observed that the overpotential for oxygen reduction at a carbon electrode was reduced by several hundred millivolts compared to the uncatalyzed reduction when laccase was adsorbed. This reaction could be inhibited by azide. The term bioelectrocatalysis was introduced for such an acceleration of the electrode process by... 

Benzoquinone/ hydroquinone cytochrome b2 + laccase membrane O2 electrode 500 Scheller et al. (1987b)... 

An enzyme electrode based on coimmobilized cytochrome b2 and laccase (Scheller et al., 1987b) allows an explanation of the principle of substrate recycling in enzyme electrodes in greater detail (Fig. 100). The advantage of this system is that the cosubstrate, oxygen, as well as the analytes, hydroquinone and benzoquinone, are electrochemically active. This permits one to study different parts of the recycling process. Recycling of the analyte in the presence of the substrate of cytochrome b2, lactate, results in an increase in the sensitivity by a factor of 500 as compared with lactate-free operation. Under conditions that are optimal for laccase the analyte is almost completely in the oxidized state, i.e. it... 

Electrons can be transferred between an enzyme and electrode directly, termed direct electron transfer (DET), or through a mediator, termed mediated electron transfer (MET). Enzymes capable of DET contain a reactive metal center or other redox center, such as flavin adenine dinucleotide (FAD), fixed in the active site. When the active site is located within a short distance of the electrode (less than —20 A), electrons are transferred between them through electron tunneling [16]. Only a few classes of enzymes have demonstrated DET, notably cytochrome c, some hydrogen-ases, peroxidases, oxidases, and laccases [17,18]. In BFCs, laccases and oxidases are the most commonly found enzymes capable of DET. 

Laccase was immobilized alone or co-immobilized with GOD or cytochrome b2 by entrapment in gelatin. The layer had a thickness of 20-30 The enzyme membranes contained 120 U/cm laccase, 56 U/cm GOD and 30 U/cm of cytochrome b2, respectively . For preparation of the sensor, the mono-or bi-enzyme layer was fixed on the dialysis membrane (17 pm thick) of the modified oxygen electrode, which comprised a platinum indicator electrode of 0.5 mm diameter covered by a dialysis membrane held by an 0-ring. 

In another recycling system we co-immobolized cytochrome bz and laccase in a gelatin layer in front of an oxygen electrode. In the presence of the cytochrome bz substrate, lactate, BQ is reduced to H2Q. Therefore, this enzyme substitutes the cathode of the previous cycling system (Figure 6). However, the substrate recyling is not restricted to the phase boundary (as with electrochemical recyling) but it proceeds in the total volume of the enzyme layer. At the optimum pH of cytochrome b2 (pH 6.5) and lactate saturation a maximum amplification of 500 has been obtained. 

---