Polyurethane (PU) is highly resistant to biodegradation, primarily due to the intrinsic stability of its urethane bond. Aside from a small number of amidases reported to hydrolyze (poly)urethane bonds, several promiscuous esterases have also been found to catalyze PU degradation. In this study, we clarified the ligand-free crystal structure of Aes72, an esterase enzyme that exhibits promiscuous hydrolytic activity toward carbamate and amide bonds, at a high resolution of 1.80 Å. We investigated the catalytic mechanism underlying urethane bond cleavage by Aes72 using multiscale quantum mechanics/molecular mechanics (QM/MM) simulations. Our findings indicate that the reaction mechanism consists of four concerted elementary steps, with the nucleophilic attack (step i) identified as the rate-determining step. The subsequent structure-guided engineering of Aes72 yielded several enhanced single mutants, ultimately resulting in a superior double mutant, F276A/L141I. This variant exhibited approximately a two-fold increase in catalytic efficacy toward bis(4-hydroxybutyl) (methylenebis(4,1-phenylene)) dicarbamate (BMC) hydrolysis and significantly enhanced degradation performance on two distinct polyether-based PU materials compared to the wild-type enzyme. Our findings provide essential mechanistic insights into the structure–function relationship of the promiscuous esterase Aes72 in PU degradation and demonstrate its potential applicability in bio-based plastic recycling.
Liu et al. (2026) studied this question.