Immobilization of Laccase on Nanoporous Gold: Comparative Studies on the Immobilization Strategies and the Particle Size Effects

Abstract

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.

Direct electron transfer of Trametes hirsuta laccase adsorbed at unmodified nanoporous gold electrodes

“…On changing the immobilization conditions to vacuum deposition of the ThLc solution on a bare, vacuum dried nanoporous gold electrode, followed by covering the electrode with a P017-epoxy cap, significantly higher currents of 7 µA (Fig 1B) were obtained. As reported previously, the linkages formed by chemisorption of the NH 2 groups of the lysine residues of ThLc on nanoporous gold are as strong as those for -SH groups on gold [32]. However, a P017-epoxy cap was also applied [33] to prevent any possible leaching of the enzyme from the gold surface.…”

Nanoporous Gold Electrodes and Their Applications in Analytical Chemistry

“…These SAMs can act as simple modifiers [68] or be used as linkers to attach various molecules to its surface such as proteins, enzymes, antibodies, or photosystem I [65,66,90,[106][107][108][109]. Because of the very high surface areas of nanoporous gold, a significantly larger amount of the modifier can be immobilized on its surface relative to a planar gold electrode.…”

“…A schematic of the immobilization of lipase onto nanoporous gold is shown in Figure 9 [139]. Nanoporous gold has already been shown to be a good support for the immobilization of a wide variety of proteins/enzymes including laccase [106,108,134,138], glucose Figure 9: A schematic of the immobilization of lipase onto nanoporous gold [139]. Reprinted from [139].…”

“…In a later study, the effect of three immobilization strategies (physical, electrostatic, and covalent) on the loading and reaction kinetics for the laccase-catalyzed oxidation of 2,6-dimethoxyphenol has also been studied [108]. The authors showed that physical and covalent immobilization gave similar loadings, whereas the electrostatic immobilization was lower by almost a half.…”

Laccase Immobilization by Chelated Metal Ion Coordination Chemistry

“…Among all these methods, adsorption is relatively simple and less expensive, so it may have a higher commercial potential than the other methodologies. Some studies have demonstrated that adsorption is preferable to other techniques for the immobilization of some particular laccases [10]. Recently, metal-based supports have been receiving emerging attention for metal-chelated enzyme adsorption [9].…”