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

Plastic waste accumulation presents an environmental and human health crisis. With current recycling technologies recovering only ∼9% of plastics globally, there is an urgent need for sustainable solutions. While enzymatic strategies for polyethylene terephthalate degradation have made significant progress, analogous approaches for other plastics, like nylon, remain underdeveloped. In particular, the persistence of cyclic nylon oligomers has received limited attention, with only one distinct enzyme (NylA) reported decades ago, exhibiting poor catalytic performance. To address this critical gap, using genome mining, novel amidases were identified with enhanced activity and thermal stability. Herein, we report the discovery and characterization of a lactam hydrolase from Thermopolyspora flexuosa , the first thermostable NylA orthologue ( T m = 72°C ± 0.3°C). Biochemical analyses reveal that Tfl NylA hydrolyzes a range of lactams, including cyclic nylon byproducts, with particularly high specificity and turnover for laurolactam. Substrate scope analysis and structural modeling revealed key molecular features governing enzyme‐substrate compatibility, explaining the preferential activity of Tfl NylA. Co‐incubation of Tfl NylA and TvgC with nylon film increased nylon dimer production, underscoring the potential of enzyme synergy for enhanced plastic degradation. This thermostable NylA variant provides an ideal starting point for enzyme engineering efforts to develop robust catalysts for nylon waste remediation.