A Click Chemistry Approach to Site-Specific Immobilization of a Small Laccase Enables Efficient Direct Electron Transfer in a Biocathode

Enzymatic biofuel cells and biosensors critically depend on efficient electron transfer between immobilized redox enzymes and the electrode. For direct electron transfer (DET) electrodes, the distance between the enzymatic active site and the electrode dictates electron harvesting efficiency, with empirical studies showing an optimum distance of approximately 20 Å. Since most proteins are much larger than 20 Å, strategic orientation of the enzyme on the electrode is crucial. In this study, we sought to enable site-specific electrode immobilization of a model small laccase from Streptomyces coelicolor (SLAC) through the in vivo incorporation of a non-natural amino acid with enabling chemistry, 4-azido-L-phenylalanine (AzF), into different sites in the vicinity of the catalytic center. AzF-containing laccase variants were immobilized onto a cyclooctyne-functionalized multi-walled carbon nanotube electrode via a copper-free cyclooctyne-azide cycloaddition reaction. One of the AzF-containing variants transferred electrons to the electrode with an efficiency, 28.7%, approaching the maximum possible within system-imposed constraints. The catalytic current generated was stable, with only ~14% reduction in current following 8 days at ambient temperature. This report provides a facile strategy for the site-specific directional immobilization of enzymes on electrodes for biofuel cell and biosensor development.