Peptide-Inspired One-Step Synthesis of Surface-Functionalized Fe₃O₄ Magnetic Nanoparticles for Oriented Enzyme Immobilization and Biocatalytic Applications

Immobilization of enzymes on magnetic nanoparticles (MNPs) is an effective way to ensure their recycling. However, MNPs are usually prepared and modified step by step before immobilization as the conditions are too harsh to maintain the three-dimensional structures and functions of the target enzymes. Herein, peptide-inspired magnetic nanocomposites (K5C@Fe3O4) were mildly prepared, where the proteins in the composites could functionalize efficiently for immobilization of the target dual enzymes without prior purification. The one-pot preparation of the K5C@Fe3O4 was achieved by the SpyCatcher-fused elastin-like polypeptides (ELPs-SpyCatcher, K5C) for biomimetic mineralization of the precursors at 30 °C without vacuum. Then, K5C@Fe3O4 were adopted to immobilize and purify the SpyTag-tagged target dual enzymes (xylanase–SpyTag–lichenase, X–T–L) directly from the crude cell lysate. Characterizations of the composites and activity assay of the dual enzymes confirmed that the X–T–L was successfully immobilized by a spontaneous reaction between SpyCatcher and SpyTag, with high immobilization yield (>90%), immobilization efficiency (>70%), and activity recovery (>70%). Besides, the immobilized dual enzymes showed excellent catalytic efficiency and reusability (>65%, 10 times). The peptide-inspired MNPs represent a bioactive hybrid material for biocatalytic applications and will shed light on covalently oriented enzyme immobilization. They will have exciting potentials in functional materials.