Improving enzyme immobilization: A new carrier-based magnetic polymer for enhanced covalent binding of laccase enzyme

In the quest for effective enzyme immobilization methods, this study focuses on synthesizing carrier-based magnetic polymer to enhance the covalent binding of T. versicolor laccase. Utilizing chitosan (CS) and alginate (ALG) composites, modified with Fe₃O₄ magnetic nanoparticles (MNPs), we aimed to improve the enzyme's stability, reusability, and performance under varying conditions. The ionic gelation method was employed to prepare CS-ALG and CS-ALG-Fe₃O₄ MNPs composites, resulting in an 84% and 91% immobilization efficiency. The immobilized enzyme demonstrated superior thermal stability, retaining 48 % activity for CS-ALG-laccase and 67 % for CS-ALG-Fe₃O₄ MNPs-laccase at 70 °C, compared to 29 % for the free enzyme. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy were used to characterize the composites, revealing significant morphological changes and successful enzyme integration. Kinetic studies indicated that immobilization increased the Vmax to 141 μmol/min for CS-ALG-laccase and 111 μmol/min for CS-ALG-Fe₃O₄ MNPs-laccase, while slightly reducing substrate affinity (Km) to 1.42 mM^-1 and 1.32 mM^-1, respectively. The immobilized laccase retained higher activity after ten reaction cycles (81 % activity for CS-ALG-Fe₃O₄ MNPs-laccase) and during prolonged storage (75 % activity retention), showcasing its potential for industrial applications. Additionally, the enzyme exhibited increased resistance to various organic solvents, enhancing its practical utility.