Enhancing the Catalytic Activity of Candida antarctica Lipase B (CALB) for the Synthesis of Moxifloxacin Intermediates by Loop Engineering

This study addressed the issue of insufficient activity in CALB lipase during the catalytic synthesis of key chiral intermediates for moxifloxacin. A structure-guided protein engineering strategy was employed to systematically modify its functional domains. Through molecular dynamics simulations of CALB-I189K, multiple regions exhibiting high conformational flexibility were preliminarily identified. Subsequently, by integrating 3D structural alignment with active site pocket distance analysis, the functionally most critical region (143–146) was selected. A site-directed saturation mutation library was constructed specifically targeting this region. Building upon the previously reported CALB-I189K, a mutant I189K/L144R/A146K was ultimately obtained through high-throughput screening combined with chiral HPLC validation. This mutant maintains excellent stereoselectivity (E = 206.52) while enhancing catalytic efficiency (kcat/Κm) to 273.73 min−1·mM−1, approximately 4.5-fold that of I189K. At a substrate concentration of 1 M, it achieves 50% conversion within 2.6 h, demonstrating kinetic resolution capabilities approaching industrial standards. Molecular simulation analysis indicates that the L144R and A146K mutations synergistically enhance catalytic performance primarily by optimizing spatial distances between catalytic residues. This study not only provides a high-performance catalyst for the efficient biosynthesis of moxifloxacin chiral intermediates but also offers new insights for enzyme rational design based on dynamic structural information.