Novel bacterial urethanases expand the enzymatic landscape for polyurethane degradation

We report the identification and characterization of six novel bacterial urethanases (urethanase 10 [u10], urethanase 11 [u11], urethanase 13 [u13], urethanase 15 [u15], urethanase 16 [u16], and urethanase [u17]), expanding the enzymatic repertoire for polyurethane (PUR) depolymerization. Among them, u15 showed the highest catalytic efficiency on the model substrate di-urethane ethylene methylenedianiline (DUE-MDA; k cat /K M = 51.8 ± 0.1 s −1 mM −1 ), motivating its selection for structural analysis. High-resolution crystallography revealed key active-site architecture underlying u15's substrate binding and specificity, while docking and dynamics simulations elucidated structural determinants of urethane bond hydrolysis and identified a stable, catalytically productive binding orientation. Because 2,4- and 2,6-toluene diisocyanate (TDI)-based PURs dominate flexible foam production, one of the largest PUR waste streams, we assessed hydrolysis of a representative flexible PUR foam (Flex Foam), where u15 emerged as the most active candidate, releasing ∼0.5 mM TDA monomers, corresponding to 1.3% of the theoretical maximum content. Collectively, these results establish the structural framework of amidase signature enzymes as a foundation for advancing sustainable, scalable biocatalytic PUR recycling.