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Cloning and Expression of Ophb Gene Encoding Organophosphorus Hydrolase from Endophytic Pseudomonas Sp. Bf1-3 Degrades Organophosphorus Pesticide Chlorpyrifos

Shah Md. Asraful Islam
Department of Plant Pathology, Patuakhali Science and Technology University

Abstract/ Executive Summary:

Chlorpyrifos is an organophosphate pesticide that has adverse effect on animals and plants. We isolated endophytic bacterial strain, Pseudomonas sp. BF1-3, from balloon power 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.

Keywords: Chlorpyrifos, Biodegradation, Endophytic bacteria, Organophosphorus hydrolase, Pseudomonas sp. BF1-3

Location: Department of Environmental Biotechnology, Gyeongsang National University, Jinju 660-701, South Korea

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Micro organism

Objectives:

To clone an ophB gene that encodes organophosphorus hydrolase enzyme (OphB) from balloon power root living Pseudomonas sp. BF1-3. This enzyme was capable of degrading organophosphate pesticide, chlorpyrifos.

To express ophB gene in Escherichia coli DH5α.

To observe chlorpyrifos degradation pattern by E. coli DH5α harboring ophB for evaluating their potential to degrade chlorpyrifos and to purify and characterize OphB enzyme.

Methodology:

Bacterial strains and growth conditions The endophytic bacterial strain was isolated from balloon power root. E. coli DH5α, E. coli BL21 (DE3) and recombinant E. coli cells were cultured at 37°C in Luria-Bertani (LB) and M9 medium supplemented with the appropriate antibiotics (ampicillin 50 μg mL^-1 and kanamycin 50 μg mL^-1). M9 medium and all antibiotics were purchased from Sigma Chemical Co. Extraction of genomic DNA from isolated strain The isolated endophytic bacteria were cultured in test tube containing 3 ml TSA medium by shaking in a rotary shaker at 180 rpm for 12 h and the cultures were centrifuged at 13,000 rpm for 5 min at 4°C. The pellet was subjected to genomic DNA extraction using the G-spinTM Genomic DNA Extraction Kit. The extracted DNA was used as a template for PCR to amplify 16S rDNA. PCR ampli-cation and sequencing of 16S rDNA Ampli-cation of 16S rDNA fragments of extracted endophytic bacterial genomic DNA were conducted by PCR ampli-cation. Bacterial 16S rDNA was amplied by PCR using the universal primers based on accession number AL009126 of Bacillus subtilis 168, 877F (5-CGG AGA GTT TGA TCC TGG-3) and 878R (5-GGC TAC CTT GTT ACG ACT T-3 were used with Super-Therm DNA polymerase. After amplifying 16S rDNA was cloned into pGEM-T easy vector. Recombinant plasmid was extracted. Based on 16S rDNA sequence we have identifed the isolated strain as Pseudomonas sp. BF1-3. The accession number of this 16S rDNA is available in nucleotide sequence database under the accession number KJ849233. Cloning and sequencing of ophB gene PCR ampli?cation of ophB from the genomic DNA of Pseudomonas sp. BF1-3 was conducted. The sense primer 2350F, 5-CGT CGT CGG CTG GGC AGG GT-3 and anti- sense primer 2351R, 5-GCG TGC GGC CTA CCT CGT TG-3 were used for the template DNA of Pseudomonas sp. BF1-3. PCR was conducted with Super-Therm DNA polymerase, 1.5 mM MgCl₂, 2 mM dNTP in a -nal volume of 50 μl for 30 cycles (denaturation at 95°C for 30 s, annealing at 50 1C for 30 s and extension at 72°C for 1 min and 30 s) followed by -nal incubation at 72°C for 10 min. The anticipated PCR product of 1270 bp was isolated after agarose gel electrophoresis using a gel extraction kit. Ampli?ed fragments were cloned into the pGEM-T Easy vector and transformed into E. coli DH5α. Nucleotide sequence data of ophB is available in the GenBank database under the accession number HM191722. Bacterial growth and degradation of chlorpyrifos Recombinant E. coli DH5α harboring ophB was cultured in 3 mL LB medium. Finally, 100 mL culture suspensions (8.0 cfu/mL) of E. coli DH5α harboring ophB was inoculated into 50 mL of M9 medium, where chlorpyrifos (100 mg L^-1) was supplemented as only source of carbon instead of glucose. E. coli DH5α was also cultured as control in similar M9 medium. At periodic intervals, three individual asks were sacrificed and their contents were used to determine bacterial growth and degradation of chlorpyrifos separately for ensuring accuracy. Growth was measured as an increase in absorbance at 600 nm using spectrophotometer. Inoculated culture of E. coli DH5α with chlorpyrifos (100 mg L^-1) was used as the negative control. Residual concentrations of chlorpyrifos were analyzed by HPLC. Expression and puri?cation of the enzyme (OphB) For high expression of enzyme, the PCR products generated with primers viz., 2478F 5-TAAT GGA TCC GCC GCA CCG CAG GT-3 (sense, BamHI sites are indicated by underline) and 2479R 5-ATTA AAG CTT CTT GGG GTT GAC GAC-3 (antisense, HindIII sites are indicated by underline) for ophB from Pseudomonas sp. BF1-3 was cloned into expression vector pET-28a, resulting in the addition of a C-terminal (His)₆ tag. E. coli BL21 (DE3) carrying pET-28a/ophB were grown at 37°C to mid-log phase in LB medium containing 50 μg mL^-1 kanamycin. Expression was then induced by adding IPTG to a ?nal concentration of 0.5 mM, and further growth was continued for 5 h. The cells were harvested by centrifugation (6000g, 10 min) and washed twice with 10 mM Tris–HCl buffer (pH 7.0). The cells were resuspended in the same buffer and stored at 20°C. The frozen cells were mixed with 50 mM Tris–HCl buffer (pH 7.5) containing 1 mg of bovine DNase I and incubated at 37°C for 30 min. Triton X-100 was added to the suspension to attain a nal concentration of 2.5%. The supernatant was collected and stored at 4°C. The solubilized recombinant OphB with His-tag was applied on a HisTrap kit. Purifcation of expressed His₆-tagged protein was carried out. The purified protein sample was analyzed by SDS-PAGE. The protein concentration was determined. Characterization of the enzyme (OphB) Chlorpyrifos is an ester like compound named as O,O-diethyl O-(3,5,6- trichloro-2-pyridinyl) ester. We did not use directly chlorpyrifos as substrate for OphB for esterase activity. Instead of chlorpyrifos we used another ester, ρ-nitrophenol-β-butyric acid (ρ-NPB) as the substrate. This substrate was reliable for colorimetric determination of the OphB enzyme activity. Therefore, this activity was determined by a spectrophotometeric method using ρ-nitrophenol-β-butyric acid (ρ-NPB). The hydrolysis rate of ρ-NPB at 37°C was measured in 50 mM sodium phosphate buffer (pH 7) at 420 nm. One unit of esterase was defined as amount of enzyme required to release 1 μmol of ρ-NP per minute under the assay conditions. The effects of pH and temperature on the esterase activity were examined with the puri?ed recombinant enzyme. To determine the effect of temperature on the enzymatic activity, samples were incubated at temperatures ranging from 15 to 65°C for 1 h. The effects of pH on the esterase activity were determined for pH 5–11 at 37°C using puri?ed enzyme. All assays were performed in triplicate. High performance liquid chromatography High performance liquid chromatography (HPLC) was performed in order to determine the chlorpyrifos degradation by the recombinant E. coli DH5α harboring ophB. Sample for HPLC operation was prepared. At periodic intervals, three individual asks were sacrificed and their contents were used to determine the degradation of chlorpyrifos separately. Five mL of the culture (containing chlorpyrifos 100 mg/L) was collected and centrifuged. One mL of supernatant was mixed with 1 mL of methanol for preparation of HPLC sample. Above mixed solution was filtered through a 0.45 mm Minipore PVDF filter for HPLC analysis. Injection volume was the 20 μl of filtered sample. The analysis of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) was carried out on HPLC using C18 column. The mixture of 0.5% acetic acid and methanol (1:4 v/v) was eluted with a low rate of 1 mL/min at 30°C. Target compounds, CP and TCP, were measured on 214 nm of UV detector. In HPLC analysis, CP was detected at approximately 9.30 min TCP was detected approximately at 5.5 min. The calibration curves for CP and TCP were made from the serial dilutions of the samples dissolved in 100% methanol. Above serial standard solutions were filtered through a 0.45 mm Mini-pore PVDF ?lter for HPLC analysis. Injection volume was the 20 μl of serial standard solutions.

Source of Information: Ecotoxicology and Environmental Safety, Year:2014, Volume: 108, Page: 135–141

Article Link (Online Journal): http://dx.doi.org/10.1016/j.ecoenv.2014.06.023

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Endophytic Pseudomonas sp. BF1-3 is supposed to be a nice candidate for bioremediation of chlorpyrifos contaminated environment. However, field studies are needed for the potential application of Pseudomonas sp. BF1-3. Moreover, other pesticide degrading endophytic bacteria to explore the pesticide degrading gene, genetic engineering of those genes for increasing their pesticide degrading capability which will provide a endophyte leading pesticide contamination free environment.

Knowledge Management: Journal