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Graphite electrodes were modified with triangular (AuNTrs) or spherical (AuNPs) nanoparticles and further modified with fructose dehydrogenase (FDH). The present study reports the effect of the shape of these nanoparticles (NPs) on the catalytic current of immobilized FDH pointing out the different contributions on the mass transfer–limited and kinetically limited currents. The influence of the shape of the NPs on the mass transfer–limited and the kinetically limited current has been proved by using two different methods: a rotating disk electrode (RDE) and an electrode mounted in a wall jet flow-through electrochemical cell attached to a flow system. The advantages of using the wall jet flow system compared with the RDE system for kinetic investigations are as follows: no need to account for substrate consumption, especially in the case of desorption of enzyme, and studies of product-inhibited enzymes. The comparison reveals that virtually identical results can be obtained using either of the two techniques. The heterogeneous electron transfer (ET) rate constants (kS) were found to be 3.8 ± 0.3 s−1 and 0.9 ± 0.1 s−1, for triangular and spherical NPs, respectively. The improvement observed for the electrode modified with AuNTrs suggests a more effective enzyme-NP interaction, which can allocate a higher number of enzyme molecules on the electrode surface. Graphical abstractThe shape of gold nanoparticles has a crucial effect on the catalytic current related to the oxidation of D-(-)-fructose to 5-keto-D-(-)-fructose occurring at the FDH-modified electrode surface. In particular, AuNTrs have a higher effect compared with the spherical one. Electronic supplementary material The online version of this article (10.1007/s00216-019-01944-6) contains supplementary material, which is available to authorized users.

d-Fructose dehydrogenase (FDH) contains a flavin adenine dinucleotide (FAD) in subunit I and three heme c moieties (1c, 2c, and 3c from the N-terminus) as the electron transfer relaying sites. The electron transfer in direct electron transfer-type bioelectrocatalysis of FDH is proposed to proceed in sequence from FAD, through heme 3c, to heme 2c without going through heme 1c. In order to improve the performance of the bioelectrocatalysis, we constructed a variant (M450QΔ1cFDH) in which 143 amino acid residues involving heme 1c were removed and M450 as the sixth axial ligand of heme 2c was replaced with glutamine to negatively shift the formal potential of heme 2c. The M450QΔ1cFDH variant was adsorbed on a planar gold electrode. The variant-adsorbed electrode produced a clear sigmoidal steady-state catalytic wave of fructose oxidation in cyclic voltammetry. The limiting current density was 1.4 times larger than that of the recombinant (native) FDH. The half-wave potential of the wave shifted by 0.2 V to the negative direction. M450QΔ1cFDH adsorbed rather homogeneously in orientations suitable for DET-type bioelectrocatalysis.

In this paper, an efficient direct electron transfer (DET) reaction was achieved between fructose dehydrogenase (FDH) and a glassy-carbon electrode (GCE) upon which anthracene-modified single-walled carbon nanotubes were deposited. The SWCNTs were activated in situ with a diazonium salt synthesized through the reaction of 2-aminoanthracene with NaNO2 in acidic media (0.5 M HCl) for 5 min at 0 °C. After the in situ reaction, the 2-aminoanthracene diazonium salt was electrodeposited by running cyclic voltammograms from +1000 to −1000 mV. The anthracene-SWCNT-modified GCE was further incubated in an FDH solution, allowing enzyme adsorption. Cyclic voltammograms of the FDH-modified electrode revealed two couples of redox waves possibly ascribed to the heme c1 and heme c3 of the cytochrome domain. In the presence of 10 mM fructose two catalytic waves could clearly be seen and were correlated with two heme cs (heme c1 and c2), with a maximum current density of 485 ± 21 μA cm–2 at 0.4 V at a sweep rate of 10 mV ...

In this paper we present a new method to electrodeposit highly porous gold (h-PG) onto a polycrystalline solid gold electrode without any template. The electrodeposition is carried out by first cycling the electrode potential between +0.8 and 0 V in 10 mM HAuCl4 with 2.5 M NH4Cl and then applying a negative potential for the production of hydrogen bubbles at the electrode surface. After that the modified electrode was characterized in sulfuric acid to estimate the real surface area ( Areal) to be close to 24 cm2, which is roughly 300 times higher compared to the bare gold electrodes (0.08 cm2). The electrode was further incubated overnight with three different thiols (4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MPh), and 4-aminothiophenol (4-APh)) in order to produce differently charged self-assembled monolayers (SAMs) on the electrode surface. Finally a fructose dehydrogenase (FDH) solution was drop-cast onto the electrodes. All the modified electrodes were investigated by cyclic voltammetry both under nonturnover and turnover conditions. The FDH/4-MPh/h-PG exhibited two couples of redox peaks for the heme c1 and heme c2 of the cytochrome domain of FDH and as well as a well pronounced catalytic current density (about 1000 μA cm-2 in the presence of 10 mM fructose) due to the presence of -OH groups on the electrode surface, which stabilize and orientate the enzyme layer on the electrode surface. The FDH/4-MPh/h-PG based electrode showed the best analytical performance with an excellent stability (90% retained activity over 90 days), a detection limit of 0.3 μM fructose, a linear range between 0.05 and 5 mM, and a sensitivity of 175 ± 15 μA cm-2 mM-1. These properties were favorably compared with other fructose biosensors reported in the literature. The biosensor was successively tested to quantify the fructose content in food and beverage samples. No significant interference present in the sample matrixes was observed.

d-Fructose dehydrogenase (FDH), a heterotrimeric membrane-bound enzyme, exhibits strong activity in direct electron transfer- (DET-) type bioelectrocatalysis. We constructed a variant (Δ1cFDH) 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 Δ1cFDH using cyclic voltammetry. A clear DET-type catalytic oxidation wave of d-fructose was observed at the Δ1cFDH-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 1c. In addition, the limiting current density of Δ1cFDH 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. Keywords: Fructose dehydrogenase, Direct electron transfer, Flavohemoprotein, Orientation, Protein engineering

d -Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 catalyzes the two-electron oxidation of d -fructose to 5-keto- d -fructose, and it is widely used in biofuel cells and biosensors. In this study, methoxy-substituent-functionalized carbon electrodes are constructed by electrochemical oxidation of methoxy-aniline derivatives on Ketjen Black (KB)-modified electrodes to improve the immobilization and bioelectrocatalysis of FDH. It is proposed that the specific interaction between FDH, especially the heme c moiety, and methoxy substituent(s) of amines on carbon electrode increases the proportion of the productively oriented FDH molecules to the total FDHs. Consequently, the limiting catalytic current density of the d -fructose oxidation increases to as much as 23 ± 2 mA cm−2 in FDH/2,4-dimethoxyaniline/KB/glassy carbon electrode, for example.

d-Fructose dehydrogenase (FDH), a flavoprotein-cytochrome c complex, exhibits high activity in direct electron transfer (DET)-type bioelectrocatalysis. One of the three types of heme c in FDH is the electron-donating site to the electrodes, and another heme c is presumed to not be involved in the catalytic cycle. In order to confirm the electron transfer pathway, we constructed three mutants in which the sixth axial methionine ligand (M301, M450, or M578) of one of the hemes was replaced with glutamine, which was selected with the expectation that it would shift the formal potential of the hemes in the negative direction. An M450Q mutant successfully reduced the overpotential by approximately 0.2 V, giving a limiting current close to that of the native FDH. In contrast, an M301Q mutant remained almost unchanged and an M578Q mutant drastically decreased DET-type catalytic activity. The results indicate that the electron transfer in the native FDH occurs in sequence from the flavin, through the heme c with M578, to the heme c with M450 (as the electron-donating site to the electrodes), without going through the heme c with M301. The M450Q mutant will be useful for biofuel cells because of the decreased overpotential. Keywords: Fructose dehydrogenase, Direct electron transfer, Electron transfer pathway, Overpotential, Axial ligand, Heme c

We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl2, addition of MgCl2 did not show any particular influence, whereas addition of monovalent cations (Na+ or K+) led to a slight linear increase in the maximum response current. To complement the amperometric investigations, spectrophotometric assays were carried out under homogeneous conditions in the presence of a 1-electron non-proton-acceptor, cytochrome c, or a 2-electron-proton acceptor, 2,6-dichloroindophenol (DCIP), respectively. In the case of cytochrome c, it was possible to observe a remarkable increase in the absorbance up to 200% when 10 mM CaCl2 was added. However, by further increasing the concentration of CaCl2 up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl2 concentration. Addition of MgCl2 or of the monovalent cations shows, surprisingly, no effect on the electron transfer to the electron acceptor. Contrary to the case of cytochrome c, with DCIP none of the cations tested seem to affect the rate of catalysis. In order to correlate the results obtained by amperometric and spectrophotometric measurements, CD experiments have been performed showing a great structural change of FDH when increasing the concentration CaCl2 up to 50 mM, at which the enzyme molecules start to agglomerate, hindering the substrate access to the active site probably due to a chelation reaction occurring at the enzyme surface with the glutamate/aspartate residues. Graphical AbstractFructose dehydrogenase (FDH) consists of three subunits, but only two are involved in the electron transfer process: (I) 2e−/2H+ fructose oxidation, (II) internal electron transfer (IET), (III) direct electron transfer (DET) through 2 heme c; FDH activity either in solution or when immobilized onto an electrode surface is enhanced about 2.5-fold by adding 10 mM CaCl2 to the buffer solution, whereas MgCl2 had an “inhibition” effect. Moreover, the additions of KCl or NaCl led to a slight current increase Electronic supplementary material The online version of this article (10.1007/s00216-018-0991-0) contains supplementary material, which is available to authorized users.

Fructose dehydrogenase (FDH, EC 1.1.99.11) is a bacterial membrane bound flavocytochrome oxidoreductase, which has been widely employed to develop direct electron transfer (DET) and mediated electron transfer (MET) based electrode platforms [1]. Most commonly FDH from Gluconobacter japonicus has been studied. FDH is a heterotrimer consisting of a catalytic dehydrogenase domain, DHFDH (subunit I), where D-(−)-fructose oxidation occurs at the covalently flavin cofactor (FAD), followed by an internal electron transfer (IET) to the cytochrome domain (CYTFDH, subunit II), which contains three heme c moieties coordinated by the enzyme scaffold, and a subunit III, which is not involved in the ET process. We have recently investigated [2-4] how to optimize orientation and the DET reaction between FDH and electrodes. In [2] we report on the influence of pH and monovalent/divalent cations on the catalytic current response, the IET, and the structure of FDH by using amperometry, spectrophotometry, and circular dichroism. Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the DHFDH and CYTFDH. In [3] we present a new method to electrodeposit highly mesoporous gold onto a polycrystalline solid gold electrode without any template. The electrodeposition is carried out by first cycling the electrode potential between +0.8 and 0 V in 10 mM HAuCl4 with 2.5 M NH4Cl and then applying a negative potential for the production of H2 bubbles at the electrode surface. This mesoporous gold electrode was modified with 4-mercaptophenol onto which FDH was orientated to optimize the DET reaction. In [4] an efficient DET reaction was also achieved between FDH and anthracene-modified single-walled carbon nanotubes (SWCNT) deposited onto a glassy-carbon electrode. The SWCNTs were activated in situ with a diazonium salt synthesized through the reaction of 2-aminoanthracene with NaNO2 in 0.5 M HCl for 5 min at 0° C. After the in situ reaction, the 2-aminoanthracene diazonium salt was electrodeposited by running cyclic voltammograms from +1000 to −1000 mV. The anthracene-SWCNT-modified GCE was further incubated in an FDH solution to allow FDH to adsorb. Cyclic voltammograms of the FDH-modified electrode revealed two couples of redox waves ascribed to the heme c 1 and heme c 3 of the CYTFDH. In the presence of 10 mM fructose two catalytic waves could clearly be seen and were correlated with two heme c:s, with a maximum current density of 485±21 μA cm − 2 at 0.4 V at a sweep rate of 10 mV s − 1. References: [1] S. Kawai, T. Yakushi, K. Matsushita, Y. Kitazumi, O. Shirai, K. Kano, Electrochem. Commun., 38, 28-31 (2014). [2] P. Bollella, Y. Hibino, K. Kano, L. Gorton, R. Antiochia, Anal. Bioanal. Chem., 410, 3253-3264 (2018). [3] P. Bollella, Y. Hibino, K. Kano, L. Gorton, R. Antiochia, Anal. Chem ., 90, 12131-12136 (2018). [4] P. Bollella, Y. Hibino, K. Kano, L. Gorton, R. Antiochia, ACS Catal ., 8, 10279-10289 (2018).

Colony colour, as assessed using waterproof colour cards, is an accurate, economical and rapid way of field monitoring coral bleaching. This technique, however, has never been applied to zoanthids, nor have variations in colony colour been directly correlated with microscopic observation of Symbiodinium condition. In this study, we examined 12 colonies of the common reef zoanthid Palythoa tuberculosa at two sites (Odo and Miyagi) in Okinawa, Japan, for one year to observe the relationship between colour card scores, Symbiodinium morphological condition and sea surface temperature (SST); Symbiodinium types within the study specimens were also examined. Colonies became paler during periods of rapid SST increase and significant correlations between P. tuberculosa colony colour and internal zooxanthellar condition were found for 5 out of the 12 colonies monitored. Results were more pronounced at the Odo site (significant correlations for four out of six colonies) as compared to the Miyagi site (a sig...

Zoanthids of the genus Palythoa are common in coral reef environments worldwide, particularly in the intertidal zone. However, their taxonomy remains problematic, resulting in an incomplete understanding of their diversity. Palythoa caesia Dana, 1846 is found in Fiji, Australia, and the Indian Ocean, while P. tuberculosa (Esper, 1805) has been reported from India, the Red Sea, Singapore, Madagascar, and Japan. The lack of obvious characters differentiating the two species, their wide distributions and high levels of intraspecific variation raise the possibility that these species are in fact one. Based on specimens from Australia, the Red Sea, and Japan, we used three DNA markers (mitochondrial cytochrome oxidase I, 16S ribosomal DNA, and the nuclear internal transcribed spacer region of ribosomal DNA) combined with morphological analyses of tentacle numbers, and cnidae to re-examine the identity of these two taxa. Phylogenetic results showed sequences from all specimens for all markers formed one monophyly, and morphological results showed little differentiation between the two putative taxa. Overall, it is apparent these two taxa are the same species, and the senior synonym P. tuberculosa should be used for specimens for the entire Indo-Pacific region.