Glucose biosensor based on cellobiose dehydrogenase

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.

The fast and reliable measurement of the glucose concentration in human blood is a necessity for diabetes patients. A great improvement for the patient`s well-being can be achieved by less invasive sampling methods, for example by the application of a miniaturised continuous sensor for a longer period (at present only one week). These glucose sensors have the potential to increase the number of analyses and the accuracy of the data and thereby ensure an optimal insulin dosage. To develop an improved biosensor for continuous glucose measurement a new glucose recognition element - the enzyme cellobiose dehydrogenase (CDH) - was investigated. The enzyme transfers electrons obtained from the oxidation of glucose directly to the electrode, without otherwise necessary redox mediators. Sensors based on this principle of direct electron transfer need less components for the sensor assembly and are therefore easier and cheaper to build. Additionally, faster (and more frequent) measurements are possible due to lower mass-transfer restrictions by polymers of membranes. Appropriate CDHs were screened for and found in the fungi Myriococcum thermophilum and Corynascus thermophilus. Both CDHs were thoroughly characterised. The difficulty to apply CDH under physiological conditions is its adaption from its natural, acidic environment to a pH of 7.4 (the pH in blood). CDH from M. thermophilum was engineered by rational design to work at pH 7.4. To that purpose a homology model of the protein structure was calculated, amino acids for site-directed mutagenesis selected and mutated by genetic engineering. The obtained enzyme variants were purified and again characterised. By combination of several (up to 7) mutations the enzyme was able to transfer electrons efficiently at physiological pH. To avoid cross-reactions with maltose, which might interfere with the glucose measurement, the catalytic centre of M. thermophilum CDH was also altered and the conversion of maltose stopped. In a first study, the unmodified CDH of C. thermophilus could already prove its usefulness as a recognition element for blood glucose determinations. A very low glucose detection limit of 0.05 mM, a linear range from 0.1 to 30 mM and a sensitivity of 222 nA M -1 cm-2 were achieved. The genetically modified M. thermophilum CDH shows even better characteristics and will soon be tested in a glucose biosensor.