Preparation optimization and stability comparison study of alkali-modified biochar immobilized laccase under multi-immobilization methods

Alkali modified biochar has attracted many interests as a potential enzyme immobilization carrier. In this paper, the preparation of alkali-modified immobilized laccases under multi-immobilization methods (adsorption: A/[email protected]; adsorption-crosslinking: A-C/[email protected]; covalent bonding: CB/[email protected]) were optimized via Design-Expert for the first time, and the stability of alkali-modified immobilized laccases was compared with other literatures. In addition, SEM, BET, FT-IR and XRD analyses were used to characterize the prepared immobilized enzyme samples. The results showed that the three kinds of immobilized laccases have improved the stability to different degrees relative to free laccase. In the pH range of 3–7, temperature range of 25–55 °C, and storage for more than 30 days, after 5 reaction cycles, the relative activity of the laccase immobilized on three different supports can still be greater than 50%. In addition, CB/[email protected] obtained the highest Vmax (0.94 U/mg), specific activity (0.66 U/mg) and activity recovery (66.20%); A-C/[email protected] obtained the highest immobilization yield (67.40%) and enzyme loading (180.81 mg/g); and A/[email protected] obtained the lowest Km of 140 μM. The above results are comparable to other reports, aiming to provide a reference for practical application of different methods of immobilized laccase in the industrial field.