Lipase immobilization via dynamic defect-engineered ZIF-8: A bioactivity-enhanced platform for high-efficiency screening of herbal lipase inhibitors

Lipase immobilization plays a pivotal role in enhancing enzyme stability, reusability, and practical applicability in ligand fishing, a technique used to identify bioactive compounds from traditional Chinese medicine (TCM). However, conventional methods often lead to significant activity loss due to suffering from enzyme leakage, conformational distortion, and mass transfer resistance. In this study, we present a dynamic defect-engineering (DDE) strategy within zeolitic imidazolate framework-8 (ZIF-8) to overcome these limitations. The DDE process enables adaptive formation of transport channels driven by enzyme–framework interactions, facilitating the migration of lipase from surface adsorption to intracrystalline encapsulation. Brunauer–Emmett–Teller (BET) analysis confirmed successful encapsulation, showing a reduced specific surface area of 565.1 m 2 /g for lipase@ZIF-8, indicative of enzyme incorporation within the defect-engineered framework. This structural optimization significantly improved biocatalytic performance, achieving 82 % activity retention and 65 % immobilization yield, compared to 44 % activity retention in conventional in situ encapsulation. Utilizing the preserved catalytic activity, we established a lipase-based ligand fishing platform, which successfully identified 20 potential inhibitors (10 anthraquinones and 10 flavonoids) from Cassiae Semen and Citri Reticulatae Pericarpium using HPLC-Q-TOF-MS/MS analysis. Overall, this work demonstrates the effectiveness of the DDE strategy in enhancing lipase immobilization within ZIF-8, offering a robust and efficient platform for the high-throughput screening of lipase inhibitors from natural products and advancing the application of enzyme-based ligand fishing in TCM research.