Trylons: Polyamide Surrogate Substrates Enable High-Throughput Screening of Nylon-Degrading Enzymes

The aminohexanoate oligomer hydrolase enzymes, NylCs, have emerged as lead biocatalysts for their potential to depolymerize nylon in a bioremediation context. Protein engineering efforts have thus far improved thermostability and activity, but conversion of the polymer to smaller oligomers remains low. With the intent to accelerate engineering efforts, we report the design, synthesis, and application of polyamide surrogate substrates to a rapid, high-throughput screen of NylC activity by continuous light scattering measurements. Evaluation of a small panel of diamide substrates revealed unprecedented specificity for the amide bonds recognized and hydrolysed by NylCs. We further demonstrate that the active substrates, Trylon-6 and Trylon-66, act as surrogates for nylon hydrolysis, where activity of NylCs towards Trylon is predictive of their activity towards bulk nylon film. Finally, we apply the surrogate screen to four mutant libraries of NylC_K TS, where activity of the libraries (nearly 100 variants each) was determined in 20 min. From this screen, we report substitution tolerances at positions 146, 189, 192, and 305, identifying residues near the active site of NylC enzymes that are necessary for amidase activity.