Zhou_2026_J.Hazard.Mater_514_142617

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 degreesC 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 degreesC to 85 degreesC), exceptional long-term stability (>70% activity retained after 28 days at 60 degreesC), 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.