Engineering Magnetic Nanobiocatalysts via Mussel Foot Protein-Mediated Laccase Immobilization for Enhanced Performance and Reusability

Abstract

Robust, reusable, and environmentally benign enzyme-based catalysts are promising for advancing more sustainable chemical processes. Laccase, a multicopper oxidoreductase, can transform a diverse range of phenolic and aromatic pollutants in water; however, limitations in operational stability and reusability hinder application scalability. Here, we report a bioinspired immobilization strategy using microbially derived mussel foot protein-5 (MFP) to anchor laccase onto superparamagnetic iron oxide nanoparticles (IONPs, similar to 8 nm). MFP's high DOPA content, strong metal oxide affinity, and cationic nature enable multipoint electrostatic binding to laccase (pI approximate to 3.5), achieving up to 98% immobilization yield with 80-110% activity recovery, surpassing cationic surfactant ligands and far exceeding anionic or silica coatings (<40%). Ultrasonication-assisted immobilization increased catalytic turnover to 706.7 U g(-1) (compared to 642.8 U g(-1) for the free enzyme). The MFP-IONP-laccase nanobiocatalyst showed 1.6-fold higher catalytic efficiency, a reduced K-M (31.9 mu M), >50% lower thermal deactivation at 45 degrees C, and a 20 kJ mol(-1) higher activation energy, reflecting enhanced stability. The system retained 71.9 +/- 8.7% activity after eight reuse cycles and achieved similar to 20% greater methyl orange removal than free laccase. Taken together, this bioinspired, magnetically recoverable platform provides a scalable route to high-efficiency, reusable, and thermodynamically stable oxidoreductase systems for a wide range of catalytic applications.