Artificial Electron Mediators Research Articles

Specific interaction of resorufin to outer-membrane cytochrome OmcE of Geobacter sulfurreducens: A new insight on artificial electron mediators in promoting extracellular electron transfer

Bioelectrochemical system (BES) is a unique biotechnology for wastewater treatment and energy recovery, and extracellular electron transfer (EET) between microbe and electrode is the key to optimize the performance of BESs. Resazurin is an effective artificial compound that can promote EET in BESs, but the way how it transports electrons is not fully understood. In this study differential pulse voltammetry revealed that the redox potential of resorufin (RR) (intermediate of resazurin reduction, actual electron mediator) within Geobacter sulfurreducens biofilm was positively shifted by 100 mV than that of free RR, and this shift was attenuated by the mutation of outer-membrane cytochrome gene omcE but not by omcS and omcZ mutation, indicating that RR specifically interacted with OmcE. By using heterologously expressed OmcE monomers in Escherichia coli, it was found that RR bonded with OmcE monomers with a moderate intensity (dissociation constant of 720 nM), and their interaction obviously increased the content of α helix in OmcE monomers. Biomolecular analysis indicated that heme II of OmcE monomer might be the binding site for RR (binding energy of -7.01 kJ/mol), which were favorable for electron transfer within OmcE-RR complex. Comparative transcriptomics showed that RZ addition significantly upregulated the expression of omcE, periplasmic cytochrome gene ppcB, and outer-membrane genes omaB, ombB and omcB, thus, it was hypothesized that OmcE-bound RR might serve as potential electron acceptor of OmbB-OmaB-OmcB porin complex which passes electrons across outer membrane. Our work demonstrated a new pathway of artificial electron mediators in facilitating EET in Geobacter species, which may guide the application of electron mediator in improving the performance of BESs.

Identifying a key spot for electron mediator-interaction to tailor CO dehydrogenase’s affinity

Fe‒S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), employ sophisticated artificial electron mediators like viologens to serve as potent biocatalysts capable of cleaning-up industrial off-gases at stunning reaction rates. Unraveling the interplay between these enzymes and their associated mediators is essential for improving the efficiency of CODHs. Here we show the electron mediator-interaction site on ChCODHs (Ch, Carboxydothermus hydrogenoformans) using a systematic approach that leverages the viologen-reactive characteristics of superficial aromatic residues. By enhancing mediator-interaction (R57G/N59L) near the D-cluster, the strategically tailored variants exhibit a ten-fold increase in ethyl viologen affinity relative to the wild-type without sacrificing the turn-over rate (kcat). Viologen-complexed structures reveal the pivotal positions of surface phenylalanine residues, serving as external conduits for the D-cluster to/from viologen. One variant (R57G/N59L/A559W) can treat a broad spectrum of waste gases (from steel-process and plastic-gasification) containing O2. Decoding mediator interactions will facilitate the development of industrially high-efficient biocatalysts encompassing gas-utilizing enzymes.

A highly sensitive and stable electrochemical nitrate biosensor

We report an electrochemical nitrate biosensor incorporating the enzyme nitrate reductase (NR) from the fungus Neurospora crassa entrapped within a polymeric chitosan matrix on a glassy carbon (GC) electrode. The artificial electron mediator anthraquinone sulfonate (AQ) is utilized as an electron transfer partner (co-substrate) for NR. The NR enzyme was effectively immobilized within the chitosan matrix without the need for a dialysis membrane, and exhibited high sensitivity and selectivity for nitrate using either rotating disk cyclic voltammetry or constant potential amperometry methods. A particular feature was the longevity of the biosensor which retained more than 70% activity over a period of 3 months. The practical application of the biosensor was demonstrated by the determination of nitrate concentrations in lake and river water samples without any pre-treatment, and the results were validated with a reliable spectroscopic assay.

Artificial Electron Mediator with Nanocubic Architecture Highly Promotes Microbial Electrosynthesis from Carbon Dioxide

Microbial electrosynthesis (MES) is an emerging strategy for converting electrical energy into chemical energy. Many efforts have been made toward the enhancement of cathode-microorganism interacti...

Elucidation of the intra- and inter-molecular electron transfer pathways of glucoside 3-dehydrogenase

Glucoside 3‑dehydrogenase (G3DH) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase that catalyzes the oxidation of the hydroxy group on the C-3 position of pyranose and shows broad substrate specificity by oxidizing many saccharides. Due to unique site specificity and wide substrate specificity, G3DHs can be used for synthesis of sugar derivatives, anodic catalysis in biofuel cells, multi-sugar analysis using enzyme electrode, and for enzymatic detection of 1,5‑anhydro‑d‑glucitol, a clinical marker for diabetes. However, few studies have focused on the fundamental biochemical properties of G3DH, including its electron transfer pathway. In this study, we isolated the G3DH gene from Rhizobium radiobacter, a homologue of marine bacterial G3DH, and reported that the isolated gene fragment contains the genes encoding the G3DH catalytic subunit (subunit I), G3DH hitch-hiker subunit (subunit II), and cytochrome c-like molecule (CYTc). Furthermore, we report the recombinant expression of G3DH from R. radiobacter in Escherichia coli, the characterization of recombinant G3DH and the investigation of the molecular electron pathway of G3DH. We first prepared the G3DH subunit I-II complex using a co-expression vector for both subunits. The G3DH subunit I-II complex showed dye-mediated G3DH activity toward methyl‑α‑d‑glucoside (MαG). Electron paramagnetic resonance (EPR) and inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses revealed that subunit I contains an iron-sulfur cluster. We, then, prepared recombinant CYTc and revealed that it is capable of accepting electrons from the catalytic subunit of G3DH by absorption spectrum analysis. These results suggested that R. radiobacter G3DH possesses an iron‑sulfur cluster that may play an important role in the electron transfer from FAD to cytochrome c like molecule, which is an external electron acceptor of G3DH. Furthermore, we demonstrated that CYTc mediate the electron transfer from G3DH to electrode without the artificial electron mediator.

Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase

l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9–12% due to oxygen interference. The Ala96Leu mutant maintained 56–69% of the response current at the same l-lactate level and minimized the relative bias error to −19% from −49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations.

Mediator Preference of Two Different FAD-Dependent Glucose Dehydrogenases Employed in Disposable Enzyme Glucose Sensors.

Most commercially available electrochemical enzyme sensor strips for the measurement of blood glucose use an artificial electron mediator to transfer electrons from the active side of the enzyme to the electrode. One mediator recently gaining attention for commercial sensor strips is hexaammineruthenium(III) chloride. In this study, we investigate and compare the preference of enzyme electrodes with two different FAD-dependent glucose dehydrogenases (FADGDHs) for the mediators hexaammineruthenium(III) chloride, potassium ferricyanide (the most common mediator in commercial sensor strips), and methoxy phenazine methosulfate (mPMS). One FADGDH is a monomeric fungal enzyme, and the other a hetero-trimeric bacterial enzyme. With the latter, which contains a heme-subunit facilitating the electron transfer, similar response currents are obtained with hexaammineruthenium(III), ferricyanide, and mPMS (6.8 µA, 7.5 µA, and 6.4 µA, respectively, for 10 mM glucose). With the fungal FADGDH, similar response currents are obtained with the negatively charged ferricyanide and the uncharged mPMS (5.9 µA and 6.7 µA, respectively, for 10 mM glucose), however, no response current is obtained with hexaammineruthenium(III), which has a strong positive charge. These results show that access of even very small mediators with strong charges to a buried active center can be almost completely blocked by the protein.

Electrochemical characterization of direct electron uptake in electrical microbially influenced corrosion of iron by the lithoautotrophic SRB Desulfopila corrodens strain IS4

Corrosion of iron infrastructure in anoxic environments is commonly ascribed to the metabolic activity of sulfate-reducing bacteria (SRB), which can influence electrochemical processes on the metallic surface. The present study characterizes electron transfer processes at the electrode/microorganism interface of the specialized lithoautotrophic SRB Desulfopila corrodens strain IS4 that is capable of taking up electrons directly from elemental iron, thereby leading to severe corrosion. Electrochemical analysis of the highly corrosive SRB is performed in bio-electrochemical cells operated with artificial seawater under anoxic conditions, complemented by infrared spectroelectrochemical analysis and environmental SEM observations. In order to provide clear insights into electron transfer processes, sterile controls and a non-corrosive hydrogenotrophic control strain are analyzed in parallel. Remarkably, the electron uptake from electrodes by D. corrodens strain IS4 is not restricted to iron serving as the sole electron donor for the microbial metabolism, but instead proceeds also on graphite and doped germanium cathodes. Consequently, a well-defined electrochemical analysis of the electron uptake mechanism is possible, since corrosion precipitates typically observed on iron electrodes are absent on both of the other cathode materials. Direct electron transfer in the absence of artificial electron mediators is achieved at a potential of −0.4V vs. SHE under neutral conditions. Electrochemical and infrared spectroelectrochemical analysis indicated c-type cytochromes as the redox active components associated with the outer membrane to be involved in the direct electron uptake from reduced surfaces by D. corrodens strain IS4.

Selective microbial electrosynthesis of methane by a pure culture of a marine lithoautotrophic archaeon

Reduction of carbon dioxide to methane by microorganisms attached to electrodes is a promising process in terms of renewable energy storage strategies. However the efficient and specific electrosynthesis of methane by methanogenic archaea on cathodes needs fundamental investigations of the electron transfer mechanisms at the microbe–electrode interface without the addition of artificial electron mediators. Using well-defined electrochemical techniques directly coupled to gas chromatography and surface analysis by scanning electron microscopy, it is shown that a pure culture of the marine lithoautotrophic Methanobacterium-like archaeon strain IM1 is capable to utilize electrons from graphite cathodes for a highly selective production of methane, without hydrogen serving as a cathode-generated electron carrier. Microbial electrosynthesis of methane with cultures of strain IM1 is achieved at a set potential of −0.4V vs. SHE and is characterized by a coulomb efficiency of 80%, with rates reaching 350nmold−1cm−2 after 23days of incubation. Moreover, potential step measurements reveal a biologically catalyzed hydrogen production at potentials more positive than abiotic hydrogen evolution on graphite, indicating that an excessive supply of electrons to strain IM1 results in proton reduction rather than in a further increase of methane production.

Engineering glucose oxidase to minimize the influence of oxygen on sensor response

Glucose oxidase (GOx) is an important industrial enzyme and is recognized as the gold standard for monitoring blood glucose. However, due to its inherent oxidase property, the presence of oxygen affects electrochemical measurements of venous blood glucose employing artificial electron mediators. We therefore attempted to engineer Penicillium amagasakiense-derived GOx into a dehydrogenase by focusing on the amino acid residues predicted to interact with oxygen. Our rational amino acid substitution approach resulted in the construction of the Ser114Ala/Phe355Leu mutant, which has an 11-fold decrease in oxidase activity and 2.8-fold increase in dehydrogenase activity compared with wild-type GOx. As a result, the dehydrogenase/oxidase activity ratio of the engineered enzyme was 32-fold greater than that of the wild-type enzyme. The enzyme sensor constructed with Ser114Ala/Phe355Leu was considerably less affected by oxygen than the wild-type GOx-based sensor at lower glucose concentrations.

A rapid and sensitive p-benzoquinone-mediated bioassay for determination of heavy metal toxicity in water

Determining and monitoring toxicity of chemicals in water are very important for human health and country security. Electrochemical measurement of respiratory chain activity is a rapid and reliable screening of the toxicity towards microorganisms. Here, we report a rapid and sensitive toxicity bioassay using p-benzoquinone as the artificial electron mediator and Escherichia coli as the test organism. Four heavy metal ions, Cu(2+), Ag(+), Hg(2+) and Co(2+), are tested as the model toxicants, and the corresponding 50% respiration inhibition concentrations (IC50) are determined to be 0.95, 8.14, 11.69 and 42.76 mg L(-1), respectively. Based on the IC50 values, the descending order of toxicity is: Cu(2+) > Ag(+) > Hg(2+) > Co(2+). The presented bioassay not only provides a good foundation for further toxicity tests using E. coli, but also the potential for expanding the technique to utilize other bacteria with complementary toxicity responses, thereby allowing use of the bioassay in a wide range of applications.

Tuning Fructosyl Peptidyl Oxidase into Dehydrogenase and Its Application for the Construction of an Enzyme Electrode

Fructosyl valyl-histidine, which is released from protease digestion of the major diabetes indicator HbA1c, is recognized as a target molecule for HbA1c sensors. The flavoenzyme fructosyl amino acid oxidase (FAOD) is an enzyme catalyzing the oxidative deglycation of fructosyl amino acids, with those also possessing high activity towards fructosyl dipeptides being referred to as fructosyl peptidyl oxidases (FPOXs). The high oxygen reactivity of FAODs and FPOXs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. We successfully modified the proton relay system of Pichia N1-1 FAOD, significantly decreasing its catalytic ability to employ oxygen as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of oxygen. We expect that similar engineering of a recently discovered FPOX will produce an enzyme that is ideally suited for HbA1c sensing.

Development of Enterobacter aerogenes fuel cells: From in situ biohydrogen oxidization to direct electroactive biofilm

This study described an Enterobacter aerogenes-catalyzed microbial fuel cell (MFC) with a carbon-based anode that exhibited a maximum power density of 2.51 W/m(3) in the absence of artificial electron mediators. The MFC was started up rapidly, within hours, and the current generation in the early stage was demonstrated to result from in situ oxidation of biohydrogen produced by E. aerogenes during glucose fermentation. Over periodic replacement of substrate, both planktonic biomass in the culture liquid and hydrogen productivity decreased, while increased power density and coulombic efficiency and decreased internal resistance were unexpectedly observed. Using scanning electron microscopy and cyclic voltammetry, it was found that the enhanced MFC performance was associated with the development of electroactive biofilm on the anodic surface, proposed to involve an acclimation and selection process of E. aerogenes cells under electrochemical tension. The significant advantage of rapid start-up and the ability to develop an electroactive biofilm identifies E. aerogenes as a suitable biocatalyst for MFC applications.

Engineering of dye-mediated dehydrogenase property of fructosyl amino acid oxidases by site-directed mutagenesis studies of its putative proton relay system

The flavoenzyme fructosyl amino acid oxidase (FAOD) catalyzes the oxidative deglycation of fructosyl amino acids, model compounds of glycated proteins. The high oxygen reactivity of FAODs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. To alter their electron acceptor availability, site-directed mutagenesis was carried out on conserved residues predicted to be involved in the proton relay system (PRS) of two eukaryotic FAODs, the FAOD from the marine yeast Pichia sp. N1-1 and amadoriase II from the fungus Aspergillus fumigatus. The substitution of a single conserved Asn residue in the putative PRS, Asn47Ala of N1-1 FAOD and Asn52Ala of amadoriase II, resulted in significant loss in the catalytic ability to employ O(2) as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of O(2).

Progress of Electrochemical Biosensors Based on Nicotinamide Adenine Dinucleotide (phosphate)-Dependent Dehydrogenases

Abstract More than four hundred dehydrogenases catalyze some reactions in the presence of nicotinamide adenine dinucleotide (NAD + ) or nicotinamide adenine dinucleotide phosphate (NADP + ) as the coenzyme, so the electrochemical oxidation of NAD(P)H is quite important for the construction of NAD(P)-dependent dehydrogenase biosensors. In this article, the electrochemical oxidation reactions of coenzymes in the presence of artificial electron mediators were introduced. In addition, design and application of NAD (P)-dependent dehydrogenase biosensors were reviewed.

PQQ glucose dehydrogenase with novel electron transfer ability

PQQ glucose dehydrogenase from Acinetobacter calcoaceticus (GDH-B) is one of the most industrially attractive enzymes, as a sensor constituent for glucose sensing, because of its high catalytic activity and insensitivity to oxygen. We attempted to engineer GDH-B to enable electron transfer to the electrode in the absence of artificial electron mediator by mimicking the domain structure of the quinohemoprotein ethanol dehydrogenase (QH-EDH) from Comamonas testosteroni, which is composed of a PQQ-containing catalytic domain and a cytochrome c domain. We genetically fused the cytochrome c domain of QH-EDH to the C-terminal of GDH-B. The constructed fusion protein showed not only intra-molecular electron transfer, between PQQ and heme of the cytochrome c domain, but also electron transfer from heme to the electrode, thereby allowing the construction of a direct electron transfer-type glucose sensor.

Glucose Biosensors Based on Carbon Nanotube Nanoelectrode Ensembles

This paper describes the development of glucose biosensors based on carbon nanotube (CNT) nanoelectrode ensembles (NEEs) for the selective detection of glucose. Glucose oxidase was covalently immobilized on CNT NEEs via carbodiimide chemistry by forming amide linkages between their amine residues and carboxylic acid groups on the CNT tips. The catalytic reduction of hydrogen peroxide liberated from the enzymatic reaction of glucose oxidase upon the glucose and oxygen on CNT NEEs leads to the selective detection of glucose. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence of common interferents (e.g., acetaminophen, uric and ascorbic acids), avoiding the generation of an overlapping signal from such interferers. Such an operation eliminates the need for permselective membrane barriers or artificial electron mediators, thus greatly simplifying the sensor design and fabrication.

THE APPLICATION OF CYTOCHROMES AS THE INTERFACE MOLECULE TO FACILITATE THE ELECTRON TRANSFER FOR PQQ GLUCOSE DEHYDROGENASE EMPLOYING MEDIATOR TYPE GLUCOSE SENSOR

ABSTRACT In order to improve the sensor response of mediator-type glucose enzyme electrode, we focused on the application of the electron transfer proteins, cytochromes, as interface molecules to facilitate the electron transfer from enzyme to artificial electron mediator. In this paper we used cytochrome c and cytochrome b 562 for the improvement of sensor signal of glucose enzyme sensor employing glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH). When sensors were operated using either potassium ferricyanide or 1-methoxy-5-methylphenazinium methylsulfate (mPMS) as the artificial electron mediator, the response was over 30-fold greater with the co-immobilization of either cytochrome c or cytochrome b 562 than with PQQGDH alone. The impact of the cytochrome co-immobilization was dependent on the amount of cytochromes, indicating that these cytochromes facilitated the electron transfer from the PQQGDH redox center to the artificial electron mediators used in the sensor system. These results demonstrated the future application of cytochromes as an essential component for the improvement of sensor response in the redox enzyme-based amperometric sensors.

Electrochemical Oxidation of Cholesterol Catalyzed by Cholesterol Oxidase with Use of an Artificial Electron Mediator

Several redox dyes of phenazine and phenothiazine derivatives including 1-methoxy-5-methylphenazinium methosulfate (MPMS), thionin, azure A, and azure C work well as electron acceptors for reduced cholesterol oxidase (EC 1.1.3.6) (ChOx). Kinetic parameters and equilibrium constants of the enzymatic oxidation of cholesterol obtained suggest that the molecular size and/or hydrophobicity of the redox dyes determines their abilities as the electron acceptors. The electrochemical sensitivity to cholesterol in the detection system using ChOx and the above-mentioned redox compounds is determined by the electron acceptability of the redox compounds, and the employment of either MPMS or thionin allows electrochemical determination of cholesterol at a low electrode potential (0 V vs SCE) in the concentration range 0.25-2.5 mmol dm(-3), which covers that in normal human blood.

Highly selective biosensing of lactate at lactate oxidase containing rhodium-dispersed carbon paste electrodes

A remarkably selective lactate biosensor, based on the immobilization of lactate oxidase within a rhodium-dispersed carbon paste is described. The effective catalytic action of the rhodium microparticles towards the generated hydrogen peroxide or consumed oxygen allows tuning of the operating potential to a region where unwanted reactions are prevented. Contributions from all major easily oxidizable substances are eliminated, without the need for membrane barriers or artificial electron mediators. The detection limit is 1.5 × 10 −5 M and the response time is 8 s. A fast and sensitive flow injection operation is illustrated.