Electrostatic-guided substrate flip enables complete di-ester bond hydrolysis of phthalate esters by a bacterial...

Phthalate esters (PAEs), as major endocrine disruptors, pose severe risks to human health and environment due to their toxicity. Enzymatic hydrolysis offers a promising detoxification route, but the incomplete enzymatic cleavage of di-ester bonds remains a bottleneck for PAEs hydrolysis. This is largely attributed to a limited mechanistic understanding of how type III hydrolases achieve di-ester bond hydrolysis of PAEs. Integrated molecular docking, molecular dynamics simulations, and site-directed mutagenesis revealed a novel electrostatic guidance mechanism of a representative type III hydrolase PAEIIIase in this work. Specifically, residues E188 and H399 mediated the spatial flip of the mono-ester intermediate via electrostatic interactions, while Y118 stabilized substrates through hydrogen bond and π-π stacking to facilitate sequential ester-bond cleavage by PAEIIIase. Guided by the catalytic mechanism, the homologous type I hydrolase CarB152 was successfully converted into a type III hydrolase CarB152-F118Y capable of complete di-ester bond hydrolysis through targeted engineering. Furthermore, homology screening identified a superior enzyme Est909, which exhibited 116.8% higher hydrolytic activity toward di(2-ethylhexyl) phthalate compared to PAEIIIase. To enhance practical applicability, a robust sodium alginate-based whole-cell immobilization system was constructed. The system demonstrated robust performance, maintaining >90% substrate degradation across 10 consecutive cycles, coupled with a hydrolysis efficiency exceeding 4.0 g/(L·d). Collectively, this study significantly advanced the enzymatic mechanistic understanding of complete di-ester bond cleavage, provided a promising engineering strategy for functionally upgrading closely related homologs, expanded the enzymatic toolbox for PAEs degradation, and established a technically feasible biocatalytic platform with potential applicability in sustainable PAEs remediation.