KnowVolution of an Efficient Polyamidase through Molecular Dynamics Simulations of Incrementally Docked Oligomeric Substrates

Abstract

Management of synthetic polymer waste is one of the most pressing challenges for society today. Enzymatic recycling of polycondensates like polyamides (PA), however, remains limited due to a lack of efficient polyamidases. This study reports the directed evolution of the polyamidase NylC_p2–TS. Key positions involved in enzyme–substrate interactions and PA 6 hydrolysis are identified through random mutagenesis and molecular dynamics (MD) simulations. The final variant, NylC–HP (NylC_p2–TS^{F134W/D304M/R330A}), exhibits a 6.9-fold increased specific activity (520 ± 1 μmol_6–AHAeq h⁻¹ mg_enzyme⁻¹) and enhanced thermal stability (T_m = 90 °C, ΔT_m = 4.2 °C), making NylC–HP the fastest polyamidase for PA 6 and PA 6,6 hydrolysis. Despite the improved reaction rate, the degree of depolymerization remains below 1%. To understand the molecular basis of achieved improvements and factors limiting the degree of depolymerization, intra- and intermolecular interactions of various enzyme-substrate complexes are analyzed by incremental docking of PA 6 tetramers and MD simulations. After optimizing the activity and stability of NylC–HP, the findings suggest that widening the substrate binding pocket is likely necessary to improve substrate accessibility to target more buried attack sites on the polymer surface and thereby enhance the degree of depolymerization.