ACS Sustain Chem Eng. 2026 Jun 8;14(24):10981-10995. doi: 10.1021/acssuschemeng.6c01404. eCollection 2026 Jun 22.

ABSTRACT

Enzymatic hydrolysis of poly-(ethylene terephthalate) (PET) releases mono-(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T _m,app) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043-5 was identified with a comparable thermostability (T _m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077^LFK, which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

PMID:42358385 | PMC:PMC13292399 | DOI:10.1021/acssuschemeng.6c01404