Munoz-Tafalla_2026_Microb.Biotechnol_19_e70432

Poly(ethylene terephthalate) (PET) hydrolases are enzymes primarily within the polyesterase-cutinase branch of the alpha/beta-hydrolase superfamily, whereas the contribution of true lipases to PET hydrolysis remains poorly explored. Here, we report the genome mining results of 18,082 extremophilic microorganisms, combined with structural modelling, enzyme-substrate simulations and experimental validation, which enables the identification of PET-active true lipases. Two true lipases, Lip(Bv) and Lip(Sh1), hydrolyse PET substrates and homologous enzymes within their respective clusters also retain PET-hydrolysing activity, supporting the existence of specific lipase lineages associated with PET hydrolysis. Structural analyses suggest that differences in loop organization and active-site accessibility may contribute to the observed PET-hydrolysing activity and hydrolysis product profiles. These lipase lineages clustered separately from 1322 putative PETase homologues from the same extremophile genomes, while all groups remained distinct from previously characterized PETases. These findings expand the evolutionary diversity of PET-hydrolysing enzymes in extremophiles.