Lee_2023_bioRxiv__

A mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low thermostability limits its industrial applications. We herein developed an engineering strategy for IsPETase to enhance PET hydrolysis activity, thermostability, and protein folding of the enzyme. Balance-directed Z1-PETase variant outperforms the stability-directed Z2-PETase variant under both mesophilic and thermophilic conditions, although Z2-PETase exhibits higher thermostability than Z1-PETase. The Z1-PETase is also superior to Fast-PETase, Dura-PETase, and LC-CICCG in terms of depolymerization rate regardless of temperature conditions we tested. Thus, maintaining a balance between PET hydrolysis activity and thermostability is essential for the developmentof high-performance PET hydrolases. In a pH-stat bioreactor, Z1-PETase depolymerized >90% of both transparent and colored post-consumer PET powders within 24 and 8 hours at 40C and 55C, respectively, demonstrating that the balance-directed IsPETase variant produced herein may be applicable in the bio-recycling of PET.