This study aimed to improve the functionality and stability of Lepidium draba peroxidase (LDP) for use in glucose biosensing. Following enzyme purification, covalent binding was optimized on chitosan- coated magnetic nanoparticles (nFe3O4-CS). Under the optimal conditions of 0.05 g nFe3O4-CS, 2.5% glutaraldehyde, and an 18 h coupling duration at 25 °C, enzyme immobilization efficiency reached 30%. The formation of the immobilized enzyme (IE) was confirmed using FT-IR, XRD, and FE-SEM. Upon immobilization, the enzyme’s affinity and catalytic efficiency for TMB were elevated by 1.7 and 11.0 times, respectively, in comparison to the free enzyme. The time course of stability against heat (at 50 °C) and storage stability (after 60 days at 4 °C) of the IE exhibited an increase of more than 225% and 230% as compared with the free enzyme. Moreover, the reusability of the IE was repeated up to 11 times, retaining 40% residual activity. Finally, the applicability of the IE was compared with that of the free enzyme; data showed linearity of the glucose detection range increased 10-fold compared to the free enzyme, and the assay time decreased 3 times relative to the free one. Overall, the improved stability of the immobilized LDP supports its potential use as a suitable enzyme for biomolecule detection in clinical practice and in various industrial applications

Keywords