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The effect of mutation on the redox potentials (E degrees') of the heme moieties in the variants of D-fructose dehydrogenase (FDH) was investigated by mediated spectroelectrochemical titrations. The replacement of the axial ligand of heme from methionine to glutamine changes the E degrees' value more negatively than that of the corresponding heme moiety in the recombinant (native) FDH (rFDH). The determined E degrees' values of non-targeted heme moieties in the variants were also shifted in a negative direction from that in rFDH. Thus, enzyme modification changes E degrees' of the heme moieties in unmodified protein regions. (C) The Author(s) 2021. Published by ECSJ.

An electrochemical d -fructose sensor was fabricated by immobilizing a variant of d -fructose dehydrogenase on a porous gold microelectrode. The enzyme-modified electrode bioelectrocatalytically oxidizes d -fructose via direct electron transfer. The catalytic current reaches a steady-state limiting value at 0.05 V vs. Ag|AgCl (sat. KCl). The temperature dependence of the sensor suggests that its response is limited by the diffusion of d -fructose. Therefore, the sensor requires no calibration. The sensitivity of the sensor, (2.0 ± 0.2) × 102 μA cm−2 mM−1, is reproducible. The upper limit of detection is ~2.0 mM and the kinetics of the bioelectrocatalytic reaction is determined the limitation. The fabricated sensors were applied in the determination of d -fructose in commercial beverages as well as honey, and compared favorably with a more commonly used photometric method.

A new mediated carbon nanotube paste (CNTP) amperometric biosensor for fructose is described. The biosensor is formed by a CNTP electrode modified with an electropolymerized film of 3,4‐dihydroxybenzaldehyde (3,4‐DHB) and is based on the activity of a commercial available D‐fructose dehydrogenase (FDH) immobilized on an immobilon membrane placed on the top of the electrode surface. Analytical parameters such as enzyme immobilization, pH, temperature, and probe lifetime were studied and optimized. The biosensor response current was directly proportional to D‐fructose concentration from 5 × 10−6 to 2 × 10−3 mol/L with a detection limit of 1 × 10−6 mol/L and a good reproducibility (RSD = 1.8%, n = 5). The biosensor was used for the determination of fructose content in three honey samples and validated with a commercial spectrophotometric enzymatic kit.

Steady-state kinetic analysis was performed on the reaction between d -fructose and ferricyanide with the quinohemoprotein fructose dehydrogenase from Gluconobacter species. The d -fructose oxidation dependence on the ferricyanide concentration resulted in a series of parallel reciprocal plots, and the reaction was assumed to proceed by a ping-pong type of mechanism. A reciprocal plot of the reduction of ferricyanide at saturating concentration of d -fructose gave a break which was considered to appear as a result of the two active centers, namely PQQ and heme e functioning. A scheme of action is proposed and the bimolecular rate constant of the d -fructose oxidation, the k cat for PQQ and the electron transfer rate between the PQQH 2 and heme c are calculated and account for 2.2 ± 0.4.10 4 M −1 s −1 , (93 ± 14) and (162 ± 7) s −1 , respectively.