KnowVolution of an efficient polyamidase through molecular dynamics simulations of incrementally docked oligomeric substrates

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 first directed evolution campaign for a 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 ± 19 µmol _6-AHAeq. h ⁻¹ mg _enzyme ⁻¹ ) and enhanced thermal stability ( T _m = 90 °C, Δ T _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. These analyses reveal that productive enzyme-substrate complexes form in a closed conformation with a single PA 6 chain. To target buried, sterically more demanding chains and thereby improve the degree of depolymerization, remodeling of the polyamidase binding site could be a promising strategy.