Zhang_2025_Bioresour.Technol_436_133035

Lipase conformational dynamics at carrier interfaces dictate biocatalytic performance, yet their structural adaptability and molecular underpinnings remain unclear. This study elucidates how reduced graphene oxide (rGO) surface hydrophobicity modulates lipase activation through spectroscopy and molecular dynamics simulations, unveiling enzyme-specific mechanisms. By tuning rGO reduction, we found that rGO-4 h optimally enhanced Candida antarctica lipase B (CALB) activity by 201.4 % through rigidity-driven stabilization, as its non-blocking lid improved structural integrity and substrate affinity rather than undergoing interfacial activation. Rhizomucor miehei lipase (RML) and Porcine phospholipase (PPL), which rely on lid-dependent activation, exhibited increased activity (122.3 % and 165.4 %, respectively) due to enhanced lid opening and hydrophobic interactions. Molecular dynamics simulations revealed that PPL's long lid required the highest flipping energy (-1112.9 kJ/mol), limiting activation. In contrast, RML's short lid had a lower energy barrier (-596.1 kJ/mol), facilitating lid flipping and interfacial activation. Spectroscopic analysis confirmed that moderate hydrophobicity preserved alpha-helix structures, whereas excessive reduction induced alpha-to-beta transitions. Radial distribution function analysis indicated that increased solvent accessibility at the active site facilitated RML and PPL activation. For CALB, enhanced van der Waals (vdW) interactions (-207.3 kJ/mol) and reduced electrostatic repulsion within the active pocket strengthened substrate binding, shortening the catalytic serine-substrate distance (2.89 A vs. 3.38 A) and improving efficiency. These findings establish that rigidity-dependent enzymes benefit from structural stabilization, whereas lid-dependent lipases require hydrophobicity-tuned flexibility for optimal activation. This study provides a structure-function framework for designing advanced immobilization platforms, with applications in biocatalysis, pharmaceuticals, and biofuel production.