Cloning of an organophosphorus hydrolase (opdD) gene of Lactobacillus sakei WCP904 isolated from chlorpyrifos-impregnated kimchi and hydrolysis activities of its gene product for organophosphorus pesticides

Abstract

Chlorpyrifos (CP) residues are absorbed from soil and often found in Korean cabbages that are being used to make kimchi. Lactobacillus sakei WCP904, harboring the organophosphorus (OP) hydrolase gene opdD, was isolated from CP-impregnated mulkimchi. The cloned gene opdD from strain CP904 comprises 825 base-pair nucleotides that encode 274 amino acids. The recombinant Escherichia coli harboring the opdD gene depleted 73% of CP after 6 days in M9 medium. In fact, the OpdD protein is a novel member of the GHSQG family of esterolytic enzymes or lactic acid bacterial Opd groups. The molecular weight of the OpdD protein was estimated to be 31 kDa using SDS-PAGE. Broad-spectrum activities of the OpdD protein were obtained against OP insecticides containing both P-O and P-S bonds. The OpdD protein exhibits maximum activity at 30°C with pH 6. No enzyme activities of the mutated OpdD (Ser116 ? Ala116) protein toward q-nitrophenyl butyrate and CP substrates were observed. These results suggested that the strain WCP904 scavenges insecticide residues from mulkimchi vegetables, thus abolishing health hazards by secreting OP hydrolase during fermentation.

Cloning, expression, and functional characterization of an organophosphates insecticides degrading gene (opdC) from a potential probiotic Lactobacillus plantarum WCP931

“…The OP insecticides degrading strain WCP931 was isolated from mulkimchi sample and identified as previously described by Haque et al (2018). The acid and gastric acid tolerance activity of the strain L. plantarum WCP931 was further confirmed previously by Cho et al (2008).…”

“…After that 10 μL of the filtrate that was mixed with methanol and passed through a 0.45-µm PVDF filter (GmbH, Dassel, Germany) and was injected into a C18 column (250 × 4.6 mm, 5 μm, Phenomenex, CA, USA) of HPLC. The concentration of the degraded insecticides residues were determined according to the peak areas in the chromatograms against standard pesticides as described Cho et al (2009a), Islam et al (2010), and Haque et al (2018). The OP insecticides degradation and its estimation were repeated in three times to obtain the standard error of the experiments.…”

“…The recombinant plasmid was digested with BamHI and HindIII and cloned into pBluscript II SK+ digested with the same restriction enzyme. The nucleotide and amino acid sequences were determined and analyzed as described by Islam et al (2010) and Haque et al (2018). The phylogenetic tree and alignment of conserved regions of the OpdC protein with other Opd enzymes and esterolytic enzymes amino acid sequences was analyzed using DNAMAN10.0 software package (Haque et al 2018).…”

“…The DpnI-treated plasmids were then transformed into E. coli DH5α according to the manufacturer's specifications. The site-directed mutagenesis procedure was adapted from as described Haque et al (2018). The predictive active site of the OpdC protein was investigated using sequential application of several bioninformatic tools.…”

“…However, its residues persist in the environment for unpredictable period of time (Tariq et al 2007) and may enter vegetables cultivated on the polluted sites and pose a great threat to the human health (Huete-Soto et al 2017). The environmental fate of CP has been extensively studied, and its half-life in soil varies from 10 to 120 days (Haque et al 2018;Getzin 1981) resulting in 3,5,6-trichloro-2-pyridinol (TCP) as the major degradation product. Hwang and Moon (2018) reported that the levels of CP residue in Korean Cabbages were ranged between 0.12~0.75 mg/kg after 32-35 days of CP (0.24~0.72 g a.i./m 2 ) treatment in different cabbage fields.…”