Wang_2026_Mol.Catal_591_115688

The escalating environmental burden of PET waste has prompted the pursuit of efficient enzymatic solutions for its degradation. This research successfully engineered the H218S/F222I variant based on a variant of the leaf and branch compost cutinase (ICCG), enhancing its PET hydrolysis capabilities through both computational and experimental studies. The variant demonstrated superior thermostability and catalytic efficiency, attributes crucial for industrial-scale PET recycling. Comprehensive kinetic analyses, utilizing both conventional and inverse Michaelis-Menten equations, underscored the variant's improved PET substrate affinity and reaction velocity. In particular, the reaction rate of ICCG-H218S/F222I are higher than that of ICCG across a range of temperatures (3070 C). Upon substrate normalization, the mutant delivered 96 % PET conversion within 24 h under high-loading conditions, substantially outperforming ICCG at 85 %, thus corroborating the engineered variants superior catalytic efficiency. Structural and QM/MM MD and free energy simulations studies elucidated the enzyme's reaction mechanisms, revealing temperature-dependent pathways that inform future enzyme optimizations. This work not only advances our understanding of PET hydrolases but also paves the way for developing more effective biocatalysts to combat plastic pollution.