Enhanced catalytic activity of polyethylene terephthalate hydrolase by structure-guided loop-focused iterative mutagenesis strategy.

The accumulation of polyethylene terephthalate (PET) waste has caused significant environmental pollution. Although biological depolymerization offers a promising solution, its efficiency remains constrained by the limited activity of PET-degrading enzymes. In this study, we designed a Structure-guided Loop-focused Iterative Mutagenesis (SLIM) strategy and rationally engineered the PET degradation enzyme ICCG for higher activity. The strategy was designed by demonstrating the critical role of the β8-α6 loop in type Ⅰ enzymes, which has currently not been reported. The best variant obtained, YITA (H183Y/L202I/I208T/T153A), exhibited 4.46-fold higher hydrolytic activity on amorphous PET at 72 °C compared to ICCG, outperforming other PET hydrolases, and exhibited superior degradation activity on real substrates such as cake containers and PET fibers. Conformational analysis revealed the key role of the remodeled β8-α6 loop in substrate binding and overall stability. Collectively, this study explores a promising approach to modifying PET hydrolase and lays a theoretical foundation for advancing bio-circular economy.