Enzyme immobilization techniques are widely used for preserving enzyme operational stability in industrial applications. As part of the present study, magnetic chitosan/carbon nanotube nanocomposites were synthesized, thiol-functionalized, and utilized for laccase of Trametes versicolor (EC 1.10. 3.2) immobilization. Under different conditions, including pH, temperatures, and chemical exposure, the immobilized laccase’s activity, kinetic parameters, reusability, and storage stability were assessed. For this purpose, magnetic CNTs-chitosan nanocomposite (CNT/MNPS/CTS) was prepared using the co-precipitation technique and then thiolated with cysteamine using glutaraldehyde as a cross-linker. The immobilization yield was 96% for non-functionalized nanocomposite, while 93% for thiol-functionalized nanocomposite. According to the results, the optimal pH for laccase immobilized on non-functionalized nanocomposite (CNT/MNPS/CTS-Lac) and thiol-functionalized nanocomposite (CNT/MNPS/CTS-Lac) was 3.5, whereas the optimal pH for the free enzyme was 2.75. The maximum activity of both immobilized enzymes was found at 45 °C, while the maximum activity of the free enzyme was observed at 40 °C. However, CNT/MNPS/CTS-Lac was reused 27 times consecutively, whereas CNT/MNPS/CTS-SS-Lac was reused only four times. The results suggest CNT/MNPS/CTS-Lac exhibits superior stability and reusability than enzymes immobilized on thiol-functionalized nanocomposite. This may be due to the breakdown of an unstable disulfide bond by which laccase was immobilized. The immobilization of laccase on chitosan/carbon nanotube magnetic nanocomposites may represent a promising approach to developing reusable and efficient tools for various applications, including wastewater treatment, remediation, and sensing.
