Engineering and Application of a Thermostable MHETase for PET Depolymerization

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 degrees C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (Tm,app) of 94.6 degrees C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ~100-fold slower unfolding rate at 65 degrees 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 (Tm,app = 96.1 degrees 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, MHT077LFK, 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.