Site‐Specific Oa AEP1 Mediated Functional Proteins Immobilization on Magnetic Nanoparticles and the Recyclings

Precise and stable immobilization of proteins on nanoparticle surfaces is essential for advancing applications in biocatalysis, biosensing, and synthetic biology. However, conventional immobilization strategies often suffer from nonspecific binding, random orientation, and loss of biological activity. Here, a chemically defined, site‐specific protein conjugation strategy is developed using the peptide ligase Oa AEP1 to catalyze ligation between N‐terminal Gly‐Leu (GL) and C‐terminal Asn‐Gly‐Leu (NGL) motifs. This approach enables controlled‐orientation and high‐efficiency immobilization of functional proteins and enzymes on Fe 3 O 4 and Fe 3 O 4 @SiO 2 magnetic nanoparticles. Fluorescent proteins and catalytically active enzymes, including TEV (Tobacco Etch Virus) protease and Taq polymerase, retain activity and recyclability following conjugation. Sequential ligation permits programmable multimeric assembly. This enzymatic platform provides a robust, reusable, and modular route to construct functional protein‐nanoparticle interfaces with broad implications for nanobiotechnology and protein engineering.