Immobilization of laccase on nanoporous gold and its enzymatic properties

Immobilization of biomolecules such as enzyme on nanomaterials has aroused wide interest in recent years. Nanoporous gold (NPG) with different pore sizes was prepared by controlling the etching time of Au/Ag alloy with concentrated nitric acid and annealing. Scanning electron microscopy and a nitrogen adsorption technique were used to characterize the NPG. The surface of NPG was activated with α-lipoic acid and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), and laccase from Trametes versicolor was then immobilized on it through chemical coupling. The effects of pore size on the specific activity and on the amount of immobilized laccase were studied. The results showed that the NPG with a smaller pore size was a better carrier for the laccase immobilization. Compared with free enzyme, the optimum pH of immobilized laccase did not change, however, the optimum temperature rose from 40°C to 60°C. Both pH and thermal stabilities were improved markedly via the immobilization. After 8 times usage, 65% of its initial activity could still remain. In addition, no obvious activity loss was observed after 1 month storage at 4°C. For the inactivated immobilized laccase, its carrier NPG could be recycled by simply immersing it into concentrated nitric acid. All these results demonstrate that NPG as a carrier for enzyme has great potential applications to biotechnology area.