Validated High-Throughput Screening System for Directed Evolution of Nylon-Depolymerizing Enzymes

Global polymer production is about to exceed 400 megatons yearly, and recycling methods enabling a climate-neutral, circular polymer economy are highly important to successfully address global challenges like environmental pollution and climate change. Polyamides (nylon-6, nylon-6,6) represent the third most-produced hydrolyzable polymer, behind polyurethanes and polyethylene terephthalate. The main challenges in developing enzymatic polyamide recycling processes are the limited number of reported polyamidases and the lack of screening systems that allow the employment of powerful directed evolution methods. Here we report the first validated high-throughput screening system to tailor polyamidases for applications in polyamide degradation. We successfully applied the screening system to detect nylon-6, nylon-6,6, and common polyurethane degradation products down to the nanomolar range in cell-free extract. One round of random mutagenesis yielded a polyamidase with a 1.9-fold improved turnover frequency (1.06 ± 0.04 s–1; NylCTSP27Q/F301L) after screening only 1700 clones. Moreover, for the first time, we determined Michaelis–Menten kinetics for polyamidases using polyamide film.