Lee_2023_J.Hazard.Mater_459_132297

The mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low stability limits its industrial applications. Here, we developed a variant, Z1-PETase, with enhanced soluble protein yield and durability while maintaining or improving activity at lower temperatures. The selected Z1-PETase not only exhibited a 20-fold improvement in soluble protein yield compared to the previously engineered IsPETase(S121E/D186H/S242T/N246D) (4p) variant, but also demonstrated a 30% increase in low-temperature activity at 40 degreesC, along with an 11 degreesC increase in its Tm(D) value. The PET depolymerization test across a temperature range low to high (30-70 degreesC) confirmed that Z1-PETase exhibits high accessibility of mesophilic PET hydrolase and rapid depolymerizing rate at higher temperature in accordance with the thermal behaviors of polymer and enzyme. Additionally, structural interpretation indicated that the stabilization of specific active site loops in Z1-PETase contributes to enhanced thermostability without adversely impacting enzymatic activity. In a pH-stat bioreactor, Z1-PETase depolymerized > 90% of both transparent and colored post-consumer PET powders within 24 and 8 h at 40 degreesC and 55 degreesC, respectively, demonstrating that the utility of this IsPETase variant in the bio-recycling of PET.