Discovery and Engineering of a Urethanase for Enhanced Depolymerization of Polyurethane

Plastics are ubiquitous in daily life, highlighting the growing need for synthetic polymer processing and recycling strategies. Recent advances in biocatalytic recycling aimed at plastics with ester bonds, such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT), and have paved the way for a biobased circular plastic economy. However, efficient enzymes capable of hydrolyzing carbamate bonds in polyurethane (PU) are still scarce. In this study, we used a phylogeny-guided enzyme mining method to identify eight carbamate hydrolases by using the recently characterized urethanase UMG-SP-2 as the search template. Among these enzymes, urethanase-5 (with 51% protein sequence identity to UMG-SP-2) demonstrated PU hydrolytic activity and was functionally characterized. After solving its crystal structure at a resolution of 2.5 Å, we analyzed its binding mode with butyl-N-[3-(butoxycarbonylamino)-4-methylphenyl]carbamate (BTC) through molecular docking and molecular dynamics (MD) simulations. Using structure-guided rational design, we generated a superior variant (P367A/L381A), which exhibited a hydrolytic activity against BTC 2.8-fold higher than that of the wild-type enzyme. When applied in the hydrolysis of commercial polyester-based PU foam, the variant produces 10.9 times more monomers than the wild type. This study thus broadened the spectrum of potent urethanases and enhanced our understanding of the structure-function relationship of urethane-hydrolyzing enzymes, facilitating the advancement of PU waste depolymerization methods.