Corynascus Thermophilus Research Articles

Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei

BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L−1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-d-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-β-d-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Determination of the Distance Between the Cytochrome and Dehydrogenase Domains of Immobilized Cellobiose Dehydrogenase by Using Surface Plasmon Resonance with a Center of Mass Based Model.

Changes in the tertiary conformation of adsorbed biomolecules can induce detectable shifts (Δθr) in the surface plasmon resonance (SPR) angle. Here it is shown how to calculate the corresponding shifts in the adsorbate’s center of mass (Δzavg) along the sensing surface normal from the measured Δθr. The novel developed model was used for determining the mean distance between the cytochrome (CYT) and flavodehydrogenase (DH) domains of the enzyme cellobiose dehydrogenase (CDH) isolated from the fungi Neurospora crassa, Corynascus thermophilus, and Myriococcum thermophilum as a function of pH, [Ca2+], and substrate concentration. SPR confirmed the results from earlier electrochemical and SAXS studies stating that the closed conformation, where the two domains are in close vicinity, is stabilized by a lower pH and an increased [Ca2+]. Interestingly, an increasing substrate concentration in the absence of any electron acceptors stabilizes the open conformation as the electrostatic repulsion due to the reaped electrons pushes the DH and CYT domains apart. The accuracy of distance determination was limited mostly by the random fluctuations between replicate measurements, and it was possible to detect movements <1 nm of the domains with respect to each other. The results agreed with calculations using already established models treating conformational changes as contraction or expansion of the thickness of the adsorbate layer (tprotein). Although the models yielded equivalent results, in this case, the Δzavg-based method also works in situations, where the adsorbate’s mass is not evenly distributed within the layer.

Exoproteome profile reveals thermophilic fungus Crassicarpon thermophilum (strain 6GKB; syn. Corynascus thermophilus) as a rich source of cellobiose dehydrogenase for enhanced saccharification of bagasse

LC-MS/MS based profiling revealed thermophilic fungus Crassicarpon thermophilum strain 6GKB as a prolific producer of cellobiose dehydrogenase (CDH; AA8). CDH is an important component of the newly discovered oxidative system comprising CDH and lytic polysaccharide monooxygenases (LPMO), known for its ability to enhance degradation of lignocellulosics. CDH from C. thermophilum was purified using culture extract harvested after 7 days of culture on cellulose, wheat bran and rice straw (CWR) based medium by employing hydrophobic interaction chromatography in tandem with ion exchange chromatography. SDS-PAGE confirmed enzyme purity and presence of a single protein band at a molecular weight of 84 kDa. The purified CDH (ctCDH) was found to be stable under optimal temperature (50 °C) and pH (5.0) conditions with t1/2 of 360 min. The Kinetic characterization of ctCDH showed similar affinity (4.5 and 4.0 mM) and specificity (2.6 × 105 and 2.5 × 105 mM−1 s−1) of the enzyme towards cellobiose and lactose, respectively. In addition, coalescence of spectral and electron acceptor studies confirmed ctCDH to be a flavocytochrome. Through this study, we further demonstrate the potential of ctCDH alone and in combination with LPMO to enhance the efficiency of commercial cellulase Cellic CTec2 to hydrolyze alkali and acid treated bagasse by upto 1.35 folds. Conclusively, ctCDH reported in this study is a potent enzyme that could be utilized for numerous biotechnological applications involving enzymatic degradation of lignocellulosic biomass.

An efficient and versatile membraneless bioanode for biofuel cells based on Corynascus thermophilus cellobiose dehydrogenase

In the present study, the effect of carbon nanotubes (CNTs) either single walled (SWCNT) or multi walled (MWCNT) on sugar oxidation by Corynascus thermophilus cellobiose dehydrogenase (CtCDH) is investigated. The current observed for the direct electron transfer (DET) communication between the enzyme and the electrode is compared to that of the mediated electron transfer (MET) obtained from CtCDH/CNT/Os-polymer modified electrodes. The CNTs provide a high surface area for the immobilization of CtCDH and the Os-polymer and increases the amount of both on the electrode surface. The amperometric current density measured for 50 mM glucose at pH 7.4 and an applied potential of 290 mV vs. SHE in DET mode is 6.90 μA cm−2, which is further improved to about 8.73 μA cm−2 when a cross-linker is applied, whereas a 14-folds increase to 97.6 μA cm−2 is observed for MET when CtCDH is immobilized in the Os-polymer and cross-linked. The current densities extracted from CVs at 150 mV vs. SHE using 20 mM glucose for CtCDH immobilized with the Os-polymer, MWCNT/Os-polymer and SWCNT/Os-polymer are 139.7, 180.3 and 328.2 μA cm−2, respectively so a remarkable increase is observed when using SWCNT for electrode modification. The bioanode also exhibits a good stability and it retains more than 50% of its activity in multicycle CV scan after five days. Thus the bioanode based on SWCNT/CtCDH/Os-polymer/cross-linker could be a promising electrode for application as anode in enzymatic biofuel cells.

Cellobiose dehydrogenase hosted in lipidic cubic phase to improve catalytic activity and stability

Lipidic cubic phase systems (LCPs) are excellent carriers for immobilized enzymes due to their biocompatibility and well-defined nanoporous structure. Lipidic cubic phases act as a convenient matrix to incorporate enzymes and hold them in the vicinity of electrode surfaces in their fully active forms. Corynascus thermophilus cellobiose dehydrogenase (CtCDH) was trapped in a monoolein cubic phase, which increased not only its stability, but also its catalytic performance with both enhanced mediated and direct electron transfer with electrodes. For studies of mediated electron transfer, three mediators with different formal potentials (E°′) were employed: horse-heart cytochrome c (cyt c), electron acceptor active with the cytochrome domain of CtCDH, and 2,6-dichlorophenolindophenol (DCPIP) as well as hexaammineruthenium(II) chloride [Ru(NH3)Cl2] both electron acceptors with the dehydrogenase domain. Ru(NH3)Cl2, having the most negative E°′ of −0.138V vs. Ag|AgCl at pH7.5, gave a catalytic current for lactose oxidation of 32.10μAcm−2 in MOPS buffer at pH7.5. The process carried out in the same solution but under direct electron conditions transfer resulted in a catalytic current of 9.22μAcm−2. Electrodes covered with CtCDH in a LCP film retained their catalytic activity after 28days showing a slightly increased current density after 6days.

Rechargeable, flexible and mediator-free biosupercapacitor based on transparent ITO nanoparticle modified electrodes acting in µM glucose containing buffers

We present a transparent and flexible self-charging biosupercapacitor based on an optimised mediator- and membrane-free enzymatic glucose/oxygen biofuel cell. Indium tin oxide (ITO) nanoparticles were spray-coated on transparent conducting ITO supports resulting in a flocculent, porous and nanostructured electrode surface. By this, high capacitive currents caused by an increased electrochemical double layer as well as enhanced catalytic currents due to a higher number of immobilised enzyme molecules were obtained. After a chemical pre-treatment with a silane derivative, bilirubin oxidase from Myrothecium verrucaria was immobilized onto the ITO nanostructured electrode surface under formation of a biocathode, while bioanodes were obtained by either immobilisation of cellobiose dehydrogenase from Corynascus thermophilus or soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus. The latter showed a lower apparent KM value for glucose conversion and higher catalytic currents at µM glucose concentrations. Applying the optimised device as a biosupercapacitor in a discontinuous charge/discharge mode led to a generated power output of 0.030mW/cm2 at 50µM glucose, simulating the glucose concentration in human tears. This represents an enhancement by a factor of 350 compared to the power density obtained from the continuously operating biofuel cell with a maximum power output of 0.086µW/cm2 under the same conditions. After 17h of charging/discharging cycles a remarkable current enhancement was still measured. The entire device was transferred to flexible materials and applied for powering a flexible display showing its potential applicability as an intermittent power source in smart contact lenses.

A Glucose/Oxygen Enzymatic Fuel Cell based on Gold Nanoparticles modified Graphene Screen-Printed Electrode. Proof-of-Concept in Human Saliva

This paper presents a new direct electron transfer based-miniaturized glucose/oxygen enzymatic fuel cell (EFC) whose operating ability has been tested in real saliva samples. The bioanode and biocathode are a graphene working electrode and a graphite counter electrode localized on the same screen printed electrode (SPE) modified with poly(vinyl alcohol) N-methyl-4(4′-formylstyryl)pyridinium methosulfate acetal (PVA-SbQ)/cellobiose dehydrogenase from Corynascus Thermophilus (CtCDH) C291Y/AuNPs and with Trametes Hirsuta laccase (ThLac)/AuNPs, respectively.In order to optimize the bioanode, several CDH immobilization procedures were adopted, such as drop-casting, use of Nafion membrane or PVA-SbQ photopolymer. The photopolymer showed the best performance in terms of stability and reliability. As biocathode a partially optimized laccase electrode was employed with the variant that the used nanomaterials allowed to reduce the overpotential of O2/H2O redox reaction catalyzed by Trametes Hirsuta Laccase (ThLac), drop-casted onto the gold nanoparticles (AuNPs) modified SPE.The performances of bioanode and biocathode were tested separately, initially immobilizing the two enzymes onto separated graphene SPEs. An efficient direct electron transfer was achieved for both elements, obtaining an apparent heterogeneous electron transfer rate constant (ks) of 0.99±0.05s−1 for CtCDH C291Y and 5.60±0.05s−1 for ThLac. Both electrodes were then assembled in a two compartment EFC obtaining a maximal power output of 5.16±0.15μWcm−2 at a cell voltage of 0.58V and an open circuit voltage (OCV) of 0.74V. Successively, the bioanode and biocathode were assembled in a non-compartmentalized EFC and a remarkable 50% decrease of the maximum power output at the value of 2.15±0.12μWcm−2 at cell voltage of 0.48V and an OCV of 0.62V at pH 6.5 was registered. In order to reduce the cell dimensions in view of its possible integration in biomedical devices, the bioanode and biocaythode were realized by immobilization of both enzymes onto the same SPE. The so miniaturized EFC delivered a maximal power output of 1.57±0.07μWcm2 and 1.10±0.12μWcm−2 with an OCV of 0.58V and 0.41V in a 100μM glucose solution and in human saliva, respectively.

A Third Generation Glucose Biosensor Based on Cellobiose Dehydrogenase Immobilized on a Glassy Carbon Electrode Decorated with Electrodeposited Gold Nanoparticles: Characterization and Application in Human Saliva.

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.

Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems

BackgroundCellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity.ResultsWe compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability.ConclusionsWhereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris.

Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

AbstractCellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence and in the presence of substrates, on graphite electrodes and thiolic self‐assembled monolayer (SAM)‐modified Au electrodes. Seven different thiols with different spacer lengths were used, containing ‐COOH, ‐OH, and ‐NH2 end groups. The length and head functionality of the thiol govern the efficiency of the DET reaction and indicate different DET properties of CtCDH and HiCDH

Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris

Abstract Novel xylanase (EC 3.2.1.8) is in great demand due to its industrial significance. In this study, we have developed and characterized a novel xylanase-producing yeast strain. This mature xylanase gene xyn11A consists of 870 base pairs and belongs to GH11 family. The gene sequence was optimized and synthesized, and was then cloned into yeast vector pGAPZαA under the control of the constitutive GAP promoter. SDS-PAGE analysis indicates that Xyn11A is extracellularly expressed as a glycosylated protein in P. pastoris. Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase retained more than 90% of its activity after incubation at 50 °C and 60 °C for up to 1 h. Xyn11A is also stable over a wide range of pH (2.0–11.0). Most metal ions tested such as copper (Cu2+) and lead (Pb2+) have little inhibitory effects on Xyn11A. It is also resistant to pepsin and proteinase K digestion, retaining 80% and 90% of its activity after digestion at 37 °C for 1 h, respectively. Those superior properties make Xyn11A a robust xylanase with great potential for industrial use. To the best of our knowledge, this is the first report of xylanase from the fungus Corynascus thermophilus.

Dual Biosensor for Simultaneous Monitoring of Lactate and Glucose Based on Thin‐layer Flow Cell Screen‐printed Electrode

AbstractA simple and robust monitoring system for lactate and glucose detection is presented. The system is based on a suitable integrated screen‐printed electrode in thin layer flow cell design (TLFCL) to perform flow injection analysis. The TLFCL is consisted of a two working and one counter electrodes made of carbon and a silver pseudoreference electrode. Both working electrodes (WE) are first modified by graphene nanomaterials. Glucose sensor based on cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) and lactate sensor based on lactate oxidase (LOx) are made on the first and second WE respectively. Both enzymes are entrapped in a photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA‐SbQ). The electrocatalytic signal of both sensors did not show any cross‐talking, whereas the linear quantification ranges of both glucose and lactate sensors are large enough for wine monitoring.

Improved DET communication between cellobiose dehydrogenase and a gold electrode modified with a rigid self-assembled monolayer and green metal nanoparticles: The role of an ordered nanostructuration

Efficient direct electron transfer (DET) between cellobiose dehydrogenase from Corynascus thermophilus (CtCDH) and a novel gold electrode platform, obtained by covalent linking of green AuNPs and AgNPs modified with a dithiol self-assembled monolayer, consisting of biphenyl-4,4′-dithiol (BPDT), was presented. The green AuNPs and AgNPs were synthesized using quercetin as reducing agent at room temperature. TEM experiments showed that the AuNPs and AgNPs were circular in shape with an average diameter of 5 and 8nm, respectively. Cyclic voltammetry of CtCDH immobilized onto the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE electrode platforms were carried out and compared with naked AuE, BPDT/AuE, AuNPs/AuE, and AgNPs/AuE. A pair of well-defined redox waves in neutral pH solution due to efficient DET of CtCDH was present with both MNPs/BPDT/AuE platforms. No DET communication was found with platforms without MNPs linked to BPDT. The apparent heterogeneous electron transfer rate constants (kS) of CtCDH were calculated to be 21.5±0.8s−1 and 10.3±0.7s−1, for the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE platforms, respectively.The modified electrodes were successively used to develop an eco-friendly biosensor for lactose detection. The CtCDH/AuNPs/BPDT/AuE based biosensor showed the best analytical performances with an excellent stability, a detection limit of 3µM, a linear range between 5 and 400µM and a sensitivity of 27.5±2.5µAcm−2mM−1. Such performances were favorably compared with other lactose biosensors reported in literature.The biosensor was successively tested to quantify lactose content in real milk and cream samples. No significant interference present in the sample matrices was observed.

Novel thin layer flow-cell screen-printed graphene electrode for enzymatic sensors

A new Screen-printed electrodes (SPE) integrated in one channel flow-cell was developed. The one channel flow-cell is attached and directly changeable with electrode. In the new flow-cell the injection is done through an “in-line luer injection port” which can be less aggressive than wall-jet flow cell for a biological recognition element immobilized on the surface of the electrode. The sample volume can be easily controlled by the operator through a syringe. In this novel thin layer flow-cell screen-printed electrodes, the working electrode was modified with graphene materials, and an enhancement of electroactive area to 388% over a standard electrode was found. This new configuration was applied to study the entrapped cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) in a photocrosslinkable PVA-based polymer. The calibration curve of lactose using optimized parameters shows a wide linear measurement ranges between 0.25 and 5mM. A good operational stability of the CtCDH-PVA-modified graphene electrode is obtained, which keeps the same initial activity during 8h and exhibits a good storage stability with a decrease of only 9% in analytical response after 3 months storage at 4◦C.

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.

Highly Efficient Membraneless Glucose Bioanode Based on Corynascus thermophilus Cellobiose Dehydrogenase on Aryl Diazonium‐Activated Single‐Walled Carbon Nanotubes

AbstractWe present an approach for electrode modification by using the oxidoreductase cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH). CtCDH is a two‐domain enzyme, in which the catalytic dehydrogenase domain (DHCDH) hosts flavin adenine dinucleotide (FAD) as cofactor and is connected through a flexible linker to a small cytochrome domain with a heme b cofactor (CYTCDH). This domain is responsible for the electron transfer from DHCDH to macromolecular electron acceptors, and is capable of direct electron transfer (DET) with electrode surfaces. CtCDH is optimal at pH values between 7 and 9 and exhibits one of the lowest apparent KM values for glucose (2.4×104 μM) in contrast to the majority of other CDHs, which have acidic optimal pH values and have very low or no activity for glucose. Glassy carbon (GC) electrodes were modified by drop‐casting single‐walled carbon nanotubes (SWCTs) and further modifying with aryl diazonium salts (DS). When adsorbed on such GC‐SWCT‐DS electrodes, CYTCDH showed efficient DET in the presence of a substrate. Six different functional groups for the aryl amines were studied and compared to non‐DS modified GC‐SWCT electrodes. The charge of the grafted aryl amines was investigated and it was found that surfaces modified with aniline, 4‐aminophenol, and 4‐aminobenzoic acid (ABA) contain negative charges at pH 7.4. On the other hand, surfaces modified with p‐phenylenediamine (PD), N,N‐dimethyl‐p‐phenylenediamine, and N,N‐diethyl‐p‐phenylenediamine remain uncharged. To date, the highest DET current density (JMAX=32.5 μA cm−2 for lactose and JMAX=16.2 μA cm−2 for glucose) for a CDH‐modified electrode at human physiological pH can be obtained by using a GC‐SWCT‐ABA CtCDH modified electrode. The use of glutaraldehyde (GA) in GC‐SWCT‐PD‐modified electrodes increases the JMAX twofold. The modified electrodes with DS showed a more negative onset potential for the catalytic current. For GC‐SWCT‐ABA modified electrodes and GC‐SWCT‐PD with GA, a change in the onset potential of −62 and −66 mV, respectively, was found compared with non‐DS modified electrodes. The prepared bioanodes show a loss in response current of 15 % after 9 days of continuous measurements from the original signal in 5 mM glucose, using 50 mM phosphate buffer solution at pH 7.40 at 37 °C.

Performance of enzymatic fuel cell in cell culture

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25μWcm−2 at 0.5V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode.

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.

Low potential biofuel cell anodes based on redox polymers with covalently bound phenothiazine derivatives for wiring flavin adenine dinucleotide-dependent enzymes

The design of biofuel cell anodes with substantially decreased potential is a prerequisite for the development of biofuel cells with large open-circuit voltage and power density. Redox polymers with covalently attached phenothiazine derivatives such of thionine acetate, toluidine blue, azure B simultaneously providing epoxide functions for covalent binding to suitably modified electrode surfaces and crosslinking were synthesized and evaluated for their ability to transfer electrons from the FAD cofactor of the flavodehydrogenase domain of cellobiose dehydrogenase from Myriococcum thermophilum (FAD-MtCDH), the flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus (FAD-CtCDH), or glucose oxidase from Aspergillus niger (GOx). Polymer/enzyme films were covalently bound via polymer bound epoxy groups to terminal amino functions introduced to graphite electrode surfaces by electrochemically induced grafting of diaminoheptane or Boc-protected ethylene diamine (EDA). The electrodes were optimized for biocatalytic glucose oxidation with respect to the hydrophilicity of the polymer backbone, the nature of the phenothiazine derivative, the pH value, as well as the relative amount of enzyme, polymer and crosslinker. Biofuel cells based on toluidine blue-modified redox polymers with integrated FAD-MtCDH, FAD-CtCDH, or GOx in combination with a bilirubin oxidase based biocathode exhibited open-circuit voltages of more than 0.7V and maximum power densities in the range of 4 to 6μWcm−2 at a pH value of 7.8.

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm(2) was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).