Abstact

Enzymatic recycling strategies employing hydrolases for the depolymerization of polyethylene terephthalate (PET) have advanced considerably in recent years. Most highly efficient PET hydrolases exhibit optimal catalytic activity under alkaline conditions, limiting their applicability under neutral or acidic environments. Here, we show that cluster 3, previously characterized within the polyesterase-lipase-cutinase family, displays optimal catalytic activity under near-neutral pH conditions, with a specific lineage represented by N1484 exhibiting maximal activity at pH 5-6. Investigation of the underlying mechanism revealed that residue replacements near the catalytic triad had no effect on pH profiles, whereas substantial differences were observed between the pH dependence of PET hydrolysis and that of the model substrate p-nitrophenyl butyrate. Altering residues around the substrate access region, particularly the negatively charged E121, shifted the pH profile and enhanced activity at alkaline pH, with analogous substitutions in homologous enzymes producing comparable effects. Variants carrying substitutions with different side-chain properties demonstrated that surface electrostatics govern optimal pH of PET hydrolases, suggesting that pH-dependent enzyme-substrate accessibility modulates apparent activity. Together, these findings provide mechanistic insights into the pH dependency of PET depolymerases and inform future enzyme engineering for industrial recycling applications.