Pastor_2025_ChemSusChem__e202500662

In this study, the three urethanases TflABH, MthABH, and OspAmd, originating from two distinct enzyme superfamilies, were identified and characterized with respect to their potential in polyurethane degradation. The substrate scope included five industrially relevant toluene diisocyanate (TDI)- and methylene diphenyl diisocyanate (MDI)-based carbamates with varied alcohol moieties, representative of intermediates from chemical PU recycling. Notably, TflABH and MthABH are the first urethanases from an esterase superfamily shown to efficiently hydrolyze at least four of the five tested PU-related substrates. Among these, TflABH displayed exceptional thermostability, with a melting temperature (Tm) at least 12 degreesC higher than those of the other urethanases evaluated. Optimal reaction conditions were established for all three enzymes, revealing pH optima of 7.0 for MthABH, 8.0 for TflABH, and 9.5 for OspAmd, while temperature optima clustered closely around 56-60 degreesC. Importantly, OspAmd demonstrated greater catalytic efficiency in the hydrolysis of MDA-MeOH, achieving conversions up to 50% after 48 h, approximately threefold higher than benchmark enzymes. These findings highlight the potential of OspAmd, in particular, as a promising biocatalyst for the enzymatic recycling of polyurethanes.