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 first directed evolution campaign for a polyamidase, NylCp2-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 (NylCp2-TSF134W/D304M/R330A), exhibits a 6.9-fold increased specific activity (520 ± 19 µmol6-AHAeq. h−1 mgenzyme−1) and enhanced thermal stability (Tm = 90 °C, ΔTm = 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.
Competing Interest Statement
The authors have declared no competing interest.