Electrical contacting of flavoenzymes and NAD(P)+-dependent enzymes by reconstitution and affinity interactions on phenylboronic acid monolayers associated with Au-electrodes

The preparation of integrated, electrically contacted, flavoenzyme and NAD(P)(+)-dependent enzyme-electrodes is described. The reconstitution of apo-glucose oxidase, apo-GOx, on a FAD cofactor linked to a pyrroloquinoline quinone (PQQ) phenylboronic acid monolayer yields an electrically contacted enzyme monolayer (surface coverage 2.1 x 10(-12) Mol cm(-2)) exhibiting a turnover rate of 700 s(-1) (at 22 2 degreesC). The system is characterized by microgravimetric quartz-crystal microbalance analyses, Faradaic impedance spectroscopy, rotating disk electrode experiments, and cyclic voltammetry. The performance of the enzyme-electrode for glucose sensing is described. Similarly, the electrically contacted enzyme-electrodes of NAD (P)(+)-dependent enzymes malate clehydrogenase, MaID, and lactate clehydrogenase, LDH, are prepared by the cross-linking of affinity complexes generated between the enzymes and the NADP(+) and NAD(+) cofactors linked to a pyrroloquinoline quinone phenylboronic acid monolayer, respectively. The MaID enzyme-electrode (surface coverage 1.2 x 10(-12) mol cm(-2)) exhibits a turnover rate of 190 s(-1), whereas the LDH enzyme-electrode (surface coverage 7.0 x 10(-12) mol cm(-2)) reveals a turnover rate of 2.5 s(-1). Chronoamperometric experiments reveal that the NAD+ cofactor is linked to the PQQ-phenylboronic acid by two different binding modes. The integration of the LDH with the two NAD(+) cofactor configurations yields enzyme assemblies differing by 1 order of magnitude in their bioelectrocatalytic activities.