Here we report the preparation and characterization of novel enzyme supports based on silica-coated Fe3O4 magnetic nanoparticles. These nanomaterials were modified at their outer silica surface with isocyanate, trimethylammonium and β-cyclodextrin moieties to immobilize laccase from Trametes versicolor through covalent, electrostatic and supramolecular interactions, respectively, with protein immobilization yields ranging from 21.7% to 53.5%. The effect of the immobilization approach on the activity, optimal working conditions, stability and reusability of the resulting biocatalysts were studied. Best results were achieved for native and adamantane-modified laccase supramolecularly immobilized on β-cyclodextrin bearing supports in terms of their catalytic properties, showing 18.0 U and 14.0 U of immobilized laccase activity per gram of support. However, high thermal stability was observed for the enzyme covalently immobilized on isocyanate-modified nanoparticles, with 14.8-fold increase in the half-life time at 65ºC in comparison with native laccase. Best reusability properties were also achieved by covalent immobilization, retaining over 88% of the initial catalytic activity after 13 cycles of magnetic reuses. All enzyme derivatives were evaluated for the catalytic degradation of methylene blue as pollutant model, showing significant reduction of the dye. In special, a 68-fold increase in the removal efficacy was observed for covalently immobilized enzyme compared to the free laccase. These results suggest high potential application of these biocatalysts in wastewater treatment.
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