Direct Electron Transfer - Direct electron transfer and electrode contacting of redox enzymes

Duration:   2009-2011

Abstract:

The possibility to realize implantable sensor-transmitter systems with a volume smaller than 1 mm³ and to measure physiologically relevant substances online, without distracting wiring of the organism, has great potential in basic science and medicine. Feasible power sources for these low power requiring systems are miniature biofuel cells with a long operating life-time and of much smaller size than batteries or capacitors. Biosensors and biofuel cells based on direct electron transfer (DET), which do not need intermediary relays – so called mediators – for electron transfer (ET) are of greatest interest in current studies. In both applications, efficient electronic communication between the enzymes and the electrode is of utmost importance to achieve low detection limits or high current densities in these miniaturized devices.

To understand the principles for efficient DET, the focus is put on two model enzymes useful for biosensors or in biofuel cells: cellobiose dehydrogenase, CDH, catalyzing the anodic oxidation of various carbohydrates, and laccase, which reduces oxygen to water in the cathodic reaction or oxidizing catecholamins and phenolic analytes on the anode. Both enzymes have been shown to be capable of DET at acidic pH, but need to be improved by: (i) enhancing the catalytic turnover under physiological conditions, (ii) increased DET rates by optimization of ET pathways, and (iii) improved packing density, orientation and binding of the enzymes and operational stability of the electrode.

For the investigation of the electron transfer mechanism, which is different for the chosen model enzymes (laccase by electron tunneling; CDH uses a mobile haem domain for DET from the buried catalytic FAD site to the electrode surface) the following tasks are envisaged: (i) improvements of the electron transfer pathways in both enzymes using comparative modeling and rational design of the involved amino acid residues, (ii) modulations of the midpoint redox potentials of the redox centers to quantify the effect of the driving force of the ET rates, and (iii) modifications of the number and position of glycosyl residues to improve the packing density and the introduction of reactive amino acid side chains for stronger and better oriented binding on the electrodes.

The research will involve recently discovered, neutral CDHs and CDH from Trametes villosa, which exhibits already high DET rates under acidic pH conditions and the well characterized acidic laccase from Trametes versicolor, which will be used as a basis for modifications derived from sequence alignments and molecular models of novel neutral laccases. After heterologous expression and purification of the enzyme variants, kinetic methods will be applied to elucidate the effect of the introduced mutations on catalytic and electron transfer properties. Successful variants will be tested using different electrode setups to evaluate the progress and structurally elucidated to provide a rational base for further improvements in an iterative process, which finally aims for efficient electron transfer under physiological conditions and high operational stability.