Discovery and rational engineering of PET hydrolase with both mesophilic and thermophilic PET hydrolase properties

Extensive research efforts have been directed towards the development of PET hydrolases with improved activity, but template enzymes used are limited. Here, the authors report a PET hydrolase from Cryptosporangium aurantiacum (CaPETase) that exhibits high thermostability and PET degradation activity at ambient temperatures and determine its crystal structure. Excessive polyethylene terephthalate (PET) waste causes a variety of problems. Extensive research focused on the development of superior PET hydrolases for PET biorecycling has been conducted. However, template enzymes employed in enzyme engineering mainly focused on IsPETase and leaf-branch compost cutinase, which exhibit mesophilic and thermophilic hydrolytic properties, respectively. Herein, we report a PET hydrolase from Cryptosporangium aurantiacum (CaPETase) that exhibits high thermostability and remarkable PET degradation activity at ambient temperatures. We uncover the crystal structure of CaPETase, which displays a distinct backbone conformation at the active site and residues forming the substrate binding cleft, compared with other PET hydrolases. We further develop a CaPETase(M9) variant that exhibits robust thermostability with a T-m of 83.2 & DEG;C and 41.7-fold enhanced PET hydrolytic activity at 60 & DEG;C compared with CaPETase(WT). CaPETase(M9) almost completely decompose both transparent and colored post-consumer PET powder at 55 & DEG;C within half a day in a pH-stat bioreactor.

Automated summary

In this study, a PET hydrolase (CaPETase) from Cryptosporangium aurantiacum with high thermostability and PET degradation activity at ambient temperatures was discovered, and its crystal structure was determined. This research is of great importance for addressing the issue of excessive PET waste.