Malthe Kjær Bendtsen, Andreas Møllebjerg, Samuel Peña‐Díaz, Rosie Graham, Nicolai Claus Petersen, Bjork Isaksen, Mathias Carstensen, Martin B. Johansen, Andreas Sommerfeldt, Allan R. Petersen, Iddi Khamisi Chuma, Cecilie Ryberg, Thomas R. Wittenborn, Virginia Gichuru, Huabing Wang, Carsten Scavenius, Alexander Sandahl, Daniel E. Otzen
ABSTRACT Better enzymes are needed to develop sustainable methods to recycle plastics with C‐X heterobonds such as polyurethane (PUR) and nylon, for which no industrial‐scale solutions exist. Current methods rely largely on sequence mining based on a small number of known enzymes. Here, we expand the pool of PURases and nylonases by bioprospecting legacy plastic waste with fluorophore plastic mimics combined with fluorescence‐assisted cell sorting (FACS). We identify 29 plastic‐degrading bacteria, from which 12 enzymes are identified by mass spectrometry and homology searches. Compared to existing enzymes, these enzymes show higher thermostability and hydrolytic activities against different high‐molecular weight PUR polymers and nylon textiles compared to previously described wildtype enzymes. To our knowledge, this is the first reported example of enzymes capable of hydrolyzing longer chains of untreated PUR and nylon as well as crosslinked PUR. This study significantly increases the number of known PURases and nylonases and provides starting points for optimization campaigns through protein engineering and for in silico discovery.