Lai_2025_Bioresour.Technol_442_133731

Phthalate esters (PAEs), widely used as plasticizers, are persistent environmental and food contaminants associated with endocrine disruption and health risks. Enzymatic degradation offers a promising solution, yet the catalytic mechanisms of the sparsely studied family VIII carboxylesterases remain largely unexplored. Here, a novel family VIII carboxylesterase HylD3 was characterized with efficient PAEs hydrolysis performance. Biochemical analysis revealed optimal activity at pH 9.5 and 45 degreesC but poor thermostability (half-life 0.12 min at 65 degreesC). Structural bioinformatics and mutagenesis uncovered a non-classical catalytic triad (S79-K82-Y185), while molecular docking and simulations showed that substrate affinity was enhanced by an enlarged active pocket and a reshaped binding energy landscape of the enzyme HylD3, which collectively provided the first molecular-level insight into PAEs hydrolysis by this enzyme family. To overcome thermostability limitations, COF-LZU1@HylD3 was prepared via rapid aqueous-phase one-pot immobilization within 10 min. The immobilized system preserved the free enzyme's catalytic properties, exhibited a 21.00-fold improvement in thermostability at 65 degreesC, and maintained 47.06 % activity after five cycles of catalysis, achieving a sustainable hydrolysis efficiency of 8.22 g/(L.d). This study advances the mechanism understanding of family VIII carboxylesterase in PAEs hydrolysis and establishes a thermostable biocatalytic platform for efficient PAEs detoxification food-related and environmental systems.