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.

Cellobiose dehydrogenases (CDHs, EC 1.1.99.18) contain a larger flavin-associated (dehydrogenase) domain and a smaller heme-binding (cytochrome) domain. CDHs from basidiomycete fungi oxidize at an appreciable level cellobiose, cellodextrins, and lactose, and those from ascomycetes may additionally oxidize some monosaccharides to their corresponding lactones at the flavin domain. CDHs are able to communicate directly with an electrode via their heme domain. In this work, different types of CDHs have been adsorbed on graphite electrodes and studied with respect to their direct electron transfer (DET) properties. Electrochemical studies were performed in the presence and absence of single-walled carbon nanotubes (SWCNTs) using lactose as substrate. In the presence of SWCNTs, the electrocatalytic current for substrate oxidation based on DET between enzyme and electrode was significantly increased. Furthermore, the onset of the electrocatalytic current was at lower potential than in the absence of SWCNTs. The highest electrocatalytic activity toward oxidation of lactose was found for CDH from the basidiomycete Phanerochaete sordida. Based on CDH from Phanerochaete sordida, an anode for biofuel cells was developed. This anode using lactose as substrate was combined with a Pt black cathode for oxygen reduction as a model for a membrane-less biofuel cell in which the processes at both electrodes occur by DET.

A powerful alternative to glucose oxidase as anode material in implantable biofuel cells is presented: Cellobiose dehydrogenase (CDH) from the ascomycete Myriococcum thermophilum (MtCDH) catalyzes the electrochemical oxidation of glucose, lactose, and cellobiose over a broad pH range. Current densities of more than 1 mA • cm -2 can be reached when MtCDH is wired to an Os redox polymer in the presence of single-walled carbon nanotubes and when lactose is used as a substrate at pH 8. In contrast to CDHs from basidiomycete fungi, which oxidize only β-1,4-linked di-and oligosaccharides efficiently, MtCDH is also able to oxidize glucose and other monosaccharides at relatively high turnover rates. The current density toward oxidation of 5 mM glucose under physiological conditions was about 100 µA • cm -2 . Outstanding properties of MtCDH are high-temperature stability; a strong discrimination of oxygen turnover (and therefore no H 2 O 2 production) in the presence of alternative electron acceptors; an ability to oxidize a range of carbohydrates, and a working pH from 3 to 10, which allows for combination with a variety of enzyme-based cathodes for oxygen reduction. The performance and stability of a membraneless glucose biofuel cell consisting of an MtCDH-modified anode and a Pt black cathode working under physiological conditions (PBS buffer, pH 7.4, 37 °C) were investigated over a period of 3 days. A maximum voltage of 500 mV, a maximum current density of almost 700 µA • cm -2 , and a maximum power density of 157 µW • cm -2 at an operating voltage of 280 mV (under oxygen purging/ nonquiescent conditions) could be obtained with glucose (100 mM) as the substrate. Furthermore, the direct and mediated electron-transfer properties of MtCDH are compared in this work. The electrocatalytic current detected for mediated electron transfer (MET) is much higher and starts at a less positive potential than that for direct electron transfer (DET). The reason is that, in MET, the Os redox polymer is able to collect the electrons from the catalytically active flavin domain, whereas DET requires the oxidation of the heme domain, which has a more positive redox potential. The electrocatalytic current densities for DET and MET are significantly increased in the presence of single-walled carbon nanotubes.