Towards the Bioremediation of Nylon Waste Materials: Genome Mining Leads to the Identification of a Thermostable Laurolactamase from Thermopolyspora flexuosa

The accumulation of plastic waste presents an ongoing environmental and human health crisis. With current recycling technologies recovering only ~9% of plastics globally, there is an urgent need for innovative and sustainable solutions. While enzymatic strategies for polyethylene terephthalate (PET) degradation have made significant progress, analogous approaches for other plastics, such as nylon, remain underdeveloped. In particular, the environmental persistence of cyclic nylon oligomers has received limited attention, with only a single distinct enzyme (NylA) reported decades ago and exhibiting poor catalytic performance. To address this critical gap, a genome mining approach was used to identify novel amidases with enhanced activity and thermal stability. Herein, we report the discovery and characterization of a thermostable lactam hydrolase from the bacterium Thermopolyspora flexuosa, representing the first thermostable NylA orthologue, with a melting temperature of 72 ± 0.3 °C. Biochemical analyses reveal that this enzyme hydrolyzes a broad range of lactams, including cyclic nylon byproducts, with particularly high specificity and turnover for laurolactam. An analysis of substrate scope trends was performed to understand the molecular features governing enzyme-substrate compatibility. Structural modeling and mutational analysis elucidated key substrate-binding feature, shedding light on the preferential activity of NylAs towards laurolactam over the cyclic nylon substrate and providing a mechanistic foundation for downstream enzyme engineering efforts. This thermostable NylA variant will serve as the ideal starting point for the development of robust enzymes capable of mitigating recalcitrant nylon waste, advancing the field of biocatalysis toward sustainable plastic remediation technologies.