Optimising direct electron transfer of cellobiose dehydrogenase for use in 3rd generation glucose biosensors
Abstract
The quantitative determination of glucose, a major participant in metabolism, has numerous applications in food technology, biotechnology, and medicine. Glucose biosensors are therefore a target of substantial research efforts, driven by estimated sales of blood-glucose meters of $6.1 billion worldwide in 2004. Several of the difficulties known from 1st and 2nd generation glucose biosensors can be circumvented by establishing direct electron transfer between the biocatalyst and the electrode. Until now, only a small number of enzymes have been found suitable for use in so called 3rd generation biosensors. One enzyme which exhibits very good direct electron transfer properties is cellobiose dehydrogenase (CDH), an extracellular fungal flavocytochrome. The properties of this enzyme, especially the one from Trametes villosa, have been used to built ultra sensitive 3rd generation lactose biosensors. Until now, it was not possible to use it for the detection of glucose for two reasons: a strong glucose discrimination, and an acidic pH optimum which prevents measurements in neutral or alkaline samples like blood. Recently, a novel CDH was discovered in the thermophilic ascomycete Myriococcum thermophilum, differing in several aspects from the well characterised basidiomycete enzymes. The enzyme is able to oxidise glucose efficiently, and in a less acidic environment. The limit of glucose detection for an graphite electrode modified with this CDH was 1 mM (pH 5.0), already below the pysiologically relevant glucose concentration of 2 to 50 mM. To improve the performance of this glucose biosensor and make it commercially viable the intramolecular electron transfer rate of M. thermophilum CDH, which limits the direct electron transfer rate, has to be increased especially at a neutral to slightly alkaline pH. Our strategy is to elucidate the highly efficient intramolecular electron transfer mechanism of T. villosa CDH and apply it to M. thermophilum CDH by site-directed mutagenesis. To increase the transfer rate at elevated pH values several promising targets have been obtained by homology modelling of published sequences, especially the ascomycte H. insolens CDH with an alkaline pH optimum. After expression and purification of the variants thermodynamic and kinetic methods will be applied to elucidate the effect of the introduced mutations on domain interaction and intramolecular electron transfer. Finally, successful candidates will be tested on various electrodes for improvement of direct electron transfer. To successfully manage this proposed task the two nominated young scientists will be assisted in their work by several undergraduate students and highly qualified national and international collaborators.
Publikationen
Bubble-free oxygenation of a bi-enzymatic system: effect on biocatalyst stability.
Autoren: Van Hecke, W; Ludwig, R; Dewulf, J; Auly, M; Messiaen, T; Haltrich, D; Van Langenhove, H Jahr: 2009
Journal articles
Comparison of direct and mediated electron transfer for cellobiose dehydrogenase from Phanerochaete sordida
Autoren: Tasca, F., Gorton, L., Harreither, W., Haltrich, D., Ludwig, R., and Nöll, G. Jahr: 2009
Journal articles
Cellobiose dehydrogenase from the ligninolytic basidiomycete Ceriporiopsis subvermispora.
Autoren: Harreither, W; Sygmund, C; Dünhofen, E; Vicuña, R; Haltrich, D; Ludwig, R; Jahr: 2009
Journal articles
Project staff
Roland Ludwig
Assoc. Prof. Dipl.-Ing. Dr. Roland Ludwig
roland.ludwig@boku.ac.at
Tel: +43 1 47654-75216
Project Leader
01.06.2007 - 30.04.2010
Dietmar Haltrich
Univ.Prof. Dipl.-Ing. Dr.techn. Dietmar Haltrich
dietmar.haltrich@boku.ac.at
Tel: +43 1 47654-75211
Project Staff
01.06.2007 - 30.04.2010