Corynascus Thermophilus Research Articles

Recombinantly produced cellobiose dehydrogenase from Corynascus thermophilus for glucose biosensors and biofuel cells

Cellobiose dehydrogenase (CDH) is an emerging enzyme in the field of bioelectrocatalysis. Due to its flexible cytochrome domain, which acts as a built-in redox mediator, CDH is capable of direct electron transfer (DET) to electrode surfaces. This rare property is employed in mediatorless "third generation" biosensors. The ability of Corynascus thermophilus CDH to oxidize glucose under physiological conditions makes it a promising candidate for miniaturized glucose biosensors or glucose powered biofuel cell anodes. We report for the first time the electrochemical application and characterization of a recombinantly produced CDH in a glucose biosensor. Recombinant CDH from C. thermophilus (rCtCDH) was expressed by the methylotrophic yeast Pichia pastoris (376 U L(-1) , 132 mg L(-1) ). A comparative characterization of rCtCDH and CtCDH shows identical pH optima, K(M) values and heme b midpoint potentials. In contrast, the specific activity of rCtCDH (2.84 U mg(-1) ) and consequently the turnover numbers were ~five-times lower than for CtCDH, which was caused by a sub-stoichiometric occupation of catalytic sites with flavin-adenin-dinukleotid (FAD). The performance of rCtCDH-modified electrodes demonstrates the suitability for electrochemical studies. This opens the possibility to engineer the substrate specificity of C. thermophilus CDH for specific carbohydrates by rational engineering or directed evolution.

Biofuel cell as a power source for electronic contact lenses

Here we present unequivocal experimental proof that microscale cofactor- and membrane-less, direct electron transfer based enzymatic fuel cells do produce significant amounts of electrical energy in human lachrymal liquid (tears). 100μm diameter gold wires, covered with 17nm gold nanoparticles, were used to fashion three-dimensional nanostructured microelectrodes, which were biomodified with Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase as anodic and cathodic bioelements, respectively. The following characteristics of miniature glucose/oxygen biodevices operating in human tears were registered: 0.57V open-circuit voltage, about 1μWcm−2 maximum power density at a cell voltage of 0.5V, and more than 20h operational half-life. Theoretical calculations regarding the maximum recoverable electrical energy can be extracted from the biofuel and the biooxidant, glucose and molecular oxygen, each readily available in human lachrymal liquid, fully support our belief that biofuel cells can be used as electrical power sources for so called smart contact lenses.

Investigation of the pH-Dependent Electron Transfer Mechanism of Ascomycetous Class II Cellobiose Dehydrogenases on Electrodes

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on various carbon and thiol-modified gold electrodes. As a result, these systems have been utilized as biocatalyst in biosensors and biofuel cell anodes. Class I CDHs, from basidiomycetous fungi, are highly specific to cellulose or lactose, and DET is only observed at pH values below 5.5. To extend the applicability of CDH-based electrodes, the catalytic properties and the behavior on electrode surfaces of ascomycetous class II CDHs from Chaetomium attrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were investigated. We found that class II CDHs have diverse properties but generally show a lower substrate specificity than class I CDHs by converting also glucose and maltose. Intramolecular electron transfer (IET) and DET at neutral and alkaline pH were observed and elucidated by steady-state kinetics, pre-steady-state kinetics, and electrochemical measurements. The CDHs ability to interact with the electron acceptor cytochrome c and to communicate with electrode surfaces through DET at various pH conditions was used to classify the investigated enzymes. In combination with stopped-flow measurements, a model for the kinetics of the pH-dependent IET is developed. The efficient glucose turnover at neutral/alkaline pH makes some of these new CDHs potential candidates for glucose biosensors and biofuel cell anodes.

Characteristics of third-generation glucose biosensors based on Corynascus thermophilus cellobiose dehydrogenase immobilized on commercially available screen-printed electrodes working under physiological conditions

In this article, we describe a third-generation amperometric glucose biosensor working under physiological conditions. This glucose biosensor consists of a recently discovered cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) immobilized on different commercially available screen-printed electrodes made of carbon (SPCEs), carboxyl-functionalized single-walled carbon nanotubes (SPCE–SWCNTs), or multiwalled carbon nanotubes (SPCE–MWCNTs) by simple physical adsorption or a combination of adsorption followed by cross-linking using poly(ethyleneglycol) (400) diglycidyl ether (PEGDGE) or glutaraldehyde (GA). The CtCDH-based third-generation glucose biosensor has a linear range between 0.025 and 30mM and a detection limit of 10μM glucose. Biosensors based on SWCNTs showed a higher sensitivity and catalytic response than the ones functionalized with MWCNTs and the SPCEs. A drastic increase in response was observed for all three electrodes when the adsorbed enzyme was cross-linked with PEGDGE or GA. The operational stability of the biosensor was tested for 7h by repeated injections of 50mM glucose, and only a slight decrease in the electrochemical response was found. The selectivity of the CtCDH-based biosensor was tested on other potentially interfering carbohydrates such as mannose, galactose, sucrose, and fucose that might be present in blood. No significant analytical response from any of these compounds was observed.

Mediatorless sugar/oxygen enzymatic fuel cells based on gold nanoparticle-modified electrodes

We report on the fabrication and characterisation of a gold-nanoparticle (AuNP)-based mediatorless sugar/oxygen biofuel cell (BFC) operating in neutral sugar-containing buffers and human physiological fluids, such as blood and plasma. First, Corynascus thermophilus cellobiose dehydrogenase (CtCDH) and Myrothecium verrucaria bilirubin oxidase (MvBOx), used as anodic and cathodic bioelements, respectively, were immobilised on gold electrodes modified with 20nm AuNPs. Detailed characterisation and optimisation of a new CDH/AuNP-based bioanode were performed and the following fundamental parameters were obtained: (i) the redox potential of the haem-containing centre of the enzyme was measured to be 75mV vs. NHE, (ii) the surface coverage of CtCDH was found to be 0.65pmolcm−2 corresponding to a sub-monolayer coverage of the thiol-modified AuNPs by the enzyme, (iii) a turnover number for CtCDH immobilised on thiol-modified AuNPs was calculated to be ca. 0.5s−1, and (iv) the maximal current densities as high as 40μAcm−2 were registered in sugar-containing neutral buffers. Second, both biomodified electrodes, namely the CtCDH/AuNP-based bioanode and the MvBOx/AuNP-based biocathode, were combined into a functional BFC and the designed biodevices were carefully investigated. The following characteristics of the mediator-, separator- and membrane-less, miniature BFC were obtained: in phosphate buffer; an open-circuit voltage of 0.68V, a maximum power density of 15μWcm−2 at a cell voltage of 0.52V and in human blood; an open-circuit voltage of 0.65V, a maximum power density of 3μWcm−2 at a cell voltage of 0.45V, respectively. The estimated half-lives of the biodevices were found to be >12, <8, and <2h in a sugar-containing buffer, human plasma, and blood, respectively. The basic characteristics of mediatorless sugar/oxygen BFCs were significantly improved compared with previously designed biodevices, because of the usage of three-dimensional AuNP-modified electrodes.

Investigation of the mediated electron transfer mechanism of cellobiose dehydrogenase at cytochrome c-modified gold electrodes

The present study reports on the comparison of direct and mediated electron transfer pathways in the interaction of the fungal enzyme cellobiose dehydrogenase (CDH) with the redox protein cytochrome c (cyt c) immobilised at a modified gold electrode surface. Two types of CDHs were chosen for this investigation: a basidiomycete (white rot) CDH from Trametes villosa and a recently discovered ascomycete from the thermophilic fungus Corynascus thermophilus. The choice was based on the pH-dependent interaction of these enzymes with cyt c in solution containing the substrate cellobiose (CB). Both enzymes show rather similar catalytic behaviour at lower pH, dominated by a direct electron exchange with the electrode. With increasing pH, however, also cyt c-mediated electron transfer becomes possible. The pH-dependent behaviour in the presence and in the absence of cyt c is analysed and the potential reaction mechanism for the two enzymes with a different pH-behaviour is discussed.

Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes.

Putative cellobiose dehydrogenase (CDH) genes are frequently discovered in various fungi by genome sequencing projects. The expression of CDH, an extracellular flavocytochrome, is well studied in white rot basidiomycetes and is attributed to extracellular lignocellulose degradation. CDH has also been reported for plant-pathogenic or saprotrophic ascomycetes, but the molecular and catalytic properties of these enzymes are currently less investigated. This study links various ascomycetous cdh genes with the molecular and catalytic characteristics of the mature proteins and suggests a differentiation of ascomycete class II CDHs into two subclasses, namely, class IIA and class IIB, in addition to the recently introduced class III of hypothetical ascomycete CDHs. This new classification is based on sequence and biochemical data obtained from sequenced fungal genomes and a screening of 40 ascomycetes. Thirteen strains showed CDH activity when they were grown on cellulose-based media, and Chaetomium atrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were selected for detailed studies. In these strains, one or two cdh-encoding genes were found that stem either from class IIA and contain a C-terminal carbohydrate-binding module or from class IIB without such a module. In several strains, both genes were found. Regarding substrate specificity, class IIB CDHs show a less pronounced substrate specificity for cellobiose than class IIA enzymes. A pH-dependent pattern of the intramolecular electron transfer was also observed, and the CDHs were classified into three groups featuring acidic, intermediate, or alkaline pH optima. The pH optimum, however, does not correlate with the CDH subclasses and is most likely a species-dependent adaptation to different habitats.

A third generation glucose biosensor based on cellobiose dehydrogenase from Corynascus thermophilus and single-walled carbon nanotubes

A third generation glucose biosensor working under physiological conditions with a linear range of 0.1-30 mM, a detection limit of 0.05 mM, and a sensitivity of 222 nA µM(-1) cm(-2) has been developed by co-adsorption of cellobiose dehydrogenase (CDH) from the ascomycete Corynascus thermophilus (CtCDH) and oxidatively shortened single-walled carbon nanotubes (SWCNTs).

A Direct Electron Transfer‐Based Glucose/Oxygen Biofuel Cell Operating in Human Serum

AbstractWe report on the fabrication and characterisation of the very first direct electron transfer‐based glucose/oxygen biofuel cell (BFC) operating in neutral glucose‐containing buffer and human serum. Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase were used as anodic and cathodic bioelements, respectively. The following characteristics of the mediator‐, separator‐ and membrane‐less, a priori, non‐toxic and simple miniature BFC, was obtained: an open‐circuit voltage of 0.62 and 0.58 V, a maximum power density of ca. 3 and 4 μW cm–2 at 0.37 and 0.19 V of cell voltage, in phosphate buffer and human serum, respectively.

Efficiency of New Fungal Cellulase Systems in Boosting Enzymatic Degradation of Barley Straw Lignocellulose

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.