Xi_2025_Int.J.Biol.Macromol_335_149285

Polyethylene terephthalate (PET) exhibits considerable environmental persistence, largely due to the accumulation of its intermediate degradation product, mono(2-hydroxyethyl) terephthalate (MHET), which hinders the overall hydrolytic efficiency of PETase. In this study, we identified and characterized a novel bifunctional hydrolase, CHeMHETase, capable of hydrolyzing both MHET and bis(2-hydroxyethyl) terephthalate (BHET). Compared to IsMHETase, the spatial distances between H518-S215 and H518-D482 in CHeMHETase are slightly increased, potentially expanding the active-site pocket to better accommodate the bulkier BHET substrate. Structural and electrostatic surface comparisons with IsPETase reveal that CHeMHETase retains a highly conserved catalytic triad, oxyanion hole, and disulfide bond network. The alpha-helical lid domain, critical for catalytic function, is also preserved. Additionally, CHeMHETase displays a more acidic and heterogeneous surface charge distribution, which may facilitate the binding of small polar substrates. Interaction analysis further indicates that the strength of enzyme-substrate interactions is a key determinant of catalytic efficiency. To improve PET depolymerization, a one-pot degradation system integrating leaf-branch compost cutinase (LCC) with CHeMHETase was developed. This integrated approach achieved a PET degradation rate of 68.4 % without intermediate product separation, representing an 18 % improvement over LCC alone.