Li_2025_Int.J.Biol.Macromol__145862

Enzymatic degradation of polyethylene terephthalate (PET) provides a sustainable and promising strategy for the recycling of plastic waste. Herein, we employed site-directed mutagenesis and machine learning methods to further enhance the performance of an efficient mutant of IsPETase, FAST-PETase-N212A, and resulting in seven variants with enhanced activity. We also found that the alpha3-beta5 loop containing the T140D mutation plays a significant role in both type I and type II cutinases. Subsequently, we determined the complex structures of two activity-elevated mutants with the PET analogue mono(2-hydroxyethyl)terephthalic acid, revealing a different binding mode. Finally, to facilitate the industrial application of PET hydrolases, we exploited the industrial strain Pichia pastoris to express the activity-enhanced mutants. Compared with E. coli-produced proteins, these mutants expressed by P. pastoris exhibited higher activity and thermal stability.