Expanding the Enzymatic Landscape for Polyurethane Degradation of Novel Bacterial Urethanases

Rotilio, L.; Oestergaard, R. R.; Thiesen, E. M.; Paiva, P.; Johansen, M. B.; Sommerfeldt, A.; Sandahl, A.; Keller, M. B.; Siebenhaar, S.; Otzen, D. E.; Fernandes, P. A.; Ramos, M. J.; Westh, P.; Morth, J. P.

doi:10.64898/2026.02.11.705263

bioRxiv · 10.64898/2026.02.11.705263

Expanding the Enzymatic Landscape for Polyurethane Degradation of Novel Bacterial Urethanases

Rotilio, L.·Oestergaard, R. R.·Thiesen, E. M.·Paiva, P.·Johansen, M. B.·Sommerfeldt, A.·Sandahl, A.·Keller, M. B.·Siebenhaar, S.·Otzen, D. E.·Fernandes, P. A.·Ramos, M. J.·Westh, P.·Morth, J. P.

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Abstract

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of five novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Two candidate enzymes, u17 u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Among them, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. To further assess enzymatic performance on a realistic industrial material, the panel was then tested on a generic flexible foam substrate derived from 2,4- and 2,6-toluene diisocyanate (TDA), where u15 and u17 emerged as the most active candidates. Collectively, we benchmark the structural framework presented by enzymes in the amidase signature family as a strong foundation for further optimization aiming at advancing sustainable and scalable biocatalytic recycling of polyurethanes.

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Rotilio, L., Oestergaard, R. R., Thiesen, E. M., Paiva, P., Johansen, M. B., Sommerfeldt, A., Sandahl, A., Keller, M. B., Siebenhaar, S., Otzen, D. E., Fernandes, P. A., Ramos, M. J., Westh, P., Morth, J. P.. 2026-02-11. Expanding the Enzymatic Landscape for Polyurethane Degradation of Novel Bacterial Urethanases. https://doi.org/10.64898/2026.02.11.705263

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