Elucidation of the substrate specificity of Type III phthalate esters esterases from Acinetobacter sp. DY-1

Phthalate esters (PAEs) are pervasive environmental pollutants that require efficient biodegradation. The substrate specificity of PAEs-degrading microorganisms hinges critically on the catalytic selectivity of microbial esterases. To elucidate the molecular basis of the specificity of Type III PAEs esterases, we developed a chromogenic assay and further integrated machine learning-driven enzyme mining with functional characterization and molecular dynamics analysis. A novel microorganism, Acinetobacter sp. DY-1, was isolated with diverse PAEs-degrading capacities. The esterase 1952 from strain DY-1 exhibited broad substrate specificity and high catalytic efficiency, with a marked preference for long-chain PAEs, such as DEHP. Molecular dynamics simulations revealed stable hydrophobic interactions underpinning 1952s affinity for DEHP. Through targeted mutagenesis, we identified key residues (Leu64, Leu68, and His229) that are essential for substrate binding and catalysis. Their substitution with polar residues drastically reduced DEHP degradation, while the G178Y mutant enhanced activity via π-stacking interactions. This study deciphers the structural determinants of PAE esterase specificity, enabling precise enzyme engineering.