Structural Analysis of Nylon Hydrolase and Enzymatic Approach to Hydrolyze Polyamide Nylon

With the growing social demands for the Sustainable Development Goals (SDGs), there is increasing concern about the environmental pollution of nonbiodegradable polymers, and the development of plastic resource recycling circulation and/or new biodegradable plastics is desired. To find a way to reduce the environmental burden caused by polyamides, we have developed enzymatic approaches that enable various aliphatic nylons, including nylon6 and nylon66, to monomers using endo-type 6-aminohexanoate oligomer hydrolase (nylon hydrolase NylC). NylC is initially expressed as an inactive precursor, but the precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of α (27 kDa) and β (9 kDa) subunits. X-ray crystallographic analysis revealed that four αβ heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. Mutations at the subunit interfaces drastically affect subunit assembly and protein stability. We verified the thermal stability of NylC mutants based on the amino acid sequence and three-dimensional structure and finally obtained a quadruple mutant that showed high thermal stability (Tm = 88 °C). By customization of the enzymatic reaction and the prechemical treatment conditions toward nylon polymers, the efficiency of the hydrolytic reaction was remarkably increased, and they succeeded in converting more than 80% of polymeric nylons into monomers. In addition, we developed a measurement system using a thin nylon film to evaluate the reaction rate. This system has a high potential to be used as a high-throughput screening for the research development of biodegradable polyamides and nylon hydrolases with enhanced enzymatic activity.