Agaricus Meleagris Research Articles

A novel membraneless β-glucan/O2 enzymatic fuel cell based on β-glucosidase (RmBgl3B)/pyranose dehydrogenase (AmPDH) co-immobilized onto buckypaper electrode

A novel membraneless β-glucan/O2 enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus β-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction.The fuel for the bioanode was produced by enzymatic hydrolysis of β-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel.The maximum power density of the assembled β-glucan/O2 biofuel cell reached 26.3 ± 4.6 μWcm−2 at 0.36 V in phosphate buffer containing 0.5 % (w/v) β-glucan at 40 °C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that β-glucan can be employed as fuel in an enzymatic biofuel cell.

Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes.

Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252 prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N319, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.

Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Multigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucose–methanol–choline (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.

Yeast surface display of dehydrogenases in microbial fuel-cells

Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of −28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm−2 using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm−2 using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

A Versatile Bioanode with Improved Current Density and the Coulombic Efficiency through a Cascade Reaction

To improve communications between enzymes and electrodes, many different methods were developed including the use of diffusional electron mediators, the “wiring” of enzymes through immobilization in redox polymers, the functionalization of enzymes with electron relays, and the use of nanomaterials. Recently a new promising strategy was reported, in which the glycoprotein glucose oxidase (GOx) was directly electrically contacted with the electrodes after removal of the glycosylating layer. Although a lot of advances in the electrical communication between enzymes and electrodes has made it possible to fabricate new biosensors and biofuel cells, the technologies still face challenging issues, such as development of long-term stable miniaturized implantable amperometric biosensors and biofuel cells. Our approach to the development of a glucose biofuel cell anode is based on the combination of different redox enzymes with complementary oxidation positions thus forming a reaction cascade to oxidize the substrate at more than one position. Improvements in current density and coulombic efficiency of a glucose oxidizing electrode were realized by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) with either glucose dehydrogenase from Glomerella cingulata (GcGDH) or cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH). The mixed enzyme electrode oxidizes glucose in several combinations at the C-1, C-2 and C-3 positions of the pyranose ring. This concerted action of enzymes increases (i) the coulombic efficiency by extracting more than 2 e- per substrate molecule and (ii) the current density of the electrode when the mass-transfer of substrates becomes rate limiting. The electrodes were investigated with flow injection analysis (FIA) using different substrates under physiological conditions (pH 7.4). These investigations showed that the product of one enzyme can be used as substrate for the other enzyme and maximally 6 e- can be gained from the oxidation of one glucose molecule using mixed enzyme electrodes like AmPDHb/GcGDH/Os-polymer 2 or the AmPDHa/MtCDH/Os-polymer 1. We propose a bioanode for use in biofuel cells with an increased current density and coulombic efficiency obtained by a cascade reaction catalyzed by redox enzymes with a different site-specificity for glucose.

Deep Oxidation of Multiple (Poly)Saccharides at a Bi-Enzymatic Bioelectrode

As perhaps the most commonly utilized fuel for enzymatic fuel cells (EFCs), glucose oxidation is largely under-exploited. Theoretically, the oxidation of glucose and its downstream metabolites is able to deliver a maximum of 24e- to a bioanode, however the vast majority of EFCs reported to date only perform a single 2e- oxidation of glucose. Commercially available glucose oxidase (GOx) from Aspergillus sp. oxidizes the anomeric (C1) carbon of glucose. Recently, mediated bioelectrocatalysis of pyranose dehydrogenase from Agaricus meleagris (AmPDH) via an osmium redox hydrogel has been demonstrated, whereby AmPDH is reported to be able to oxidize glucose and other saccharides at combinations of their respective C1/C2/C3 positions.1 Another attractive property of an EFC is the ability to utilize a single biocatalyst to oxidize a range of fuels. We recently reported the use of an engineered commercially-available glucose oxidase with broader substrate specificity at an electrode surface that resulted in an EFC configuration that was able to oxidize multiple (poly)saccharides, including lactose from dairy milk and starch.2 This study reports the ability of a ferrocene redox hydrogel to mediate bioelectrocatalytic saccharide oxidation by AmPDH. Further, AmPDH and broad substrate specificity glucose oxidase were combined to yield an EFC that was able to operate on multiple (poly)saccharides while theoretically increasing the extent of oxidation of each substrate, resulting in a more-versatile EFC with greater efficiency.

Reaction of pyranose dehydrogenase from Agaricusmeleagris with its carbohydrate substrates.

Monomeric Agaricus meleagris pyranose dehydrogenase (Am PDH) belongs to the glucose–methanol–choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. Am PDH can double oxidize various mono‐ and oligosaccharides at different positions (C1 to C4). To study the structure/function relationship of selected active‐site residues of Am PDH pertaining to substrate (carbohydrate) turnover in more detail, several active‐site variants were generated, heterologously expressed in Pichia pastoris, and characterized by biochemical, biophysical and computational means. The crystal structure of Am PDH shows two active‐site histidines, both of which could take on the role as the catalytic base in the reductive half‐reaction. Steady‐state kinetics revealed that His512 is the only catalytic base because H512A showed a reduction in (k cat/KM)glucose by a factor of 105, whereas this catalytic efficiency was reduced by two or three orders of magnitude for His556 variants (H556A, H556N). This was further corroborated by transient‐state kinetics, where a comparable decrease in the reductive rate constant was observed for H556A, whereas the rate constant for the oxidative half‐reaction (using benzoquinone as substrate) was increased for H556A compared to recombinant wild‐type Am PDH. Steady‐state kinetics furthermore indicated that Gln392, Tyr510, Val511 and His556 are important for the catalytic efficiency of PDH. Molecular dynamics (MD) simulations and free energy calculations were used to predict d‐glucose oxidation sites, which were validated by GC‐MS measurements. These simulations also suggest that van der Waals interactions are the main driving force for substrate recognition and binding.

A Versatile Bioanode with Improved Current Density and the Coulombic Efficiency through a Cascade Reaction

To improve communications between enzymes and electrodes, many different methods were developed including the use of diffusional electron mediators, the “wiring” of enzymes through immobilization in redox polymers, the functionalization of enzymes with electron relays, and the use of nanomaterials. Recently a new promising strategy was reported, in which the glycoprotein glucose oxidase (GOx) was directly electrically contacted with the electrodes after removal of the glycosylating layer. Although a lot of advances in the electrical communication between enzymes and electrodes has made it possible to fabricate new biosensors and biofuel cells, the technologies still face challenging issues, such as development of long-term stable miniaturized implantable amperometric biosensors and biofuel cells. Our approach to the development of a glucose biofuel cell anode is based on the combination of different redox enzymes with complementary oxidation positions thus forming a reaction cascade to oxidize the substrate at more than one position. Improvements in current density and coulombic efficiency of a glucose oxidizing electrode were realized by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) with either glucose dehydrogenase from Glomerella cingulata (GcGDH) or cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH). The mixed enzyme electrode oxidizes glucose in several combinations at the C-1, C-2 and C-3 positions of the pyranose ring. This concerted action of enzymes increases (i) the coulombic efficiency by extracting more than 2 e- per substrate molecule and (ii) the current density of the electrode when the mass-transfer of substrates becomes rate limiting. The electrodes were investigated with flow injection analysis (FIA) using different substrates under physiological conditions (pH 7.4). These investigations showed that the product of one enzyme can be used as substrate for the other enzyme and maximally 6 e- can be gained from the oxidation of one glucose molecule using mixed enzyme electrodes like AmPDHb/GcGDH/Os-polymer 2 or the AmPDHa/MtCDH/Os-polymer 1. We propose a bioanode for use in biofuel cells with an increased current density and coulombic efficiency obtained by a cascade reaction catalyzed by redox enzymes with a different site-specificity for glucose. Key words: Bio-anode, glucose dehydrogenase, Os-polymers, pyranose dehydrogenase, cellobiose dehydrogenase, coulombic efficiency

Bioelectrochemical Behavior of the Composite PVP-Os/chitosan as a Mediator with Different Types of Enzymes at Graphite Electrode

Chitosan was cross-linked to an osmium redox polymer, poly(4-vinylpyridine) osmium bipyridyl [PVP-Os-(bpy)2-Cl], to form PVP-Os-(bpy)2-Cl/chitosan composite known to make a porous and hydrophilic film with an enzyme. In this work we demonstrate such a composite is a useful trestle, for hosting various sugars oxidizing enzymes to construct biosensors. Glucose sensing ability has been proven with the following glucose oxidizing redox enzymes; Aspergillus niger glucose oxidase (AnGOX), Myriococcum thermophilum cellobiose dehydrogenase, (MtCDH), glycosylated Agaricus meleagris pyranose dehydrogenase (gAmPDH), fragmented deglycosylated Agaricus meleagris pyranose dehydrogenase (fdgAmPDH), and Aspergillus sp. glucose dehydrogenase (AspGDH), as well as recombinant Glomerella cingulata glucose dehydrogenase (rGcGDH).

Pyranose dehydrogenase ligand promiscuity: a generalized approach to simulate monosaccharide solvation, binding, and product formation.

The flavoenzyme pyranose dehydrogenase (PDH) from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD) simulations is reported. Free energy calculations according to the one-step perturbation (OSP) method revealed the solvation free energies (ΔGsolv) of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG) of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from K m values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD) revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔGbind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

Agaricus meleagris pyranose dehydrogenase: Influence of covalent FAD linkage on catalysis and stability

Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose–methanol–choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability.

A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39–0.40s−1) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, “wired” to graphite electrodes with two different osmium redox polymers: [Os(4,4′-dimethyl-2,2′-bipyridine)2(poly(vinylimidazole))10Cl]+ [Os(dmbpy)PVI] and [Os(4,4′-dimethoxy-2,2′-bipyridine)2(poly-(vinylimidazole))10Cl]+ [Os(dmobpy)PVI]. When “wired” with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N75, N175, and N252 were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

Effect of deglycosylation on the mediated electrocatalytic activity of recombinantly expressed Agaricus meleagris pyranose dehydrogenase wired by osmium redox polymer

The effect of deglycosylation of pyranose dehydrogenase (PDH) obtained from Agaricus meleagris (Am) and recombinantly expressed in Pichia pastoris on its electrocatalytic activity was investigated. Glycosylated (gAmPDH) and deglycosylated PDH (dgAmPDH) were immobilised on spectrographic graphite (G) simultaneously with an osmium redox polymer (Os-RP) using poly(ethylene glycol)(400) diglycidyl ether (PEGDGE) as cross-linking agent. The amperometric response to glucose, recorded at G/(Os-RP)-gAmPDH and G/(Os-RP)-dgAmPDH bioelectrodes, was optimised under flow injection conditions concerning the applied potential, enzyme loading, working pH and flow rate. The G/(Os-RP)-dgAmPDH bioelectrode is characterised by better kinetic and electroanalytical parameters compared with the G/(Os-RP)-gAmPDH bioelectrode: (i) a higher value of the maximum catalytic current density, jmax=(146.6±2.6)μAcm−2vs. jmax=(80.9±1.9)μAcm−2; (ii) a lower value of the apparent Michaelis–Menten constant, KMapp=2.4±0.1⁡mMvs. KMapp=7.5±0.3⁡mM; (iii) a higher slope of the linear domain, (43.6±1.1)μAcm−2mM−1vs. (9.74±0.16)μAcm−2mM−1. Additionally, the time dependent decay of the amperometric response to glucose shows a slightly better operational stability for the G/(Os-RP)-dgAmPDH bioelectrode than that for the G/(Os-RP)-gAmPDH. The enzyme deglycosylation induces significant changes in the order of substrate selectivity for gAmPDH and dgAmPDH.

Improving the Current Density and the Coulombic Efficiency by a Cascade Reaction of Glucose Oxidizing Enzymes

Improvements in current density and coulombic efficiency of a glucose oxidizing electrode were realized by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) with glucose dehydrogenase from Glomerella cingulata (GcGDH). The mixed enzyme electrode oxidizes glucose in several combinations at the C-1, C-2 and C-3 positions of the pyranose ring. This concerted action of enzymes increases (i) the coulombic efficiency by extracting more than 2e- per substrate molecule and (ii) the current density of the electrode when the mass-transfer of substrates becomes rate limiting. The electrodes were investigated with flow injection analysis (FIA) using different substrates under physiological conditions (pH 7.4). These investigations showed that the product of one enzyme can be used as substrate for the other enzyme and maximally 6e- can be gained from the oxidation of one glucose molecule using mixed enzyme electrode AmPDH/GcGDH/Os-polymer. We propose a bioanode for use in biofuel cells with an increased current density and coulombic efficiency obtained by a cascade reaction catalyzed by redox enzymes with a different site-specificity for glucose.

Optimization of a Membraneless Glucose/Oxygen Enzymatic Fuel Cell Based on a Bioanode with High Coulombic Efficiency and Current Density

After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. The optimized biofuel cell with the AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 μW cm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.

Molecular dynamics simulations give insight into d-glucose dioxidation at C2 and C3 by Agaricus meleagris pyranose dehydrogenase

The flavin-dependent sugar oxidoreductase pyranose dehydrogenase (PDH) from the plant litter-degrading fungus Agaricus meleagris oxidizes d-glucose (GLC) efficiently at positions C2 and C3. The closely related pyranose 2-oxidase (P2O) from Trametes multicolor oxidizes GLC only at position C2. Consequently, the electron output per molecule GLC is twofold for PDH compared to P2O making it a promising catalyst for bioelectrochemistry or for introducing novel carbonyl functionalities into sugars. The aim of this study was to rationalize the mechanism of GLC dioxidation employing molecular dynamics simulations of GLC–PDH interactions. Shape complementarity through nonpolar van der Waals interactions was identified as the main driving force for GLC binding. Together with a very diverse hydrogen-bonding pattern, this has the potential to explain the experimentally observed promiscuity of PDH towards different sugars. Based on geometrical analysis, we propose a similar reaction mechanism as in P2O involving a general base proton abstraction, stabilization of the transition state, an alkoxide intermediate, through interaction with a protonated catalytic histidine followed by a hydride transfer to the flavin N5 atom. Our data suggest that the presence of the two potential catalytic bases His-512 and His-556 increases the versatility of the enzyme, by employing the most suitably oriented base depending on the substrate and its orientation in the active site. Our findings corroborate and rationalize the experimentally observed dioxidation of GLC by PDH and its promiscuity towards different sugars.Electronic supplementary materialThe online version of this article (doi:10.1007/s10822-013-9645-7) contains supplementary material, which is available to authorized users.

The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.

Recombinant pyranose dehydrogenase—A versatile enzyme possessing both mediated and direct electron transfer

The catalytical properties of glycosylated pyranose dehydrogenase (gPDH) and deglycosylated PDH (dgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris were studied. Both gPDH and dgPDH were “wired” to an osmium redox polymer on graphite electrodes mounted in a flow-injection system. The current from oxidation of glucose by immobilised gPDH and dgPDH was compared using flow injection amperometry and cyclic voltammetry. An increase in the current density was observed for dgPDH (190μAcm−2) compared with that for gPDH (90μAcm−2) due to the improved electron transfer between the active site and the electrode. Additionally, the ability of dgPDH for direct electron transfer (DET) was discovered, which is rather unique among FAD-containing enzymes. The ability to oxidise a variety of sugars at a rather low potential makes dgPDH attractive for construction of biofuel cells with high power output.

Mutual enhancement of the current density and the coulombic efficiency for a bioanode by entrapping bi-enzymes with Os-complex modified electrodeposition paints

A bioanode with high current density and coulombic efficiency was developed by co-immobilization of pyranose dehydrogenase from Agaricus meleagris (AmPDH) with the dehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophiles (recDHCtCDH) expressed recombinantly in Escherichia coli. The two enzymes were entrapped in Os-complex modified electrodeposition polymers (Os-EDPs) with specifically adapted redox potential by means of chemical co-deposition. AmPDH oxidizes glucose at both the C2 and C3 positions whereas recDHCtCDH oxidizes glucose only at the C1 position. Electrochemical measurements reveal that maximally 6 electrons can be harvested from one glucose molecule at the two-enzyme anode via a cascade reaction, as AmPDH oxidizes the products formed from of the recDHCtCDH catalyzed substrate oxidation and vice versa. Furthermore, a significant increase in current density can be obtained by combining AmPDH and recDHCtCDH in a single modified electrode. We propose the use of this bioanode in biofuel cells with increased current density and coulombic efficiency.