Cloning and expression of ophB gene encoding organophosphorus hydrolase from endophytic Pseudomonas sp. BF1-3 degrades organophosphorus pesticide chlorpyrifos

• Endophytic Pseudomonas sp. BF1-3 was isolated from balloon flower root.
• Pseudomonas sp. BF1-3 degrades chlorpyrifos.
• ophB gene encoding a chlorpyrifos degrading protein was cloned into E. coli DH5α.
• E. coli DH5α harboring ophB degraded about 97% chlorpyrifos within 9 days.

Chlorpyrifos is an organophosphate pesticide that has adverse effect on animals and plants. We isolated endophytic bacterial strain, Pseudomonas sp. BF1-3, from balloon flower root which can hydrolyze chlorpyrifos. A gene (ophB) encoding a protein involved in chlorpyrifos degradation from this strain was cloned into Escherichia coli DH5α for confirming enzyme activity. After sequencing, total 1024 bp nucleotide sequences were found in the open reading frame of ophB. The chlorpyrifos degradation patterns by E. coli DH5α (ophB) were observed. During incubation in minimal salt (M9) medium supplemented with chlorpyrifos (100 mg L−1), the E. coli DH5α harboring ophB degraded about 97% initial chlorpyrifos (100 mg L−1) and accumulated 86 mg L−1 3,5,6-trichloro-2-pyridinol (TCP) within 9 days. In addition, optical density (OD) of E. coli DH5α (ophB) culture at 600 nm was increased from 0.172 to 1.118 within 2 days of inoculation in the chlorpyrifos supplemented M9 medium. The estimated molecular weight of purified OphB protein was determined to be 31.4 kDa by SDS-PAGE. The OphB enzyme was most active at pH 8 and an optimal temperature around 35 °C. These results indicate that endophytic bacteria are supposed to be useful for biological control of environments contaminated with pesticides.