Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer

Abstract

Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode 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 amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine 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 the electron-transfer subunit (i.e., β subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (-)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH redox wave and a stabilized bioelectrocatalytic current of more than 100 μA, 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 applied to other redox enzymes having a buried active site.

Electroenzymatic Nitrogen Fixation Using a MoFe Protein System Immobilized in an Organic Redox Polymer

“…[11,14] Accordingly, the measured bioelectrocatalytic current is solely due to the enzyme attached as a protein monolayer within the electron tunneling distance. [2,10,[14][15][16] An approach to improve the loading of wired nitrogenase on electrodes is to form an electron-conducting matrix to entrap and stabilize nitrogenase at the electrode interface by employing redox relays tethered to a polymer network, which are in principle able to efficiently wire any orientation of a redox enzyme to the electrode. Hence, electron transfer (ET) becomes independent of the electrode-enzyme distance and orientation via a mediated charge transfer.…”

Selectively Probing Neurochemicals in Living Animals with Electrochemical Systems

“…Biosensors to detect these species usually employ an enzyme to firstly undergo a catalytic reaction with the analyte, generating electrochemical active products that can be detected on the electrode surface. As such, enzyme serves as a key bio‐recognition element, providing electrochemical biosensors with attributes of specificity and sensitivity [10b,c,30] …”

Enzymatic Bioreactors: An Electrochemical Perspective

“…Some methods of enzyme immobilisation can improve DET [35]. For example, Lee et al used a gold-binding peptide (GBP) to bind glucose dehydrogenase which facilitates enzyme orientation on the surface of the gold electrode, decreasing the distance between flavin adenine dinucleotide (FAD) and the electrode surface [70]. Cross-linked hydrogels can be used for enzyme immobilisation on the surface of an electrode.…”