β-sheet Engineering of IsPETase for PET Depolymerization

The enzymatic depolymerization of polyethylene terephthalate (PET) offers a sustainable approach for the recycling of PET waste. Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface. Here, we report the systematic engineering of whole β-sheet regions in the core of IsPETase (a PETase from Ideonella sakaiensis) via a fluorescent high-throughput screening assay. Twenty-one beneficial substitutions were obtained and iteratively recombined. The best variant, DepoPETase β, with an increase in the melting temperatures (T_m) of 22.9 °C, exhibited superior depolymerization performance and enabled complete depolymerization of 100.5 g of untreated post-consumer PET (pc-PET; 0.26% W_enzyme/W_PET enzyme loading) in liter-scale bioreactor at 50 °C within 4 d. Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region, a more tightly packed structure of the core sheets and the surrounding helix, and improved binding of PET to the active sites. This study not only demonstrates the importance of engineering strategy in the β-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.