Acid Hydrolyzed Nylon-6 Turbidity as a Novel, Efficient, and Adaptable Assay for Nylonase Activity

The development of enzymes for plastic recycling is reliant on the ability to identify and engineer novel biocatalysts. Nylon-6 is a plastic for which there is great importance for recycling and valorization due to its use in textiles, automotive components, and engineered materials. High-throughput screening is increasingly the preferred method for enzyme engineering, and while high-throughput assays exist for nylonase activity, they suffer from a variety of pitfalls including dependence on complex instrumentation, utilization of nonrepresentative model substrates, inconsistent product derivatization, and sensitivity to pH and protein concentrations. Limitations in high-throughput nylonase screening correspondingly limit the number of variants that can be tested and thus hamper efforts to improve the relatively small number of nylonases known. Here, we demonstrate the utilization of acid oligomerized nylon-6 (AON6) to assay the performance of nylon-6 hydrolyzing enzymes in a manner that is compatible with purified protein and cell lysate while also allowing for variation in pH, solid loading, and enzyme concentration.