Reconstitution of the quinoprotein glucose dehydrogenase from its apoenzyme on a gold electrode surface modified with a monolayer of pyrroloquinoline q

Abstract

A gold electrode surface was modified with a chemisorbed monolayer of cystamine, and the cystamine amino groups were used for covalent immobilization of pyrroloquinoline quinone (PQQ). The spacer length between the electrode surface and the immobilized PQQ was increased using glutaraldehyde and 1,8-diaminooctane as additional spacers. Both kinds of PQQ modified electrodes (with short and long spacers) exhibited a reversible electrochemical process with the redox potential −0.125 V (vs. SCE) at pH 7.0. The electrode modified with the long spacered PQQ was used as a support for the immobilization of glucose dehydrogenase (GDH) apoenzyme having affinity for PQQ which is its native cofactor. After the anchorage of the GDH apoenzyme, this electrode exhibited enzymatic activity for glucose oxidation. Electrocatalytic oxidation of glucose was obtained only in the presence of a solubilized electron transfer mediator (2,6-dichlorophenolindophenol) which has a more positive redox potential than the immobilized PQQ. Therefore, although the enzymatic activity was reconstituted by the interaction of the apoenzyme binding site with the long spacered PQQ immobilized as a monolayer on the electrode surface, there was no direct electron transfer between the electrode surface and the PQQ redox site inside the holoenzyme. Attempts to reconstitute the holoenzyme from the electrode surfaces modified with the short spacered PQQ as well as with the long spacers without PQQ at the end resulted in electrodes without any catalytic activity.