Modulating interfacial conformational dynamics of lipase on bifunctional microenvironments for sustainable biodiesel conversion

Sequential coupling of hydrophobic orientation and rapid covalent locking. • Immobilized TLL exhibits >4000% relative activity via conformational tuning. • Interfacial microenvironment regulates partitioning and methanol tolerance. • Biocatalyst demonstrates exceptional durability and efficient conversion. Efficient multiphase bioprocessing for sustainable lipid valorization and biodiesel synthesis relies on robust solid–liquid biointerfaces. Their rational design is fundamentally constrained by severe reaction–diffusion constraints and the intrinsic activity–stability trade-off. Herein, we report a synergistic “orient-and-lock” strategy on bifunctional mesoporous SBA-15 to decouple these limitations. Hydrophobic domains first drive the oriented adsorption and “lid-opening” hyperactivation of Thermomyces lanuginosus lipase, followed by rapid isocyanide-mediated covalent anchoring to kinetically trap this metastable state without compromising structural flexibility. Molecular dynamics simulations elucidate a critical microenvironmental compartmentalization effect. This spatial partitioning selectively enriches lipophilic substrates while isolating polar inhibitors through localized hydrogen-bond networks and hydrophobic compartments, thereby effectively reducing substrate transfer resistance and minimizing active site blockage. Consequently, the immobilized biocatalyst exhibits an unprecedented 40-fold enhancement in apparent specific activity and a 57-fold increase in catalytic efficiency ( k cat / K m ) compared to the free enzyme. Its excellent tolerance to methanol enables a highly efficient one-step methanol addition reaction. The system demonstrates exceptional operational robustness, retaining 80% activity over 25 consecutive cycles and achieving a 96.8% biodiesel yield, establishing a synergistic paradigm of conformational engineering and transport regulation for high-performance heterogeneous biocatalysts. Download: Download high-res image (141KB) Download: Download full-size image