D-fructose Dehydrogenase Research Articles

Enhancement of direct electron transfer by aromatic thiol modification with truncated d-fructose dehydrogenase

A heterotrimeric membrane-bound d-fructose dehydrogenase (FDH) from Gluconobacter japonicus, composed of subunits L, C, and S, shows strong direct electron transfer (DET)-type bioelectrocatalytic performance in the two-electron oxidation of d-fructose. The electrode-active site of the enzyme is the heme c moiety in the subunit C. A previous study reported the construction of the subunit L/S subcomplex (ΔC FDH) lacking the subunit C. In this work, we attempted to realize the DET-type reaction of ΔC FDH using porous gold electrodes functionalized with several thiols, and investigated the influence of the thiols on DET. Kinetic analysis of the steady-state catalytic waves revealed that the electrode-active site of ΔC FDH is the [3Fe-4S] iron-sulfur cluster in the L subunit. The experimental results demonstrated that uncharged aromatic thiols on the electrode enhance the DET-type reaction of ΔC FDH. Based on the pH-dependent profile of the DET-type activity and the surface conditions of the modified thiols evaluated by electrochemical reductive desorption, we suggest an enhancement mechanism for DET by aromatic thiols.

Structural and electrochemical elucidation of biocatalytic mechanisms in direct electron transfer-type D-fructose dehydrogenase

D-Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of intense direct electron transfer (DET)-type bioelectrocatalysis, was investigated using protein engineering, electrochemistry, structural biology, and bioinformatics. DET-type reactions have led to an increase in energy efficiency, biocompatibility, and design freedom in bioelectrochemical devices, such as biofuel cells, biosensors, biosupercapacitors, and bioreactors. However, one of the greatest challenges for this reaction is the limited number of the applicable enzymes. The creation of variants with altered substrate specificities using template enzymes is a promising solution to address this problem. For creating variants using FDH as a template, it is important to understand the biocatalytic mechanisms. Recently, the structure of FDH has been analyzed using cryo-electron microscopy (cryo-EM) single particle analysis, and the three amino acid residues (N1146, H1147, and N1190) may be critical for substrate recognition by FDH. In this study, we aimed to understand the mechanisms of the biocatalytic reaction of FDH and discuss the roles of these residues. In addition, we validated the structures of the in-silico variants by comparing them to the cryo-EM structures. Furthermore, for the variants of N1146 involved in D-fructose recognition, we have verified the changes in specificity for other substrates.

Giant Carbon Nano-Test Tubes as Versatile Imaging Vessels for High-Resolution and In Situ Observation of Proteins.

Cryogenic electron microscopy is one of the fastest and most robust methods for capturing high-resolution images of proteins, but stringent sample preparation, imaging conditions, and in situ radiation damage inflicted during data acquisition directly affect the resolution and ability to capture dynamic details, thereby limiting its broader utilization and adoption for protein studies. We addressed these drawbacks by introducing synthesized giant carbon nano-test tubes (GCNTTs) as radiation-insulating materials that lessen the irradiation impact on the protein during data acquisition, physical molecular concentrators that localize the proteins within a nanoscale field of view, and vessels that create a microenvironment for solution-phase imaging. High-resolution electron microscopy images of single and aggregated hemoglobin molecules within GCNTTs in both solid and solution states were acquired. Subsequent scanning transmission electron microscopy, small-angle neutron scattering, and fluorescence studies demonstrated that the GCNTT vessel protected the hemoglobin molecules from electron irradiation-, light-, or heat-induced denaturation. To demonstrate the robustness of GCNTT as an imaging platform that could potentially augment the study of proteins, we demonstrated the robustness of the GCNTT technique to image an alternative protein, d-fructose dehydrogenase, after cyclic voltammetry experiments to review encapsulation and binding insights. Given the simplicity of the material synthesis, sample preparation, and imaging technique, GCNTT is a promising imaging companion for high-resolution, single, and dynamic protein studies under electron microscopy.

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 μA mM−1cm−2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

The Redox Potential Measurements for Heme Moieties in Variants of D-Fructose Dehydrogenase Based on Mediator-assisted Potentiometric Titration

The effect of mutation on the redox potentials (E°′) 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°′ value more negatively than that of the corresponding heme moiety in the recombinant (native) FDH (rFDH). The determined E°′ 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°′ of the heme moieties in unmodified protein regions.

Diffusion-limited electrochemical d-fructose sensor based on direct electron transfer-type bioelectrocatalysis by a variant of d-fructose dehydrogenase at a porous gold microelectrode

Abstract 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.

5-Keto-D-fructose production from sugar alcohol by isolated wild strain Gluconobacter frateurii CHM 43.

CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

Special Collection on Bioelectrochemistry

Special Collection on Bioelectrochemistry

Ultimate downsizing of d-fructose dehydrogenase for improving the performance of direct electron transfer-type bioelectrocatalysis

d-Fructose dehydrogenase (FDH), a membrane-bound heterotrimeric enzyme, shows strong activity in direct electron transfer (DET)-type bioelectrocatalysis. An FDH variant (Δ1c2cFDH) which lacks 199 amino acid residues including two heme c moieties from N-terminus was constructed, and its DET-type bioelectrocatalytic performance was evaluated with cyclic voltammetry at Au planar electrodes. A DET-type catalytic current of d-fructose oxidation was clearly observed on Δ1c2cFDH-adsorbed Au electrodes. Detailed analysis of the steady-state catalytic current indicated that Δ1c2cFDH transports the electrons to the electrode via heme 3c at a more negative potential and at more improved kinetics than the recombinant (native) FDH.

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 (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.

Array of biosensors for discrimination of grapes according to grape variety, vintage and ripeness

A bioelectronic tongue based on nanostructured biosensors specific for the simultaneous detection of sugars and phenols has been developed. The array combined oxidases and dehydrogenases immobilized on a lipidic layer prepared using the Langmuir-Blodgett technique where Glucose oxidase, d-Fructose dehydrogenase, Tyrosinase or Laccase were imbibed. A phthalocyanine was co-immobilized in the sensing layer and used as electron mediator. The array thus formed has been used to analyze grapes and provides global information about the samples while providing specific information about their phenolic and their sugar content. Using Principal Component Analysis (PCA) the array of voltammetric biosensors has been successfully used to discriminate musts prepared from different varieties of grapes (Tempranillo, Garnacha, Cabernet-Sauvignon, Prieto Picudo and Mencía). Differences could be also detected between grapes of the same variety and cultivar harvested in two successive vintages (2012 and 2013).Moreover, the ripening of grapes could be monitored from veraison to maturity due to the changes in their phenolic and sugar content. Using Partial Least Squares (PLS-1) analysis, excellent correlations have been found between the responses provided by the array of biosensors and classical parameters directly related to phenols (total polyphenol index, TPI) and sugar concentration (degree Brix) measured by chemical methods with correlation coefficients close to 1 and errors close to 0. It is also worthy to notice the good correlations found with parameters associated with the pH and acidity that can be explained by taking into account the influence of the pH in the oxidation potentials of the phenols and in the enzymatic activity.This bioelectronic tongue can assess simultaneously the sugar and the phenolic content of grapes and could be used to monitor the maturity of the fruit and could be adapted easily to field analysis.

Interaction between d-fructose dehydrogenase and methoxy-substituent-functionalized carbon surface to increase productive orientations

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±2mAcm−2 in FDH/2,4-dimethoxyaniline/KB/glassy carbon electrode, for example.

Designing Macro-Meso Pore Structure of MgO-Templated Carbon for Det-Based Enzyme Electrodes

Porous carbon materials, including carbon nanotubes and carbon blacks, and mesoporous carbons, carbon cryogel and carbon aerogel, have attracted attention for improving the performance of enzymatic electrode. Carbon nanotubes and carbon blacks aggregate and create mesospaces suitable for enzymatic reactions. However, much of the mesospaces is unused because the pore size is random and controlling the pore structure and pore size is difficult. In contrast, carbon cryogel or aerogel has a well-controlled mesopore size distribution with an average pore diameter ranging from 10 to 100 nm. However, the procedure of carbon gel production is time-consuming and not suitable for mass production. We focused on a new mesoporous carbon material, MgO-templated carbon (MgOC) as an alternative to carbon gels. The mesopore size can be controlled by alternating the MgO template size[1]. One of the striking advantages of MgOC over the previously reported mesoporous carbons is its simple and cost-effective production procedure. The effects of the pore structure of MgOCs on the direct electron transfer (DET) reaction of redox enzyme, D-fructose dehydrogenase, and bilirubin oxidase, were investigated[2]. We found two effects of optimizing pore structure. First, the macropores of MgOC can enhance current production. When the pore size of MgOC was much larger than the size of enzyme, the MgOC could adsorb a sufficient amount of enzyme even on the pores formed inside the MgOC particle structure, and the enzymes adsorbed inside the MgOC worked well as an electrocatalyst. In contrast, when the pore size of MgOC was comparable to the size of enzymes, the enzymes could not penetrate the MgOC particles through the inter-connecting pores, and only the enzyme adsorbed on the mesopores formed at the surface of the carbon particles produced catalytic current. Second, the mesopores which were close to the size of enzymes improve thermal stability of electrode. When enzymes molecules were embedded in the carbon mesopores with pore size comparable to that of enzymes, an enhanced contact area between enzymes and the MgOC surface led to strong adsorption of enzymes on the MgOC surface as well as to enzyme stabilization by preventing the denaturation, aggregation and collision of enzyme molecules. Furthermore, we developed the novel MgOC which have hierarchical structure composed by meso-macro pores. This material improved current density and thermal stability of enzyme modified electrodes. Reference [1] T. Morishita, T. Tsumura, M. Toyoda, J. Przepiórski, A. W. Morawski, H. Kanno, and M. Inagaki, Carbon, 48, 2690 (2010) [2] H. Funabashi, K. Murata, S. Tsujimura, Electrochemistry, 83(5), 372 (2015).

Bioelectrochemical characterization of the reconstruction of heterotrimeric fructose dehydrogenase from its subunits

d-Fructose dehydrogenase (FDH) is a heterotrimeric-membrane-bound enzyme capable of direct electron transfer (DET)-type bioelectrocatalysis. Subunit II contains three heme C moieties and is presumed to play a key role in the DET reaction because the subunit I/III subcomplex (without subunit II) lacks DET-type activity. We constructed an expression system for subunit II. A non-turnover signal from subunit II was not observed on voltammograms, and a subunit II-adsorbed electrode did not exhibit DET-type activity in the presence of the subunit I/III subcomplex and fructose. Gel filtration column chromatography indicated that subunit II formed multimeric complexes with four or more subunits. The aggregation of subunit II seemed to interfere with its direct communication with the electrodes. In contrast, when subunit II was mixed with the subunit I/III complex in a solution, they formed a 1:1 full complex, and the complex recovered DET-type bioelectrocatalytic activity. These results strongly suggest that the heme C in subunit II is the electron transfer site for the DET-type bioelectrocatalytic activity of FDH.

Lyotropic Liquid Crystalline Cubic Phases as Versatile Host Matrices for Membrane-Bound Enzymes.

Lyotropic liquid crystalline cubic mesophases can function as host matrices for enzymes because of their biomimetic structural characteristics, optical transparency, and capability to coexist with water. This study demonstrates that the in meso immobilized membrane-bound enzyme d-fructose dehydrogenase (FDH) preserves its full activity, follows ideal Michaelis-Menten kinetics, and shows improved stability compared to its behavior in solution. Even after 5 days, the immobilized FDH retained its full activity in meso, whereas a model hydrophilic enzyme, horseradish peroxidase, maintained only 21% of its original activity. We reason that the lipidic bilayers in the three-dimensional structures of cubic mesophases provide an ideal environment for the reconstitution of a membrane-bound enzyme. The preserved activity, long-term stability, and reusability demonstrate that these hybrid nanomaterials are ideal matrices for biosensing and biocatalytic fuel cell applications.

Mutation of heme c axial ligands in d-fructose dehydrogenase for investigation of electron transfer pathways and reduction of overpotential in direct electron transfer-type bioelectrocatalysis

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 0.2V, 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.

Improving the Direct Electron Transfer in Monolithic Bioelectrodes Prepared by Immobilization of FDH Enzyme on Carbon-Coated Anodic Aluminum Oxide Films

The present work reports the preparation of binderless carbon-coated porous films and the study of their performance as monolithic bioanodes. The films were prepared by coating anodic aluminum oxide (AAO) films with a thin layer of nitrogen-doped carbon by chemical vapor deposition. The films have cylindrical straight pores with controllable diameter and length. These monolithic films were used directly as bioelectrodes by loading the films with D-fructose dehydrogenase (FDH), an oxidoreductase enzyme that catalyzes the oxidation of D-fructose to 5-keto-D-fructose. The immobilization of the enzymes was carried out by physical adsorption in liquid phase and with an electrostatic attraction method. The latter method takes advantage of the fact that FDH is negatively charged during the catalytic oxidation of fructose. Thus the immobilization was performed under the application of a positive voltage to the CAAO film in a FDH-fructose solution in McIlvaine buffer (pH 5) at 25 oC. As a result, the FDH modified electrodes with the latter method show much better electrochemical response than that with the conventional physical adsorption method. Due to the singular porous structure of the monolithic films, which consists of an array of straight and parallel nanochannels, it is possible to rule out the effect of the diffusion of the D-fructose into the pores. Thus the improvement in the performance upon using the electrostatic attraction method can be ascribed not only to a higher uptake, but also to a more appropriate molecule orientation of the enzyme units on the surface of the electrodes.

Thermally reduced graphene oxide: The study and use for reagentless amperometric d-fructose biosensors

Aiming to create reagentless amperometric d-fructose biosensor, graphene based electrode materials have been synthesized by newly proposed thermal reduction of graphene oxide. The method allowed to separate and collect different fractions of thermally reduced graphene oxide (TRGO) with different physicochemical properties. The structural characteristics and surface morphologies of TRGO fractions were evaluated using SEM, XRD, TGA analysis, Raman spectroscopy and BET measurements. Three different fractions of TRGO were tested as electrode materials for d-fructose amperometric biosensors. The direct electron transfer (DET) from the active site of d-fructose dehydrogenase (FDH) to the electrode was achieved with all TRGO fractions. High values of the sensitivity (up to 14.5μAmM−1cm−2) are of the same order as these for other d-fructose sensors based on the synergistic mediated processes. The relationships between the structure of TRGO fractions and the molecular processes determining the effect of DET in bioelectrocatalysis by FDH have been studied. Stability of the d-fructose biosensors was also assessed. The best results were achieved when immobilization of FDH was performed using a crosslinking with glutaraldehyde. For the best group, after a period of 5 days the sensitivity of the biosensor for D-fructose determination decreased by less than 20%.

Amperometric fructose sensor based on ferrocyanide modified screen-printed carbon electrode

The first fructose sensor using a commercial screen-printed ferrocyanide/carbon electrodes (SPFCE) is reported here. The ferrocyanide is included in the carbon ink of the commercial screen-printed carbon electrode. The immobilization of enzyme d-fructose dehydrogenase (FDH) was carried out in an easy way. An aliquot of 10μL FDH was deposited on the electrode surface and left there until dried (approximately 1h) at room temperature. The sensor, so constructed, shows a good sensitivity to fructose (1.25μA/mM) with a slope deviation of ±0.02μA/mM and a linear range comprised between 0.1 and 1mM of fructose, with a limit of detection of 0.05mM. These sensors show good intersensors reproducibility after a previous pretreatment and a high stability. Fructose was determined in real samples as honey, Cola, fruit juices (orange, tomato, apple and pineapple), red wine, red and white grapes, musts and liquor of peach with a good accuracy.