Genome mining for NylB-type amidases to facilitate closed-loop nylon recycling
Graeme Howe
Professor, Department of Chemistry and Biomedical & Molecular Sciences
Queen’s University
April 21, 2026
11 a.m.
In-person: C2-361
Abstract:
Plastic pollution is now widely recognized as an environmental crisis with uncertain impacts on human health and wellbeing, yet plastic production levels are still anticipated to increase over the next several decades. This reflects the nature of plastics as a double-edged sword: the same durability and inertness that make these materials so useful in our society has also precluded efficient recycling of plastic wastes. While many promising developments with PET-hydrolyzing biocatalysts have occurred over the past decade, enzymes that process other plastics have not been particularly forthcoming. For instance, nylon-degrading enzymes have only recently begun to attract attention as potentially viable biocatalysts for nylon waste recycling. Recently, our group described an extremely stable amidase (TvgC) that catalyzes the endohydrolytic release of short oligomers from both nylon-6 and nylon-6,6. While engineering efforts are ongoing to improve the catalytic efficiency of TvgC, we envisioned that exohydrolytic (NylB-type) amidases might also be required to enable closed-loop recycling, where the pool of oligomeric products released by TvgC treatment are further hydrolyzed to the monomeric building block(s), simplifying separation of these value-added small molecules. Unfortunately, the only known NylB is both catalytically inefficient and insufficiently stable for industrial recycling efforts.
Here, sequence similarity networks were used to identify five putative NylB-type enzymes from thermophilic microorganisms. One of these proteins from an unidentified Thermomicrobiales bacterium (TmbNylB) was expressed solubly in E. coli and could be purified by immobilized metal affinity chromatography. Gratifyingly, TmbNylB was found to be thermostable (Tm ~ 59 °C) and catalytically active for significantly longer periods than the only other known NylB: at 50 °C, TmbNylB had a half-life of inactivation of t1/2 (50 °C) ~ 12 hours. In contrast, the literature enzyme loses half of its activity after only 22 minutes at 40 °C. After TmbNylB was found to exhibit both esterase and amidase activity against chromogenic p-nitrophenyl-based substrates, the substrate scope was further explored using several small molecules resembling nylon-6 and nylon-6,6 oligomers. Substrates harbouring a terminal amino group were found to be poor substrates for TmbNylB, while the presence/absence of a terminal carboxylate did not impact activity as significantly. Guided by these observations, semi-rational engineering was employed to develop a triple mutant of TmbNylB with significantly enhanced activity toward authentic nylon-6 dimers and trimers. This talk will describe our genome mining and enzyme engineering strategy, the resulting improvements in catalytic performance, and the mechanistic insights gained from kinetic analysis of additional TmbNylB point mutants that highlight the potential of NylB-type amidases in closed-loop nylon recycling.
Dr. Graeme Howe is an Associate Professor in the Departments of Chemistry and Biomedical & Molecular Sciences at Queen’s University. He received his doctorate in physical organic chemistry from the University of Toronto before moving to the University of Illinois at Urbana-Champaign to study the mechanism of an unusual phosphotransferase as an NSERC PDF. His current research focuses on the discovery, characterization, engineering, and implementation of enzymes as sustainable biocatalysts for useful chemistries. The genome mining pipeline developed by his group has delivered new biocatalysts with tailored properties to enable i) high-value synthetic transformations, ii) the chemoenzymatic production of novel therapeutics, and iii) the enzymatic degradation of PET, nylons, and polyurethanes. He serves as the Activity Lead for the OpenPlastic consortium’s efforts to find and engineer novel plastic-degrading enzymes and microbes. Dr. Howe received the Polanyi Prize in Chemistry in 2020, and in 2024, he was named an “Emerging Talent in Sustainability Research” by the Editorial Board of ChemSusChem. Recently, his work describing a hyperthermostable nylon-degrading enzyme was published in Angewandte Chemie International Edition, and, resultingly, Dr. Howe was profiled this leading venue for chemical research.