Nanoporous gold (NPG), prepared simply by dealloying Ag from Au/Ag alloy, was used in the present study as a carrier for laccase immobilization. Three immobilization strategies, i.e., physical adsorption, electrostatic attraction, and covalent coupling, were used to immobilize laccase on NPG. A detailed comparison among the three strategies was made in light of the loading, the specific activity, and the leakage of laccase. The present results indicated that the physical adsorption strategy was the best one for laccase immobilization on NPG. This was because of the potential covalent linkage between the nanoscale gold surface and the amino groups of the residue amino acids of laccase. The effects of the particle size of NPG on laccase loading and enzyme kinetics were also investigated. When the particle size of NPG got smaller, more laccase could access the inner pore and be immobilized. The kinetic study showed that the crushed NPG not only enhanced mass transfer of the substrate and its oxidation product but also favored the exposure of the active sites of the immobilized laccase to the substrate, i.e., the crushing facilitated the enhancement of the catalytic efficiency of laccase.

Nanoporous gold (NPG) with different pore sizes was obtained by simple dealloying and thermal annealing methods. The morphology of the NPG was characterized by scanning electron microscopy and nitrogen adsorption technique. Laccase was immobilized on the surface of the NPG by physical adsorption. Detailed studies were made on the effect of the pore size on laccase immobilization. NPG with pore size of 40−50 nm was demonstrated to be a suitable support for laccase immobilization. Compared with free enzyme, the optimum pH of immobilized laccase did not change; the optimum temperature, however, rose from 40 to 60 °C. Both thermal and storage stabilities of laccase improved markedly via the immobilization. Laccase immobilized on NPG (100 nm in thickness) was used for enzyme electrode construction. Direct electrochemistry of laccase on NPG supported by glassy carbon electrode (NPG/GC) was achieved with high efficiency due to the outstanding physicochemical characteristics of the NPG. The laccase-loaded NPG/GC electrode also exhibited a strong electrocatalytic activity toward O2 reduction. When stored at 4 °C for 1 month, the electrode showed no obvious changes in its response. All results presented in the paper indicated that NPG was an excellent carrier for laccase immobilization and would have potential applications in biofuel cell and/or biosensor areas.

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