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/lac@A-MB; adsorption-crosslinking: A-C/lac@A-MB; covalent bonding: CB/lac@A-MB) 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 degrees 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/lac@A-MB obtained the highest Vmax (0.94 U/mg), specific activity (0.66 U/mg) and activity recovery (66.20%); A-C/lac@A-MB obtained the highest immobilization yield (67.40%) and enzyme loading (180.81 mg/g); and A/lac@A-MB obtained the lowest K-m of 140 mu M. 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.
