Construction of a protein-engineered variant of D-fructose dehydrogenase for direct electron transfer-type bioelectrocatalysis

D-Fructose dehydrogenase (FDH), a heterotrimeric membrane-bound enzyme, exhibits strong activity in direct electron transfer- (DET-) type bioelectrocatalysis. We constructed a variant (Delta 1cFDH) that lacks 143 amino acid residues involving one heme c moiety (called heme 1c) on the N-terminus of subunit II, and characterized the bioelectrocatalytic properties of Delta 1cFDH using cyclic voltammetry. A clear DET-type catalytic oxidation wave of D-fructose was observed at the Delta 1cFDH-adsorbed Au electrodes. The result clearly indicates that the electrons accepted at the Flavin adenine dinucleotide catalytic center in subunit I are transferred to electrodes via two of the three heme c moieties in subunit II without going through heme lc. In addition, the limiting current density of A1cFDH was one and a half times larger than that of the native FDH in DET-type bioelectrocatalysis. The downsizing protein engineering causes an increase in the surface concentration of the electrochemically effective enzymes and an improvement in the heterogeneous electron transfer kinetics. (C) 2017 Elsevier By. All rights reserved.