Discovery and engineering of a compost-derived thermophilic BHETase for enhanced depolymerization of post-consumer...

The accumulation of polyethylene terephthalate (PET) waste urgently demands sustainable recycling technologies. Enzymatic depolymerization represents an eco-friendly approach, but its efficiency is frequently hampered by product inhibition, as accumulated oligomeric intermediates such as bis-(2-hydroxyethyl) terephthalate (BHET) suppress the activity of backbone-degrading PET hydrolases. Herein, we report the identification and engineering of a thermophilic BHETase from a compost-derived thermophilic bacterium and demonstrate its synergistic use with the representative cutinase ICCG for efficient depolymerization of untreated post-consumer PET bottles. Using enrichment culture at 60 °C with PET as the sole carbon source, we isolated Geobacillus sp. strain Z231. By integrating genome sequencing and secretome analysis, we identified a thermophilic esterase named gsBHETase. This enzyme exhibits remarkable BHET-hydrolyzing activity, with catalytic efficiency 5.5-fold that of ICCG and 6.3-fold that of IsPETase. The engineered variant M3 exhibited a remarkably increased optimal temperature (from 60 °C to 85 °C), exceptional long-term stability (>70% activity retained after 28 days at 60 °C), and a 2.6-fold improvement in PET-hydrolytic activity relative to the wild-type enzyme. Notably, when combined with ICCG, variant M3 boosted the degradation of untreated post-consumer PET bottles, including dye-containing green PET, by up to 3-fold. This study not only provides a robust thermophilic BHETase as a promising biocatalyst but also establishes a synergistic dual-enzyme system that overcomes the rate-limiting step of oligomer hydrolysis, paving the way for green and efficient PET biorecycling.