Biodegradation of phthalic acid esters (PAEs) by Bacillus sp. LUNF1 and characterization of a novel hydrolase capable of catalyzing PAEs

Abstract

Phthalic acid esters (PAEs), the universal plasticizers, have widely entered the environment and cause serious contamination, potentially threatening human health. Furthermore, PAEs are highly resistant to degradation due to their hydrophobicity. In the present study, a PAE degrader, Bacillus sp. LUNF1, was isolated from sewage sludge. Strain LUNF1 completely removed dibutyl phthalate (DBP) from wastewater, demonstrating its great potential for PAE bioremediation. The genetic mechanism underlying PAE degradation by strain LUNF1 was elucidated by identification of the novel PAE hydrolase DphBL1 belonging to family VII. The hydrolase was overexpressed in E. coli BL21(DE3) and purified by affinity chromatography. It displayed high hydrolytic efficiency toward DBP at 10–50 °C and pH 5.0–9.0. Importantly, DphBL1 maintained its stability in the presence of β-mercaptoethanol (β-ME), phenylmethylsulphonyl fluoride (PMSF) and various metal ions, which did not significantly inhibit DphBL1. It was also found that the catalytic triad (Ser190-His395-Glu306), oxygen hole (Gly107-Ala108) and other residues (Phe310, Ile358, Phe359, Leu309 and Leu269) were involved in interacting DBP. DphBL1 mutants with significantly reduced activity confirmed the essential role of these active residues and revealed the catalytic mechanism of DphBL1. These results will support structural modification of PAE hydrolases to improve catalytic efficiency in bioremediation, and promote the development of efficient strategies for PAE remediation in the environment.