Elsevier

Process Biochemistry

Volume 94, July 2020, Pages 340-348
Process Biochemistry

Biodegradation of organophosphorus insecticides by two organophosphorus hydrolase genes (opdA and opdE) from isolated Leuconostoc mesenteroides WCP307 of kimchi origin

https://doi.org/10.1016/j.procbio.2020.04.026Get rights and content

Highlights

  • The opdA (930 bp, 309 aa) and opdE (894 bp, 297 aa) genes were cloned from Leconostoc mesenteriodes WCP907.

  • Esc. coli harboring opdA and opdE genes depleted 72% and 83% chlorpyrifos in M9 medium after 6 days.

  • The OpdA and OpdE represented MW 35 and 33 kDa, optimum temperature 30 and 30 °C, optimum pH 6 and 7, respectively.

  • The Ser128 of OpdA and Ser129 of OpdE are the essential amino acids for catalysis.

  • The opdA and opdE are the first reports of opd gene from Leuconostoc genus.

Abstract

This study is aimed to reveal the molecular incidence of organophosphorus insecticides degradation during the fermentation of Korean food yeulmu-mulkimchi. To this end, two opdA and opdE which consist of 930 and 894 bp that encode 309 and 297 amino acids, respectively, were cloned from the Leuconostoc mesenteroides WCP307 strain that was isolated from chlorpyrifos (CP) impregnated kimchi. The Escherichia coli that harbored the opdA and opdE genes depleted a CP concentration of 72% and 83%, respectively, in an M9 medium after 6 days. The OpdA and OpdE enzymes molecular weights were estimated to be approximately 35 and 33 kDa and showed optimal activities at 30 °C with a pH of 7.0 and 6.0, respectively. However, the mutated OpdA (Ser128 Ala128) and OpdE (Ser129 Ala129) enzymes had no activities on OP insecticides and ρ-nitrophenyl butyrate substrates. In addition, the OpdA and OpdE enzymes showed profound catalytic activities against cadusafos, comnaphos, diazinon, dyfonate, ethoprophos, fenamiphos, methylparathion, and parathion insecticides. Therefore, it is assumed that OpdA and OpdE enzymes detoxified the pesticides contaminated kimchi composition like Chinese cabbages during fermentation. Furthermore, the OpdA and OpdE enzymes augmented the diversity of new LAB-opd enzymes group in nature.

Graphical abstract

We previously reported that lactic acid bacteria degraded organophosphate insecticides (OP) during yeulmu-mulkimchi fermentation. In this study, we aimed to isolate OP insecticide-degrading gene from Leu. mesenteroides WCP907. We cloned two organophosphorus hydrolase genes from Leu. mesenteroides WCP907 using a PCR-based technique and expressed them in E. coli.

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Introduction

The application of pesticides in agriculture was originally intended to enhance the production of food for mitigating the requirement of the growing worldwide population [1,2]. However, the concern over the dependence on pesticides has risen due to their persistence and hazardous nature [3,4]. Because of the extremely long persistence and toxicity of organochlorine insecticides, biodegradable and environmentally friendly insecticides have been sought to replace them. For instance, organophosphate pesticides constitute a less dangerous alternative [5,6]. In particular, chlorpyrifos (CP) is one of the most extensively applied organophosphorus (OP) insecticides. However, the residues of CP persist in the environment for an unpredictable period of time and might enter the vegetables cultivated on polluted sites. Hence, this practice poses a threat to human health [7,8]. Recently, Hwang and Moon [9] reported that the residual CP ratios in Korean cabbage were 0.93–6.01 and 0.57–2.61%, respectively. In a related study, the highest concentration of CP was found 0.13 and 0.03 mg/kg in Eggplant and chill pepper, respectively. Moreover, the other organophosphorus pesticide residues like malathion, methamidophos, chlorphenviphos as well as chlorpyrifos, diazinon, and cypermethrin, etc., were detected in Ghanaian chill pepper [10] and cocoa beans [11], respectively.

Meanwhile, several types of food processing methods like physical, biological, thermal, ozone, washing, and membrane are being studied for removing the pesticide residues. In fact, pesticide residues during fermentation may interference the growth of microorganisms [12]. Kimchi is a traditional Korean fermented food made from different vegetables that is used as a side dish with cooked rice and other dishes. The characteristics of kimchi depend on the action of lactic acid bacteria (LAB) during fermentation. Previously, we reported that LAB degraded OP insecticides during the yeulmu-mulkimchi fermentation. Indeed, we identified and demonstrated that four LABs, namely, Leuconostoc mesenteroides WCP307, Lactobacillus brevis WCP902, Lactobacillus plantarum WCP931, and Lactobacillus sakei WCP904, can utilize OPs as a source of carbon and phosphorus in a defined medium. In particular, Leu. mesenteroides WCP307 and Lac. brevis WCP902 were present at higher levels in the yeulmu-mulkimchi fermentation with CP in the immature and optimum ripening stages, respectively [13]. Recently, the genus Lactobacillus was reclassified into the genera Atopobium, Carnobacterium, Weissella, Oenococcus, and Leuconostoc. Leu. mesenteroides is the only predominant species in the early stage of kimchi fermentation [[14], [15], [16]] and is able to degrade OP insecticides, no study revealed the molecular metabolism of OP insecticides degradation of Leu. mesenteroides to date.

Yet several different types of organophosphorus hydrolase (Oph) are described. In particular, opd, opdA, opdB, ophc2, mpd, OPAA, hocA, NC OPH, adpB NC ADPase Oph are reported from representative bacteria Sphingobium/Pseudomonas, Agrobacterium, Lactobacillus, Pseudomonas/Stentrophomonas, Plesiomonas/Pseudomonas/Stentrophomonas, Alteromonas, Nocardia/Nocardioides/Pseudomonas, respectively [[17], [18], [19]]. Among them the opd gene have been much more reported than the others organophosphorus hydrolase genes that were isolated from chromosome based and plasmid DNA of different bacterial species [[20], [21], [22], [23], [24]].

This study aimed at isolating the OP insecticide-degrading gene from Leu. mesenteroides WCP307. Therefore, two organophosphorus hydrolase genes were cloned from Leu. mesenteroides WCP307 using a PCR-based technique and expressed them in Escherichia coli. The biochemical and molecular characteristics of these new LAB-opd enzymes were revealed, which constitutes the first report of the cloning of opd genes from Leu. mesenteroides WCP907.

Section snippets

Materials and reagents

Esc. coli DH5α, BL21 (DE3) and recombinant Esc. coli cells were cultured at 37 °C in a Luria-Bertani (LB) medium or LB medium and M9 medium supplemented with the appropriate antibiotics. The M9 medium and all of the antibiotics were purchased from Sigma-Aldrich, Inc. (Merck KGaA, Darmstadt, Germany) and used at the following concentrations: 50 μg/mL of ampicillin. The LB and lactobacilli MRS (MRS) media were purchased from Difco Co. (Becton Dickinson Co., Sparks, MD, USA). The pGEM-T easy

Degradation of Chlorpyrifos by the WCP307 strain in liquid culture

The lactic acid bacteria (LAB) strain WCP307 was isolated from high concentrations of insecticides mixed yeulmu-mulkimchi preparation as previously described [13]. The 16S rRNA gene sequence of the WCP307 strain was closely related with the LAB including Lactobacillus, Leuconostoc, and Weissella species. The highest level of 16S rRNA of the WCP307 strain showed 99.9% similarity with Leuconostoc mesenteroides DSM 20343T (Supplementary data Fig. S1).

The growth performance of WCP307 strain in CP

Conclusions

This study is the first to describe opd genes from Leu. mesenteroides WCP307, which increases the host diversity of OP hydrolases. Particularly, the recombinant Esc. coli harboring the opdA and opdE genes degraded approximately 72% and 83% of the initial CP concentration within 6 day, respectively. The opdA and opdE gene products showed strong degradation ability against dimethyl compounds, such as MPT, and diethyl compounds, such as CP, CM, DZ, and PT. The OpdA and OpdE enzymes do not belong

Authors statement

KMC and JHL conceived and designed the experiments. KMC, MAH, and JHL interpreted the data and wrote the manuscript. CEH, SCK, DYC, and HYL performed the experiments and analyzed the data. All authors read and approved the final manuscript.

Declaration of Competing Interest

None.

Acknowledgments

This research was supported by the Basic Science Research Program (grant number 2016R1D1A1B01009898) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education and the Research Project for Finding Materials of Forest Resources (grant number 2017023A00-1720-BA01) through the Korea Forestry Promotion Institute (KOFPI) funded by the Korea Forest Service, Republic of Korea.

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