Improved Direct Bioelectrochemical Fructose Oxidation with Surfactant-Free Heterotrimeric Fructose Dehydrogenase Variant Truncating Heme 1c and C-Terminal Hydrophobic Regions

Direct electron transfer (DET)-type bioelectrocatalysis is a coupled redox reaction between enzymatic and electrode reactions. Such mediatorless reactions are an environmentally safe approach that can be applied to various bioelectrochemical devices. We focused on fructose dehydrogenase (FDH), a membrane-bound heterotrimeric enzyme that catalyzes DET-type D-fructose oxidation. Although the overall structure was recently elucidated, its membrane-bound region has not been completely identified. Therefore, this study assumed that the heme 1c region and C-terminal hydrophobic region (CHR) were bound to the membrane. A constructed double variant (Δ1cΔCHR_FDH) was soluble without any surfactants; additionally, cryo-electron microscopy confirmed that this variant was downsized. Δ1cΔCHR_FDH exhibited a 14-fold higher catalytic current density (11 ± 1 mA cm–2) than that of the wild-type recombinant FDH (rFDH) at multi-walled carbon nanotube electrodes. Kinetic analysis of the voltammograms suggested that downsizing of the enzyme and the removal of surfactants increased the surface concentration of enzymes at the electrode. This study elucidates the membrane-binding mechanism of proteins and efficient bioelectrocatalysis overcoming the interference of surfactants.