Agarose-chitosan hydrogel-immobilized horseradish peroxidase with sustainable bio-catalytic and dye degradation properties.

Herein, we developed and characterized robust agarose-chitosan hydrogel using N‑hydroxysuccinimide (NHS) as a mild chemical cross-linker. The hydrogel offered a simple, effective and eco-friendlier support material with >90% of immobilization efficiency of horseradish peroxidase. The surface morphology and functional properties of the agarose-chitosan hydrogel with and without immobilized horseradish peroxidase were investigated by scanning electron microscopy and Fourier-transform infrared, respectively. The agarose-chitosan hydrogel-immobilized horseradish peroxidase (ACH-HRP) exhibited wide-working pH and temperature stability, and promising reusability for its substrate oxidation. The ACH-HRP preserved a better activity under acidic environments, pH 4.0 (38 vs. 5.9%), and well stabilized under alkaline conditions, retaining a 3.9-folds greater activity than a free counterpart at pH 10. With reference to a free enzyme, 1.6- and 4-fold greater catalytic activity was achieved at 50 and 70 °C, respectively, by the immobilized HRP. Further, the hydrogel displayed insignificant loss in enzyme functionality sustaining above 90% and 60% of original activity after 5 and 10 continuous cycles of use. HPLC profile corroborated the enzyme-assisted Reactive Blue 19 (RB-19) degradation, whereas UPLC/MS analysis scrutinized the dye degradation intermediates and a tentative mechanistic degradation pathway was proposed. In conclusion, the results demonstrate that ACH-HRP is a promising option for use as industrial biocatalyst in diverse biotechnological applications.