Cellobiose dehydrogenase catalyses the oxidation of aldoses--a simple reaction, a boring enzyme? No, neither for the envisaged bioelectrochemical applications nor mechanistically. The catalytic cycle of this flavocytochrome is complex and modulated by its flexible cytochrome domain, which acts as a built-in redox mediator. This intramolecular electron transfer is modulated by the pH, an adaptation to the environmental conditions encountered or created by the enzyme-producing fungi. The cytochrome domain forms the base from which electrons can jump to large terminal electron acceptors, such as redox proteins, and also enables by that path direct electron transfer from the catalytically active flavodehydrogenase domain to electrode surfaces. The application of electrochemical techniques to the elucidation of the molecular and catalytic properties of cellobiose dehydrogenase is discussed and compared to biochemical methods. The results lead to valuable insights into the function of this cellulose-bound enzyme, but also form the basis of exciting applications in biosensors, biofuel cells and bioelectrocatalysis.

We have compared the properties and resistance to DA fouling of a carbon nanotube fiber (CNTF) microelectrode to a traditional carbon fiber (CF) microelectrode. These two materials show comparable electrochemical activities for outer-sphere and inner-sphere redox reactions. Although the CNTF might have a higher intrinsic RC constant, thus limiting its high-frequency behavior, the CNTF show a significantly higher durability than the CF in terms of electrode stability. During constant oxidation of 100 μM DA, the signal measured by the CNTF microelectrode shows a 2-hour window over which no decrease in current is observed. Under the same conditions, the current obtained at the CF microelectrode decreases by almost 50 %. A model of the fouling process, assuming the formation of growing patches of insulator on the surface, has been compared to the data. This model is found to be in good agreement with our results, and indicates a growth rate of the patches in the 0.1 - 2 nm s−1 range.

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

The catalytic properties of cellobiose dehydrogenase (CDH) from the ascomycete fungus Myriococcum thermophilum adsorbed on a graphite electrode were investigated for a large variety of carbohydrate substrates. The effects of applied potential, pH and buffer composition were tested and optimized, and the most suitable conditions were used to evaluate the detection limit, linear range, and sensitivity of the sensor for different carbohydrates in the flow injection mode. Subsequently, the long term stability of the modified electrodes was determined. Additionally, the direct and mediated electron transfer between the active site of the enzyme and the electrode has been investigated by amperometric flow injection measurements in the absence and presence of the mediator 1,4-benzoquinone in the presence of cellobiose or lactose.

We 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.