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

A sensitivehigh-throughput screening system enables directeddepolymerase evolution to leverage sustainable polyamide and polyurethanerecycling. Global polymer production is about to exceed 400 megatonsyearly,and recycling methods enabling a climate-neutral, circular polymereconomy are highly important to successfully address global challengeslike 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 challengesin developing enzymatic polyamide recycling processes are the limitednumber of reported polyamidases and the lack of screening systemsthat allow the employment of powerful directed evolution methods.Here we report the first validated high-throughput screening systemto 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 nanomolarrange in cell-free extract. One round of random mutagenesis yieldeda polyamidase with a 1.9-fold improved turnover frequency (1.06 & PLUSMN;0.04 s(-1); NylC(TS) (P27Q/F301L)) after screening only 1700 clones. Moreover, for the first time,we determined Michaelis-Menten kinetics for polyamidases usingpolyamide film.