Engineering a thermostable MHET-active esterase through active-site compaction for efficient dual-enzyme PET...

Efficient enzymatic recycling of polyethylene terephthalate (PET) is frequently hindered by the accumulation of mono-(2-hydroxyethyl) terephthalate (MHET), a soluble intermediate that competitively suppresses PET hydrolase activity. Although MHET-hydrolysing enzymes can overcome this limitation, these enzymes often exhibit poor catalytic performance or thermal compatibility with highly active PETases. In this study, we characterized a previously uncharacterized MHET-active esterase from Saccharothrix carnea and enhanced its catalytic and thermal properties through structure-guided semirational engineering. Sequence and structural analyses place this enzyme in the polyesterase-lipase-cutinase family rather than the specialized tannase-family MHETases. In contrast to the active-site expansion strategy commonly applied to polymer-degrading PET hydrolases, we investigated whether active-site compaction could improve catalytic preorganization for MHET. Mutational analysis revealed that F210I reshaped the substrate entrance region, whereas N213M compacted the inner binding pocket. Combining these mutations increased the catalytic efficiency by 63.2-fold towards MHET compared with the wild-type enzyme. To further improve the thermal stability, an engineered disulfide bond was introduced, generating variant MT10, which maintained a 59.9-fold increase in k_cat/K_m, together with a 9.0 °C increase in melting temperature. Molecular dynamics simulations suggested that pocket remodelling promoted a more stable productive binding mode for MHET and shortened the catalytic distance between Ser131 and the substrate. In coupled reactions with FAST-PETase at 50 °C, MT10 efficiently reduced MHET accumulation and enhanced terephthalic acid production from PET films. Collectively, these findings demonstrate that active-site compaction combined with thermostabilization is an effective strategy for engineering MHET-active esterases and improving enzymatic PET depolymerization cascades.