Direct Electron Transfer at Cellobiose Dehydrogenase Modified Anodes for Biofuel Cells Federico Tasca, † Lo Gorton, † Wolfgang Harreither, ‡ Dietmar Haltrich, ‡ Roland Ludwig, § and Gilbert No ¨ll* ,† Department of Analytical Chemistry, Lund UniVersity, P.O. Box 124, SE-221 00 Lund, Sweden, DiVision of Food Biotechnology, Department of Food Sciences and Technology, BOKU-UniVersity of Natural Recources and Applied Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria, and Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria ReceiVed: March 10, 2008; ReVised Manuscript ReceiVed: April 17, 2008 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. Introduction Redox enzymes catalyze the oxidation or reduction of a specific substrate or a group of substrates usually with similar structural and electronic properties. Depending on their enzy- matic function these enzymes can be applied in biosensors 1–3 or biofuel cells. 4–7 While for biosensor applications, high sensitivity and substrate selectivity have to be reached, biofuel cells require modified electrodes working with high current densities. At the same time, long-term stability is desired. This is in contrast to biosensors which have to be cheap and are often designed for fast response time and single use. For both applications, the electrochemical addressability of the redox enzymes has to be achieved. This is possible by the use of redox mediators. 8,9 When enzymes are “wired” to osmium redox polymers, the electron transfer (ET) is mediated by flexible Os 2+/3+ redox centers which are able to transport the charge from the electrochemically active part of the enzyme to the electrode. 7,10 As an alternative to mediated electron transfer (MET), some enzymes are able to communicate directly with the electrode. The waiving of redox mediators in biofuel cell or biosensor applications simplifies the electrode architecture and reduces the amount of components which have to be optimized. Furthermore, the maximum cell voltage of biofuel cells is usually decreased by the use of redox mediators (for thermodynamic reasons). 7 Currently, direct electron transfer (DET) has been reported only for about 50 out of 1060 redox enzymes know today. 2,11–13 In recent years, the sensitivity of amperometric biosensors consisting of enzymes embedded in osmium redox polymers could be improved by the introduction of carbon nanotubes. 14–16 During investigations of redox en- zymes with respect to biosensor or biofuel cell applications, it also turned out that the rate of DET can be increased by single-walled 17–20 or multiwalled 21–29 carbon nanotubes. In this contribution, the DET properties of different types of cellobiose dehydrogenase (CDHs) in the presence and absence of single- walled carbon nanotubes (SWCNTs) are compared with respect to potential applications in biofuel cells. CDHs have been used previously for the development of amperometric biosensors based on DET. 30–33 CDHs are extracellular enzymes produced by a variety of fungi from the phyla Basidiomycota and Ascomycota. 13,34 The enzymes exist usually as monomers containing a larger flavin-associated (dehydrogenase) domain and a smaller heme containing (cytochrome) domain. 34 The cofactors in the flavin and heme domain are flavin adenine dinucleotide (FAD) and heme b. 34 CDHs oxidize cellobiose, cellodextrins, and lactose at the flavin domain. 30–34 When CDHs are immobilized on electrodes, oxidation of a substrate at the flavin domain is followed by intramolecular ET to the heme domain, which is able to communicate with the electrode via DET. 13,30–33 Alternatively, the electrons can be transferred to the electrode from either the flavin or the heme domain by MET. 13,30 Whereas MET has been shown for the intact enzyme as well as for the separated flavin domain, efficient DET requires the presence of the heme domain. 13,30,35 We have screened five different types of CDH with respect to their DET properties after coadsorption with SWCNTs on graphite electrodes. The CDHs from the basidiomycete fungi Phanerochaete sordida 31,32 * Corresponding author. Phone: +46 46 222 0103. Fax: +46 46 222 4544. E-mail: Gilbert.Noll@analykem.lu.se. New address: Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany † Lund University. ‡ BOKU-University of Natural Recources and Applied Life Sciences Vienna. § Reseach Centre Applied Biocatalysis. J. Phys. Chem. C 2008, 112, 9956–9961 9956 10.1021/jp802099p CCC: $40.75  2008 American Chemical Society Published on Web 06/11/2008