Synthesis and characterization of ZrFe2O4 and ZrFe2O4@UiO-66-NH2 nanoparticles for efficient immobilization of Humicola insolens lipase: a comparative study of precipitation-crosslinking versus covalent binding methods

Abstract

This study focused on immobilizing Humicola insolens lipase onto magnetic nanoparticles of ZrFe₂O₄ and ZrFe₂O₄@UiO-66-NH₂ using precipitation-crosslinking and covalent binding methods. Characterization techniques, including FT-IR, SEM, energy-dispersive X-ray spectroscopy, X-ray diffraction, BET, DLS, and thermogravimetric analysis, confirmed the successful synthesis and functionalization of the supports. Enzyme immobilization was assessed using different buffer systems, and Tris–HCl buffer at a low concentration was chosen as the optimal medium due to its compatibility with the support structure. The precipitation-crosslinking method resulted in enzyme loadings of 260 mg g⁻¹ for ZrFe₂O₄ and 226 mg g⁻¹ for ZrFe₂O₄@UiO-66-NH₂, achieving immobilization efficiencies of about 40% and 60%, respectively. In contrast, the covalent binding technique significantly improved the enzyme loading and immobilization efficiency, with ZrFe₂O₄ achieving 305 mg g⁻¹ and 65% efficiency. ZrFe₂O₄@UiO-66-NH₂ demonstrated even greater performance, with the immobilization efficiency exceeding 80%. The reusability and thermal stability of the immobilized lipase improved markedly with covalent binding, particularly for the biocatalyst obtained by immobilizing lipase on ZrFe₂O₄@UiO-66-NH₂ nanoparticles. This biocatalyst retained over 70% of its activity after five reuse cycles and retained 40% activity at 80 °C. In contrast, the precipitation-crosslinking method led to a significant decline in activity during successive cycles, with no observable enhancement in enzyme thermal stability using this technique.