Site-Specific and Covalent Immobilization of Lipase on Natural Polyphenol-Modified Magnetic Nanoparticles for Effective Biodiesel Production

Site-specific and covalent attachment is the most desirable immobilization strategy, but classic methods typically require genetic engineering or complicated material fabrication, resulting in operational complexity and difficulty. Herein, a novel site-specific and covalent immobilization strategy based on accurately selected immobilization sites on lipase was developed. Specifically, computer-aided structural analysis of functional groups on lipase revealed that lysine residues with free amino groups were far away from the catalytic pocket and lid, which were suitable to be chosen as the best immobilization sites to effectively reduce the loss in its activity. Meanwhile, natural polyphenol-modified magnetic nanoparticles could increase the active immobilization sites, and lipase can be immobilized on them directly via a covalent reaction. This site-specific immobilization system exhibited significant enhancement in activity recovery (71.3%) compared to random immobilization (42.5 and 55.9%). As expected, experimental and computational analyses revealed that tailor-made site-specific immobilization carriers were beneficial to maintain the native catalytic pocket conformation and enhance the rigidity of the immobilized lipase, which exhibited a higher biodiesel yield (92.1%) than free and randomly immobilized lipases. Besides, the site-specifically immobilized lipase could maintain as high as 75.3% biodiesel yield after eight cycles, making it an ideal nanocatalyst for efficient production of biodiesel. Overall, the site-specific enzyme immobilization technology can provide stable catalytic activity advantages over the randomly covalent immobilization strategy, which can significantly promote green manufacturing and sustainable production.