Graphitic carbon nitride interfaces for lipase Immobilization: Molecular insights into stability and kinetic resolution

This study aims to improve catalytic stability and operational performance and reports the covalent immobilization of Lipase Eversa® Transform 2.0 (Et 2 ) onto glutaraldehyde-functionalized graphitic carbon nitride (g-C 3 N 4 ). As a metal-free material with high stability and biocompatibility, g-C₃N₄ provides a unique 2D platform for enzyme anchoring. A Taguchi experimental design was applied to optimize immobilization factors. The biocatalyst, g-C 3 N 4 -GLU@Et 2 , was characterized by FTIR, XRD, TGA, and SEM/EDS and evaluated for storage, thermal stability, pH tolerance, temperature tolerance, solvent tolerance, and reusability. Molecular docking and kinetic resolution (KR) of racemic acetate were also performed. The biocatalyst showed an immobilization yield of 98% and a recovery activity of 17.35 U/g. It maintained ~75% activity after 45 days at 4 °C, retained ~30% of its initial activity at 70 °C, and exhibited enhanced apparent activity in hydrophobic solvents, particularly hexane (+172% activity). The immobilized enzyme retained ~50% of its initial activity after three reuse cycles. The biocatalyst g-C 3 N 4 -GLU@Et 2 , in KR experiments, enabled the enantioselective hydrolysis of 1-phenylethyl acetate, achieving conversions of 99% and 50%, which is consistent with the binding interactions predicted by molecular docking. Thus, the results show that g-C 3 N 4 is an efficient macromolecular support suitable for the immobilization of lipases, enabling the development of robust, reusable, and enantioselective biocatalysts with potential applications in industrial bioprocesses and ecological systems. • Graphitic carbon nitride provides a robust macromolecular interface for lipase immobilization • Covalent multipoint attachment enhances enzyme stability against pH, temperature, and solvents • Immobilized lipase retains high activity after long-term storage and repeated use • Molecular docking correlates protein–support interactions with catalytic performance • The biocatalyst enables efficient and enantioselective kinetic resolution of racemic substrates