Mechanistic insight into esterase-catalyzed hydrolysis of phthalate esters (PAEs) based on integrated multi-spectroscopic analyses and docking simulation.

A novel PAE-hydrolyzing esterase (named Hyd) gene was screened from the genomic library of Rhodococcus sp. 2G and was successfully expressed in heterologous E. coli, which was defined as a new family of esterolytic enzymes. The purified Hyd could efficiently degrade various PAEs, displaying high activity and stability with a broad range of pH (4-10) and temperature (20-60 °C). Interaction mechanism of Hyd with dibutyl phthalate (DBP) was investigated by integrated multi-spectroscopic and docking simulation methods. Fluorescence and UV-vis spectra revealed that DBP could quench the fluorescence of Hyd through a static quenching mechanism. The results from synchronous fluorescence and CD spectra confirmed that the DBP binding to Hyd triggered conformational and micro-environmental changes of Hyd, which were characterized by increased stretching extent and random coil, and decreased α-helix and ß-sheet. Molecular docking study showed that DBP could be bound to the cavity of Hyd with hydrogen bonding and hydrophobic interaction. A novel and distinctive catalytic mechanism was proposed: two key residues Thr190 and Ser191 might catalyze the hydrolysis of DBP, instead of the conserved catalytic triad (Ser-His-Asp) reported elsewhere, which was confirmed by site-directed mutagenesis.

Biodegradation pathway of di-(2-ethylhexyl) phthalate by a novel Rhodococcus pyridinivorans XB and its bioaugmentation for remediation of DEHP contaminated soil.

A novel bacterial strain designated as Rhodococcus pyridinivorans XB, capable of utilizing various endocrine disruptor phthalates or phthalic acid (PA) as sole source of carbon and energy, was isolated from activated sludge. Under the optimal culture conditions (pH 7.08, 30.4 °C, inoculum size (OD600 nm) of 0.6) obtained by response surface methodology, di-(2-ethylhexyl) phthalate (DEHP, 200 mg/L) could be degraded by strain XB with a removal rate of 98% within 48 h. Under the observation of an atomic force microscope, it was confirmed that DEHP did not inhibit the growth of strain XB which might produce some extracellular polymeric substances as a response to DEHP stress, resulting in rapid degradation of DEHP. At initial concentrations of 50-800 mg/L DEHP, its degradation curves were well fitted with the first-order kinetic model, and the half-life of DEHP degradation varied from 5.44 to 23.5 h. The degradation intermediates of DEHP were identified by both GC-MS and high performance liquid chromatography-time of flight-mass spectrometry (HPLC-TOF-MS). Significant up-regulation was observed for the relative expression levels of genes (i.e., phthalate hydrolase, PA 3,4-dioxygenase, protocatechuate 3,4-α and 3,4-ß dioxygenase) involved in DEHP degradation determined by real-time quantitative PCR (RT-qPCR). A DEHP biodegradation pathway by strain XB was proposed based on the identified intermediates and the degrading genes. Bioaugmentation of DEHP-contaminated soils with strain XB could efficiently promote DEHP removal, offering great potential in bioremediation of DEHP-contaminated environment.

Functional genomic analysis of phthalate acid ester (PAE) catabolism genes in the versatile PAE-mineralising bacterium Rhodococcus sp. 2G.

Microbial degradation is considered the most promising method for removing phthalate acid esters (PAEs) from polluted environments; however, a comprehensive genomic understanding of the entire PAE catabolic process is still lacking. In this study, the repertoire of PAE catabolism genes in the metabolically versatile bacterium Rhodococcus sp. 2G was examined using genomic, metabolic, and bioinformatic analyses. A total of 4930 coding genes were identified from the 5.6 Mb genome of the 2G strain, including 337 esterase/hydrolase genes and 48 transferase and decarboxylase genes that were involved in hydrolysing PAEs into phthalate acid (PA) and decarboxylating PA into benzoic acid (BA). One gene cluster (xyl) responsible for transforming BA into catechol and two catechol-catabolism gene clusters controlling the ortho (cat) and meta (xyl &mhp) cleavage pathways were also identified. The proposed PAE catabolism pathway and some key degradation genes were validated by intermediate-utilising tests and real-time quantitative polymerase chain reaction. Our results provide novel insight into the mechanisms of PAE biodegradation at the molecular level and useful information on gene resources for future studies.