Hu_2025_Chembiochem__e202500065

Enzymatic hydrolysis of polyethylene terephthalate (PET) is a promising technology for advancing a circular PET economy. Several PET-degrading alpha/beta hydrolases have been identified, but the full potential of this enzyme family to catalyze PET hydrolysis remains largely unexplored. To address this, sequence similarity networks were employed to investigate the alpha/beta hydrolase fold-5 subfamily (IPR029059) for new PETases. Priority was given to sequences from thermophiles, as thermostable enzymes are likely more suitable for industrial applications. Ten enzymes with ~20% sequence identity to the well-known LCC-PETase were identified, and seven were successfully overexpressed and purified for in vitro characterization. Each enzyme catalyzed the hydrolysis of p-nitrophenyl butyrate, a mimic of trimeric PET, and emulsified PET nanoparticles. Notably, three enzymes were also capable of hydrolyzing PET films. Novel PETases exhibited melting temperatures (Tm) exceeding 55 degreesC and only modest losses of activity after incubation at 70 degreesC for 24 hours. The crystal structure of AroC (Tm = 85 degreesC) was resolved to 2.2 A, revealing several salt bridges that likely confer thermostability, and a unique loop that is conserved amongst the PETases described here. These novel enzymes will enable engineering campaigns to generate thermostable and catalytically efficient PETases for use as industrial biocatalysts.