Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial ...metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such a transcellular electron transfer might confer syntrophic metabolism that promotes growth and other activities of bacteria in the microbiota of humans and animals.
Efficient direct electron transfer (DET) between a cellobiose dehydrogenase mutant from Corynascus thermophilus (CtCDH C291Y) and a novel glassy carbon (GC)-modified electrode, obtained by direct ...electrodeposition of gold nanoparticles (AuNPs) was realized. The electrode was further modified with a mixed self-assembled monolayer of 4-aminothiophenol (4-APh) and 4-mercaptobenzoic acid (4-MBA), by using glutaraldehyde (GA) as cross-linking agent. The CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC platform showed an apparent heterogeneous electron transfer rate constant (ks) of 19.4 ± 0.6 s-1, with an enhanced theoretical and real enzyme surface coverage (Γtheor and Γreal) of 5287 ± 152 pmol cm-2 and 27 ± 2 pmol cm-2, respectively. The modified electrode was successively used as glucose biosensor exhibiting a detection limit of 6.2 μM, an extended linear range from 0.02 to 30 mM, a sensitivity of 3.1 ± 0.1 μA mM-1 cm-2 (R2 = 0.995), excellent stability and good selectivity. These performances compared favourably with other glucose biosensors reported in the literature. Finally, the biosensor was tested to quantify the glucose content in human saliva samples with successful results in terms of both recovery and correlation with glucose blood levels, allowing further considerations on the development of non-invasive glucose monitoring devices.
Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved ...performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.
•Fructose content is determined in honey and drinks with no interferences.•The biosensor method is rapid, simple with no sample pretreatment required.•The first fructose biosensor based on a graphene ...electrode is developed.•A biosensor based on a screen-printed graphene electrode is proposed.
This paper describes the development and performance of the first fructose biosensor based on a commercial screen-printed graphene electrode (SPGE). The electrode was modified with an osmium-polymer, which allowed the efficient wiring of the enzyme fructose dehydrogenase (FDH). The immobilization of both osmium-polymer and FDH was realized in an easy way. Aliquots of 10μL Os-polymer and 10μL FDH were thoroughly mixed with poly(ethylene glycol) (400) diglycidyl ether (PEDGE) and deposited on the electrode surface and left there to dry overnight. The biosensor exhibits a detection limit of 0.8μM, a linear range between 0.1 and 8mM, high sensitivity to fructose (2.15μAcm−2/mM), good reproducibility (RSD=1.9%), fast response time (3s) and a stability of 2 months when stored in the freezer.
The proposed fructose biosensor was tested in real food samples and validated with a commercial spectrophotometric enzymatic kit. No significant interference was observed with the proposed biosensor.
In this review, we analyze several types of graphene‐based sensors for glucose detection with respect to their preparation, properties and efficiency in electrochemical processes. Graphene may ...display different types of defects, which play a role in the electron transfer processes. Oxygenated groups on the edges of graphene planes reduce the graphene in‐plane conductivity, but may enhance the heterogeneous electron/proton transfer constant. Other positive effects of defects are related to the shortening of the distance between active centers and electrodes upon enzyme or protein immobilization. However, though by different mechanisms, all types of graphene enhance the electrochemical response at the electrode.
Cellobiose dehydrogenase catalyses the oxidation of aldoses—a simple reaction, a boring enzyme? No, neither for the envisaged bioelectrochemical applications nor mechanistically. The catalytic cycle ...of this flavocytochrome is complex and modulated by its flexible cytochrome domain, which acts as a built‐in redox mediator. This intramolecular electron transfer is modulated by the pH, an adaptation to the environmental conditions encountered or created by the enzyme‐producing fungi. The cytochrome domain forms the base from which electrons can jump to large terminal electron acceptors, such as redox proteins, and also enables by that path direct electron transfer from the catalytically active flavodehydrogenase domain to electrode surfaces. The application of electrochemical techniques to the elucidation of the molecular and catalytic properties of cellobiose dehydrogenase is discussed and compared to biochemical methods. The results lead to valuable insights into the function of this cellulose‐bound enzyme, but also form the basis of exciting applications in biosensors, biofuel cells and bioelectrocatalysis.
An enzyme with contacts: Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome consisting of flavodehydrogenase (DHCDH) and cytochrome (CYTCDH) domains with the ability to communicate with an electrode by direct (DET) or mediated electron transfer (MET; see picture). Bioelectrochemical techniques used to elucidate basic mechanistic and kinetic properties of CDH and pioneer potential applications in biosensors, biofuel cells and bioelectrosynthesis are presented.
•Electrode–microorganism electron transfer (ET) interactions are discussed.•Mediators for improving electrode–microorganism communication are reviewed.•Electrode materials in electrode–microorganism ...system are discussed.
Over the past decades great attention has been paid to the phenomenon of microorganisms’ ability to transfer electrons between their metabolism and solid conductive surfaces. This fact keeps potential to gain extensive reliance for applications including electricity production coupled to wastewater treatment, bioremediation, and synthesis of value products. Effective electrochemical communication directly or via various mediated systems between microorganisms and electrodes is a challenge of fundamental interdisciplinary research. The present review discusses the main critical factors affecting efficient electrochemical “wiring” living cells to conductive electrode surfaces, including the molecular mechanism that makes electron exchange possible for various microorganisms, utilization of different types of mediators and new electrode materials to enhance microbial kinetics as opposed to other bioelectrochemical systems based on purified enzymes 1,2.
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 c 1 and heme c 3 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 c 1 and c 2), with a maximum current density of 485 ± 21 μA cm–2 at 0.4 V at a sweep rate of 10 mV s–1. In contrast, for the plain SWCNT-modified GCE only one catalytic wave and one couple of redox waves were observed. Adsorbing FDH directly onto a GCE showed no non-turnover electrochemistry of FDH, and in the presence of fructose only a slight catalytic effect could be seen. These differences can be explained by considering the hydrophobic pocket close to heme c 1, heme c 2, and heme c 3 of the cytochrome domain at which the anthracenyl aromatic structure could interact through π–π interactions with the aromatic side chains of the amino acids present in the hydrophobic pocket of FDH.
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 CaCl
2
, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca
2+
and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl
2
, addition of MgCl
2
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 CaCl
2
was added. However, by further increasing the concentration of CaCl
2
up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl
2
concentration. Addition of MgCl
2
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 CaCl
2
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 Abstract
Fructose 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 CaCl
2
to the buffer solution, whereas MgCl
2
had an “inhibition” effect. Moreover, the additions of KCl or NaCl led to a slight current increase
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