PQQ-dependent Alcohol Dehydrogenase Research Articles

Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation

BackgroundBacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in Komagataeibacter spp. by promoting efficient glucose utilization.ResultsK. sucrofermentans ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation.ConclusionsThe results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in K. sucrofermentans ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.

Biotransformation of Deoxynivalenol by a Dual-Member Bacterial Consortium Isolated from Tenebrio molitor Larval Feces.

In this study, a dual-member bacterial consortium with the ability to oxidize deoxynivalenol (DON) to 3-keto-DON, designated SD, was first screened from the feces of Tenebrio molitor larvae. This consortium consisted of Pseudomonas sp. SD17-1 and Devosia sp. SD17-2, as determined by 16S rRNA-based phylogenetic analysis. A temperature of 30 °C, a pH of 8.0-9.0, and an initial inoculum concentration ratio of Devosia to Pseudomonas of 0.1 were optimal single-factor parameters for the DON oxidation activity of the bacterial consortium SD. Genome-based bioinformatics analysis revealed the presence of an intact PQQ biosynthesis operon (pqqFABCDEG) and four putative pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) genes in the genomes of Pseudomonas strain SD17-1 and Devosia strain SD17-2, respectively. Biochemical analyses further confirmed the PQQ-producing phenotype of Pseudomonas and the DON-oxidizing enzymatic activities of two of four PQQ-dependent ADHs in Devosia. The addition of PQQ-containing a cell-free fermentation supernatant from Pseudomonas activated DON-oxidizing activity of Devosia. In summary, as members of the bacterial consortium SD, Pseudomonas and Devosia play indispensable and complementary roles in SD's oxidation of DON. Specifically, Pseudomonas is responsible for producing the necessary PQQ cofactor, whereas Devosia expresses the PQQ-dependent DON dehydrogenase, together facilitating the oxidation of DON.

Four PQQ-Dependent Alcohol Dehydrogenases Responsible for the Oxidative Detoxification of Deoxynivalenol in a Novel Bacterium Ketogulonicigenium vulgare D3_3 Originated from the Feces of Tenebrio molitor Larvae.

Deoxynivalenol (DON) is frequently detected in cereals and cereal-based products and has a negative impact on human and animal health. In this study, an unprecedented DON-degrading bacterial isolate D3_3 was isolated from a sample of Tenebrio molitor larva feces. A 16S rRNA-based phylogenetic analysis and genome-based average nucleotide identity comparison clearly revealed that strain D3_3 belonged to the species Ketogulonicigenium vulgare. This isolate D3_3 could efficiently degrade 50 mg/L of DON under a broad range of conditions, such as pHs of 7.0-9.0 and temperatures of 18-30 °C, as well as during aerobic or anaerobic cultivation. 3-keto-DON was identified as the sole and finished DON metabolite using mass spectrometry. In vitro toxicity tests revealed that 3-keto-DON had lower cytotoxicity to human gastric epithelial cells and higher phytotoxicity to Lemna minor than its parent mycotoxin DON. Additionally, four genes encoding pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases in the genome of isolate D3_3 were identified as being responsible for the DON oxidation reaction. Overall, as a highly potent DON-degrading microbe, a member of the genus Ketogulonicigenium is reported for the first time in this study. The discovery of this DON-degrading isolate D3_3 and its four dehydrogenases will allow microbial strains and enzyme resources to become available for the future development of DON-detoxifying agents for food and animal feed.

Providing octane degradation capability to Pseudomonas putida KT2440 through the horizontal acquisition of oct genes located on an integrative and conjugative element.

The extensive use of petrochemicals has produced serious environmental pollution problems; fortunately, bioremediation is considered an efficient way to fight against pollution. In line with Synthetic Biology is that robust microbial chassis with an expanded ability to remove environmental pollutants are desirable. Pseudomonas putida KT2440 is a robust lab microbe that has preserved the ability to survive in the environment and is the natural host for the self-transmissible TOL plasmid, which allows metabolism of toluene and xylenes to central metabolism. We show that the P. putida KT2440 (pWW0) acquired the ability to use octane as the sole C-source after acquisition of an almost 62-kb ICE from a microbial community that harbours an incomplete set of octane metabolism genes. The ICE bears genes for an alkane monooxygenase, a PQQ-dependent alcohol dehydrogenase and aldehyde dehydrogenase but lacks the electron donor enzymes required for the monooxygenase to operate. Host rubredoxin and rubredoxin reductase allow metabolism of octane to octanol. Proteomic assays and mutants unable to grow on octane or octanoic acid revealed that metabolism of octane is mediated by redundant host and ICE enzymes. Octane is oxidized to octanol, octanal and octanoic acid, the latter is subsequently acylated and oxidized to yield acetyl-CoA that is assimilated via the glyoxylate shunt; in fact, a knockout mutant in the aceA gene, encoding isocitrate lyase was unable to grow on octane or octanoic acid.

Bioinorganic insights of the PQQ-dependent alcohol dehydrogenases.

Among the several alcohol dehydrogenases, PQQ-dependent enzymes are mainly found in the α, β, and γ-proteobacteria. These proteins are classified into three main groups. Type I ADHs are localized in the periplasm and contain one Ca2+-PQQ moiety, being the methanol dehydrogenase (MDH) the most representative. In recent years, several lanthanide-dependent MDHs have been discovered exploding the understanding of the natural role of lanthanide ions. Type II ADHs are localized in the periplasm and possess one Ca2+-PQQ moiety and one heme c group. Finally, type III ADHs are complexes of two or three subunits localized in the cytoplasmic membrane and possess one Ca2+-PQQ moiety and four heme c groups, and in one of these proteins, an additional [2Fe-2S] cluster has been discovered recently. From the bioinorganic point of view, PQQ-dependent alcohol dehydrogenases have been revived recently mainly due to the discovery of the lanthanide-dependent enzymes. Here, we review the three types of PQQ-dependent ADHs with special focus on their structural features and electron transfer processes. The PQQ-Alcohol dehydrogenases are classified into three main groups. Type I and type II ADHs are located in the periplasm, while type III ADHs are in the cytoplasmic membrane. ADH-I have a Ca-PQQ or a Ln-PQQ, ADH-II a Ca-PQQ and one heme-c and ADH-III a Ca-PQQ and four hemes-c. This review focuses on their structural features and electron transfer processes.

Engineered PQQ-Dependent Alcohol Dehydrogenase for the Oxidation of 5-(Hydroxymethyl)furoic Acid

Furan-2,5-dicarboxylic acid (FDCA) is a bio-based platform chemical with the potential to replace terephthalic acid in the production of polymers. A critical step for enzymatic and whole-cell production of FDCA from 5-(hydroxymethyl)furfural (HMF) is the transformation of 5-(hydroxymethyl)furoic acid (HMFA) into 5-formylfuroic acid (FFA). Here, we establish periplasmic pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADHs) as biocatalytic tools for the oxidation of HMFA, HMF, and 5-formylfurfural (FFF). Further, we identify several amino acid residues including the “lid loop” of the substrate channel as promising targets for future engineering steps toward a fully periplasmic oxidation pathway to FDCA.

Characterization of methylated azopyridine as a potential electron transfer mediator for electroenzymatic systems

Abstract N , N' -dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron transfer mediator for oxidation reactions catalyzed by NAD + - and pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity with MAZP was defined as (2.2 ± 0.1) × 10 5 M −1 s −1 , whereas the bimolecular rate constant of reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was determined to be (4.7 ± 0.1) × 10 4 M −1 s −1 . The use of MAZP for the regeneration of the cofactors was investigated by applying the electrochemical oxidation of the mediator. The total turnover numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD + -dependent alcohol dehydrogenase depended on the concentration of the substrate and the duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable, and the turnover number of the enzyme reached a value of 2.3 × 10 3 . In addition, oxidation of 1,2-propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded enantioselectively to yield l -lactic acid.

Harvesting Energy using Biocatalysts

<i>Harvesting Energy using Biocatalysts</i>

Membraneless enzymatic ethanol/O2 fuel cell: Transitioning from an air-breathing Pt-based cathode to a bilirubin oxidase-based biocathode

The bioelectrooxidation of ethanol was investigated in a fully enzymatic membraneless ethanol/O2 biofuel cell assembly using hybrid bioanodes containing multi-walled carbon nanotube (MWCNT)-decorated gold metallic nanoparticles with either a pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) enzyme or a nicotinamide adenine dinucleotide (NAD+)-dependent ADH enzyme. The biofuel cell anode was prepared with the PQQ-dependent enzyme and designed using either a direct electron transfer (DET) architecture or via a mediated electron transfer (MET) configuration through a redox polymer, 1,1′-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI). In the case of the bioanode containing the NAD+-dependent enzyme, only the mediated electron transfer mechanism was employed using an electropolymerized methylene green film to regenerate the NAD+ cofactor. Regardless of the enzyme being employed at the anode, a bilirubin oxidase-based biocathode prepared within a DET architecture afforded efficient electrocatalytic oxygen reduction in an ethanol/O2 biofuel cell. The power curves showed that DET-based bioanodes via the PQQ-dependent ADH still lack high current densities, whereas the MET architecture furnished maximum power density values as high as 226 ± 21 μW cm−2. Considering the complete membraneless enzymatic biofuel cell with the NAD+-dependent ADH-based bioanode, power densities as high as 111 ± 14 μW cm−2 were obtained. This shows the advantage of PQQ-dependent ADH for membraneless ethanol/O2 biofuel cell applications.

Membraneless Ethanol/O2 Biofuel Cell Using PQQ-Dependent Alcohol and Aldehyde Dehydrogenase Along with Au Nanoparticles

Introduction Among the various alternative technologies for efficient energy conversion, the use of enzymes immobilized onto the surface of electrodes as the main catalyst in biological fuel cells has been extensively reported [1]. The biofuel cell provide a means to obtain clean, renewable energy and have great potential for maybe in the future be used as alternative energy source for low power devices. Some key issues in the development of this device, such as lifetime, stability of enzymes, achievement of higher power densities, overcoming difficulties in the electron transfer between enzymes and electrodes, and the improvement of techniques for enzyme immobilization are still important objects of research. We have previously studied the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles along with different immobilization techniques [2]. Here, we investigated the use of these bioelectrodes in a complete membrane-less enzymatic biofuel cell employing bilirubin oxidase-based biocathodes that use anthracene-modified MWCNTs to allow for the electrocatalytic oxygen reduction. Experimental The PQQ-dependent enzymes ADH and AldDH were isolated and purified from Gluconobacter sp.33 (DSM 3504) as previously described [2]. MWCNTs-decorated gold nanoparticles were synthesized by the dendrimer-encapsulated Au nanoparticle method, in which the metallic species are firstly stabilized onto the NH2-functionalized PAMAM dendrimers, reduced with NaBH4, extracted with n-dodecanethiol, and finally supported onto the surface of MWCNTs for a final 2 wt% metal catalyst loading. DET bioanodes were prepared by immobilizing the purified enzymes onto Toray carbon paper along with the MWCNTs-decorated gold nanoparticles and Tbab-modified Nafion. MET bioanodes were obtained by a redox polymer based on ferrocene-modified linear polyethyleneimine (LPEI), which was prepared by the attachment of 3-(di-methyl ferrocenyl) propyl groups to ca.17% of its nitrogen atoms (FcMe2-C3-LPEI). Enzyme immobilization was achieved by pipetting 40 µL of a vortex-mixed solution containing 6.75 mg mL-1 of the redox polymer, 2.9 mg mL-1 purified PQQ-containing enzymes, and 0.15 vol% ethyleneglycoldiglycidylether (EGDGE) directly onto the carbon paper electrode containing MWCNTs-decorated gold nanoparticles. Bilirubin oxidase-based biocathodes were achieved by immobilizing the enzymes onto the same carbon paper support using Tbab-modified Nafion and anthracene-modified MWCNTs that orientate the enzymes for DET bioelectrocatalytic oxygen reduction. Biofuel cell tests were conducted in a single compartment cell containing 0.2M Tris–HCl buffer pH 7 and 1 mM CaCl2, controlled by a CH Instruments 611C potentiostat. Discussion Figure 1 show representative polarization and power curves for the membrane-less ethanol/O2 biofuel cell test in 0.2M Tris–HCl buffer pH 7, 1 mM CaCl2, and 20 mM ethanol, using the hybrid PQQ-dependent ADH/AldDH bioanode in MET architecture and the bilirubin oxidase-based biocathode. In this situation, an open circuit potential of 657 mV and maximum current and power density of 0,354 mA cm-2 and 85 µW cm-2, were obtained respectively. Complete membrane-less enzymatic biofuel cell tests employing DET bioanodes showed that this bioanode configuration still lacks of high current density, in fact, power density values as low as 12 µW cm-2were registered. Those data are mainly related to the poor electronic communication between the enzymes’ redox centers and the electrode surface, as well as the minor control of the enzyme arrangement for reaching efficient DET. In the case of the bioanodes operating via MET employing ferrocene-modified LPEI redox polymer, efficient energy conversion capability in ethanol/air biofuel cells is observed. Acknowledgements This work was supported by FAPESP, CNPq, and CAPES. References 1) M. Rasmussen, S. Abdellaoui, S.D. Minteer, Biosens. Bioelectron. 76 (2015) 91-102. 2) S. Aquino Neto, R. D. Milton, D. P. Hickey, A. R. De Andrade, S.D. Minteer, Biosens. Bioelectron. 72 (2015) 247-254. Figure 1. Polarization and power curves for the membrane-less ethanol/O2 biofuel cell test in 0.2M Tris–HCl buffer pH 7, 1 mM CaCl2, and 20 mM ethanol. Figure 1

Crystal structure of quinone‐dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates

The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

High current density PQQ-dependent alcohol and aldehyde dehydrogenase bioanodes.

In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells.

New mediators for biosensors based on PQQ-dependent alcohol dehydrogenases

A set of 10 quinoidal compounds synthesized enzymatically using laccase from Coriolopsis byrsina GRB13 have been examined as redox mediators applicable for bioelectrocatalytic systems using two types of pyrroloquinoline quinone-dependent alcohol dehydrogenases – periplasmic (s-PQQ-ADH) and membrane-bound (m-PQQ-ADH). The efficiency of the biosensors have been evaluated for both types of enzymes and varied in range from 2×10−7A for Phenazine-2,3-diamine to 26×10−7A for 2-(3-Nitroanilino)-1,4-benzoquinone. Experimental data correlated with the molecular properties of mediators using results of ab initio quantum chemical calculations. The oxidation potential of mediators as well as the maximum current (Imax) of biosensors correlated with the calculated energy of the lowest unoccupied molecular orbital (ELUMO) of mediators. In the case of Imax, this result indicates the rate-limiting character of enzymatic mediator reduction in the overall process of biosensor response. The proposed approach demonstrates that the advance determination of molecular properties of potential mediators by ab initio quantum chemical calculations might be a useful instrument in the development process of the redox enzymes based biosensors.

A unique enzyme of acetic acid bacteria, PQQ-dependent alcohol dehydrogenase, is also present in Frateuria aurantia

A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558(T). Although F. aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80% amino acid (AA) identity to AdhA and 68% AA identity to AdhB of Ga. europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.

Direct electron transfer-based bioanodes for ethanol biofuel cells using PQQ-dependent alcohol and aldehyde dehydrogenases

This paper compares the performance of a DET (direct electron transfer) bioanode containing both PQQ-ADH (pyrroloquinoline quinone-dependent alcohol dehydrogenase) and PQQ-AldDH (PQQ-dependent aldehyde dehydrogenase) immobilized onto different modified electrode surfaces employing either a tetrabutylammonium (TBAB)-modified Nafion® membrane polymer or polyamidoamine (PAMAM) dendrimers for the enzyme immobilization. The electrochemical characterization showed that the prepared bioelectrodes were able to undergo DET onto glassy carbon surface in the presence as well as the absence of multi-walled carbon nanotubes (MWCNTs); also, in the latter case a relevant shift in the oxidation peak of about 180mV vs. saturated calomel electrode (SCE) was observed. A very similar redox potential was achieved with the self-assembled bioelectrode prepared onto modified-gold surfaces with dendrimers, indicating that both methodologies provide an environment that enables the PQQ-enzymes to undergo DET. The biofuel cell tests confirmed the ease of the DET process and the enhanced performance in the presence of the carbon nanotubes. Considering the bioanodes prepared with PAMAM dendrimers, the power density values vary from 19.4μWcm−2 without MWCNTs to 25.7μWcm−2 in the presence of MWCNTs. Similarly, with the bioanodes prepared with the TBAB-modified-Nafion® polymer, the results indicate power densities of 27.9 and 38.4μWcm−2 respectively. These electrode modifications represent effective methods for immobilization and direct electrical connection of quinohemoproteins to electrode surfaces.

Induced Evolution of PQQ-Dependent Alcohol Dehydrogenase Activity in &lt;I&gt;Gluconobacter&lt;/I&gt; sp.33 for Use in Enzymatic Biofuel Cells

Induced Evolution of PQQ-Dependent Alcohol Dehydrogenase Activity in &lt;I&gt;Gluconobacter&lt;/I&gt; sp.33 for Use in Enzymatic Biofuel Cells

Characterization of PQQ-Dependent Alcohol Dehydrogenase from Gluconobacter in L-Cysteine Monolayer on Pd Surface

not Available.

Membrane-bound dehydrogenases from Gluconobacter sp.: Interfacial electrochemistry and direct bioelectrocatalysis

Although membrane-bound dehydrogenases isolated from Gluconobacter sp. (mainly PQQ-dependent alcohol and fructose dehydrogenase) have been used for preparing diverse forms of bioelectronic interfaces for almost 2 decades, it is not an easy task to interpret an electrochemical behaviour correctly. Recent discoveries regarding redox properties of membrane-bound dehydrogenases along with extensive investigations of direct electron transfer (DET) or direct bioelectrocatalysis with these enzymes are summarized in this review. The main aim of this review is to draw general conclusions about possible electronic coupling paths of these enzymes on various interfaces via direct electron transfer or direct bioelectrocatalysis. A short overview of the metabolism and respiration chain in Gluconobacter relevant to interfacial electrochemistry is given. Biosensor devices based on DET or direct bioelectrocatalysis using membrane-bound dehydrogenases from Gluconobacter sp. are described briefly with the emphasis given on practical applications of preparing enzymatic biofuel cells. Moreover, interfacial electrochemistry of Gluconobacter oxydans related to the construction of microbial biofuel cells is also discussed.

Complete Oxidation of Glycerol in an Enzymatic Biofuel Cell

AbstractGlycerol has drawn increasing attention as a prospective fuel because it has many desirable qualities and is abundant due to the fact that it is a by‐product of biodiesel production. Qualities such as nontoxicity, extremely low vapour pressure, low flammability and high energy density make glycerol very appealing as an energy source. Previous research has shown that partial oxidation of glycerol can occur at enzymatic bioanodes of biofuel cells utilising PQQ‐dependent alcohol dehydrogenase (PQQ‐ADH) and PQQ‐dependent aldehyde dehydrogenase (PQQ‐AldDH). In this paper, we describe the use of glycerol for a fuel in an enzymatic biofuel cell that utilizes a three‐enzyme cascade on the anode that can accomplish the complete oxidation of glycerol. The bioanode that was developed contained PQQ‐ADH, PQQ‐AldDH and oxalate oxidase immobilised within a tetrabutylammonium‐modified Nafion membrane. Our previous research has shown that glycerol is an effective fuel with the PQQ‐ADH and PQQ‐AldDH but still was unable to be fully oxidised. With the addition of oxalate oxidase, these glycerol/air biofuel cells have yielded power densities of up to 1.32 mW cm–2, and have the ability to operate at high fuel concentrations. The oxidation products were confirmed with 13C NMR and comprised mainly 13C‐labelled carbonate and glycerate.

Apparent Michaelis constant of the enzyme modified porous electrode

Plate-gap model of enzyme doped porous electrode was utilized in order to calculate apparent Michaelis constants ( $$K_{\rm M}^{\rm app}$$ ) and apparent maximal currents ( $$I_{\rm max}^{\rm app}$$ ) of modeled amperometric biosensor for the wide range of given reaction/diffusion parameters. It was found that $$K_{\rm M}^{\rm app}$$ of plate-gap biosensor linearly depends on $$I_{\rm max}^{\rm app}$$ when rates of enzymatic reaction are lower than critical. Theoretically predicted linear correlation between apparent parameters was observed experimentally for the case of carbon paste electrodes, which were modified by PQQ-dependent alcohol dehydrogenases. At overcritical rates (or apparent maximal currents), $$K_{\rm M}^{\rm app }$$ is practically independent on Michaelis constant of soluble enzyme. Therefore, apparent Michaelis constant can be regarded as biosensor’s topology representing parameter which, in fact, is not related to the specificity of enzyme kinetics. High and rate-independent values of $$K_{\rm M}^{\rm app}$$ indicate that reaction proceeds at substrate-exposed top layer of the gap. In this case, reaction–diffusion system formally is stratified into separate reaction (top) and diffusion (bottom) zones. Topology of such reaction–diffusion system reminds “inverted” planar electrode, which contains diffusion layer below reaction layer. The net effect of plate-gap topology of working electrode on apparent Michaelis constant is similar to the effect of diffusion layer covering enzymatic planar electrode.