Nylon Analogue Substrates Allow for Continuous Quantification of Polyamidase Activity in Nylon-Degrading Enzymes

Nylon-hydrolyzing enzymes have been of increased interest recently in the context of bioremediation. The aminohexanoate oligomer hydrolases (NylCs) are thus far the most promising biocatalysts identified to this end. Protein engineering has been used to increase the thermal stability of these enzymes, but relatively little work has been done to improve their catalytic activity, due in part to a lack of high-throughput assays. Herein we report the design, synthesis, and enzymatic hydrolysis of polyamide analogue substrates mimicking various nylon architectures. We observed hydrolysis of diamide analogues 2, 4, and 5 in a continuous and quantitative manner via a light-scattering assay, which is amenable to a high-throughput screen in 96-well plates. The reaction products were characterized by liquid chromatography-coupled mass spectrometry, revealing insight into the structural elements required for recognition of substrates by NylC enzymes. The assay may be performed in minutes at elevated temperatures, allowing for efficient screening of thermostable nylonase enzymes. The activity of the NylC enzymes towards substrate 2 correlated to their corresponding enzymatic hydrolysis of Nylon-6 film, indicating that these substrates are surrogates for bulk nylon hydrolysis. Finally, we demonstrate the applicability of this assay to cell lysate, further enabling protein engineering efforts.