Immobilized laccase on chitosan-clay beads: an efficient system for synthetic dye removal

The increasing release of synthetic dyes into aquatic environments has caused severe ecotoxicological impacts. Conventional treatment methods have shown limited efficiency, whereas enzymatic catalysis employing laccase has emerged as a sustainable alternative for dye removal. Here, we report a non-toxic immobilization strategy in which citric acid is used as the sole cross-linking agent to prepare chitosan-montmorillonite beads under mild heating, enabling efficient laccase immobilization without chemical activating agents. CuSO₄-assisted activation was employed to further enhance enzymatic performance, while eliminating the need for glutaraldehyde and other hazardous reagents. Among the supports evaluated, the CC-2/1 beads showed the highest immobilization performance (yield 93%; efficiency 210%), enabling rapid dye removal and complete degradation of bromophenol blue and phenol red within 5 min, as well as improved thermal and pH tolerance. After five reuse cycles, the immobilized enzyme retained approximately 70% of its initial activity and remained active for at least three months of storage. FTIR and SEM analysis revealed chemical and morphological changes indicative of effective immobilization. The environmental safety of the process was confirmed by acute toxicity assays using Artemia salina. A substantial reduction in toxicity was observed after treatment, indicating that the laccase-mediated oxidation products are less toxic than the parent molecules. Altogether, the results demonstrate a safe and efficient biocatalyst that combines rapid and robust degradation with proven ecotoxicity reduction, supporting its application in wastewater treatment and aligning with SDGs 6, 9, and 12.