The C-terminal Ω-Loop of Candida antarctica Lipase A Governs Active Pocket Exposure via Conformational Switching

Abstract Candida antarctica lipase A (CALA) is widely used in industrial catalysis due to its excellent thermostability and unique substrate selectivity. However, the molecular mechanism governing its active pocket exposure remains unclear, which hampers its further application. In this study, by integrating multiscale simulations and protein engineering, the C-terminal Ω-loop (Loop 3) was identified as the key structural element governing active pocket opening. Truncation or enhancement of flexibility of this loop was found to promote conformational transitions toward the open state. Enzymatic assays revealed that Loop 3 variants shift the optimal substrate from C16 to C14 without altering regioselectivity. Notably, immobilization on hydrophobic supports likely restricts Loop 3 conformation, leading to complete inactivation in anhydrous systems. These results identified Loop 3 as a critical regulator of CALA conformational dynamics, providing a structural basis for rational engineering and immobilization strategy optimization.