Rational redesign of FAST-PETase via a “locking” strategy for efficient PET depolymerization

Extensive accumulation of polyethylene terephthalate (PET) plastic waste causes serious pollution to the global environment, and developing superior PET hydrolases is vital for the enzymatic degradation and biorecycling of PET. Here, we propose a “locking” strategy for the rational redesign of FAST-PETase, one of the best-performing PETase variants reported so far, to further improve its performance. The best variant, FAST-PETase DC (A171C/S193C), exhibits 1.9-fold enhanced degradation efficiency compared with FAST-PETase at 50 °C, and a 4.1 °C increase in melting temperature ( T m ). FAST-PETase DC can almost completely depolymerize untreated post-consumer PET film within 3 d at 50 °C with periodic enzyme replenishment, two-fold faster than FAST-PETase. Molecular dynamics simulations reveal that the mutation locks Helix 5 and Loop 10 through a stable disulfide bond, and reduces the flexibility of the mutation sites and their connected regions. The structural changes consequently promote the substrate binding to the enzyme, facilitate the interaction within the catalytic triad, and rigidify the overall structure of the enzyme, leading to the improved degradation efficiency and thermostability. The study underscores the “locking” strategy as an effective way to enzyme redesign, and the engineered FAST-PETase variant is a promising hydrolase for the treatment and recycling of low-to-medium crystallinity PET plastic waste.