Characterization of Novel Urethanases and a Commercial Cutinase for the Degradation of Polyurethane

Every year, 460 metric tons of plastic are produced, yet only 10% is recycled. Due to the chemical inertness and durability of plastic materials, recycling techniques employ combustion and melting, exacerbating the global climate crisis by releasing greenhouse gases. Polyurethane (PU) is the second most abundant “hydrolysable” plastic, harboring hydrolytically sensitive functional groups in its polymer backbone. Yet only a fraction of PU waste is recycled, often through mechanical processes that downcycle waste into lower quality plastics. Enzymatic plastic degradation provides a promising alternative to traditional recycling since enzymes function under milder conditions in aqueous solvents. Recently, three mesostable urethanases identified from a functional metagenomic screen were found to hydrolyze small-molecule dicarbamates produced from the glycolysis of polyether-PU foam. Despite the utility of this discovery, the glycolytic pre- treatment prior to carbamate hydrolysis results in undesirable waste byproducts. Thus, in this work, we used sequence similarity networks to mine thermophile genomes in search of novel urethanases that act on virgin polyether polyurethane. Fifteen genes encoding putatively thermostable urethanases were identified and synthesized into overexpression constructs. Two promising candidates, WprA and TolA, were characterized on small molecule PU mimics as well as Impranilâ DLN W50 polyester PU dispersion. While both candidates possess a conserved Ser- cisSer-Lys catalytic triad characteristic of the Amidase Signature family, preliminary efforts to assay partially purified WprA demonstrated significant activity on substrates of interest. Specifically, opaque Impranil dispersions were cleared by treatment with WprA, strongly suggesting that this enzyme can hydrolyze polyester PU. To further this work, an on-plate screening method was developed to evaluate both polyester and polyether polyurethanases, using the commercial cutinase, HiC, as a proof-of-concept. Future work will involve improving WprA’s purity via cell-free synthesis and assessing its kinetic profile on model substrates and polyether- PU thermoplastics. Directed evolution efforts could expand WprA’s catalytic profile to include polyether polyurethane, which very few enzymes have been found to hydrolyze to date. Collectively, this work demonstrates the utility of bioinformatics and on-plate screening methodologies for the discovery of promising urethanases candidate worthy of further study.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2025

Author dc:creator

• Quigg, Kennedy E.

Advisor dc:contributor.supervisor

• Howe, Graeme

• Urethanase
• Polyurethane
• Plastic Degradation
• Enzyme
• Recycling
• Urethane
• Enzymatic Plastic Degradation

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/35235

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/35235