Computational Redesign of a Urethanase for Efficient Polyurethane Depolymerization

Polyurethane (PU) is one of the most widely used petroleum-based plastics, significantly contributing to the global plastic waste crisis. In recent years, enzyme-based recycling technology has emerged as a promising eco-friendly solution to plastic pollution. Several urethanases capable of hydrolyzing PU have recently been identified. However, their low activity limits their utility for efficient PU degradation. Herein, we investigated the mechanism of PU hydrolysis catalyzed by the urethanase UMG-SP1 through multiscale quantum mechanics/molecular mechanics calculations using a substrate mimic containing two carbamate bonds. The deacylation stage was identified as the rate-determining step with an estimated Gibbs free energy barrier of 19.0 kcal·mol –1, consistent with experimentally determined range of 16.8–16.9 kcal·mol –1 . The effect of active-site structure and the enzymatic electric field on catalytic activity was analyzed, and their relationships with catalytic efficiency were established. Based on these relationships, we proposed two semirational enzyme-engineering strategies that successfully identified beneficial mutations. Ultimately, we obtained several UMG-SP1 mutants with improved hydrolytic activities, including the most active variant, L126M, which exhibits a close to 6-fold improvement in depolymerizing a self-synthesized thermoplastic polyether-PU compared to the wild-type enzyme. These findings offer a semirational approach for urethanase engineering, which has substantial potential for extension to the development of other plastic hydrolases.