Development of Direct Electron Transfer Based Enzyme Electrode Using Synthetic FADDependent Glucose Dehydrogenase of Burkholderia cepacia

Abstract

Electron transfer in biological systems has been actively investigated in various fields, including photosynthesis, biofuel cells, biomass degradation, bioelectronic devices, and biosensors. Efficient electrical communication between biocatalysts and target electron acceptors are needed to optimise the performance of these systems. In most redox enzymes, the redox active site is buried deep inside the protein core, thereby requiring a long electron tunneling distance and occurring inefficient electron transfer. Significant efforts have been applied toward improving the efficiency of electrical communications between these enzymes and electron acceptors. Typical approaches involve the addition of redox mediators that facilitate electron transfer. This mechanism is referred to as mediated electron transfer (MET). Redox mediators are usually small mobile molecules with redox potentials that are intermediates between those of the electron-donating enzymes and the electron acceptors, which are often oxidative electrodes. Redox mediators assist electron transfer by diffusing in and out of the enzyme active site. Although mediators can overcome the long distance between the active site and the electrode surface, they suffer from certain disadvantages, including a high cost, potential toxicity, and destabilise the system by diffusing away over time. Ideally, the electron tunneling distance in a redox centre should be reduced to allow efficient direct electron transfer (DET), obviating the need for a redox mediator. Facilitating DET requires a detailed understanding of the enzyme–electrode interface, the intermolecular and intramolecular electron transfer mechanisms, and methodological principles, all of which critically influence the activity of an enzymatic electrode To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current is transferred to final electron acceptor due to energy loss at enzymeelectrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold binding peptide at its aminoor carboxy- terminus for direct contact between the enzyme and electrode. The fused gold binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto an Au electrode. Depending on the fused site of binding peptide to the enzyme complex, 9 combinations of recombinant GDH proteins on the electrode show significantly different direct electron transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy-terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme– electrode interface even in the absence of electron transfer subunit (i.e., β subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of c.a. (−) 0.46 V vs. Ag/AgCl was associated with the appearance of an FAD/FADH2 redox wave and a stabilized bioelectrocatalytic current of more than one hundred microamperes, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 μA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific “sticky-ended” proteins without loss of catalytic activity could be applicable to other redox enzymes having a buried active site.