Distribution and Catalytic Mechanism of a Novel Carboxylesterase Degrading Multiple Aryloxyphenoxypropionate Herbicides in Bacteria and Archaea

Abstract Aryloxyphenoxypropionate (AOPP), a widely used class of herbicides, pose significant risks to ecosystems and human health. In this study, Bacillus amyloliquefaciens L1 was isolated as a novel degrader, completely degrading 97.42 mg/L cyhalofop-butyl within 12 h via ester bond hydrolysis to cyhalofop acid. A new carboxylesterase, PnbA, was identified from strain L1, showing substrate preference for cyhalofop-butyl > clodinafop-propargyl > haloxyfop-p-methyl > quizalofop-p-ethyl. Molecular docking and mutagenesis identified S190 as a key catalytic residue. Two mutants, K411A and T412A, exhibited enhanced relative activities toward both cyhalofop-butyl (124.08 and 153.16%, respectively) and quizalofop-p-ethyl (120.97 and 171.06%, respectively) due to accelerated substrate capture and improved binding affinity. Strain L1 effectively remediated cyhalofop-butyl from contaminated soil and water matrices in natural microbial backgrounds. Homologous protein analysis revealed an overwhelming bacterial origin (99.39%) versus archaea (0.61%). This study provides novel enzymatic resources for AOPP remediation and expands the understanding of its microbial genetic basis.