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

ChemSusChem, vol. 18, pp. e202500257

Abstract

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 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-TS F134W/D304M/R330A ), exhibits a 6.9-fold increased specific activity (520 ± 19 µmol6-AHAeq. h −1 mgenzyme −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. 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 enhancing the degree of depolymerization.

Authors 6

1. Hendrik Puetz Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringer Weg 3 52074 Aachen Germany

   RWTH Aachen University: Rheinisch-Westfalische Technische Hochschule Aachen Institute of Biotechnology Worringer Weg 3 52074 Aachen GERMANY

2. Alexander-Maurice Illig Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringer Weg 3 52074 Aachen Germany

   RWTH Aachen University: Rheinisch-Westfalische Technische Hochschule Aachen Institute of Biotechnology GERMANY

3. Mariia Vorobii Aachen DWI-Leibniz-Institut fur Interaktive Materialien DWI-Leibniz-Institut fur Interaktive Materialien GERMANY

   DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI‐Leibniz Institute for Interactive Materials Forckenbeckstraße 50 52074 Aachen Germany

   DWI-Leibniz-Institut fur Interaktive Materialien DWI-Leibniz-Institut fur Interaktive Materialien GERMANY

4. Christoph Janknecht Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringer Weg 3 52074 Aachen Germany

   RWTH Aachen University: Rheinisch-Westfalische Technische Hochschule Aachen Institute of Biotechnology GERMANY

5. Francisca Contreras Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringer Weg 3 52074 Aachen Germany

   RWTH Aachen University: Rheinisch-Westfalische Technische Hochschule Aachen Institute of Biotechnology GERMANY

6. Fabian Flemig Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringer Weg 3 52074 Aachen Germany

   RWTH Aachen University: Rheinisch-Westfalische Technische Hochschule Aachen Institute of Biotechnology GERMANY