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Biodegradable properties of organophosphorus insecticides by the potential probiotic Lactobacillus plantarum WCP931 with a degrading gene (opdC)

Biodegradable properties of organophosphorus insecticides by the potential probiotic... An organophosphorus (OP) insecticide-mineralizing strain, Lactobacillus plantarum WCP931, harboring a new OP hydrolase (opdC) gene, was isolated during kimchi (Korean traditional food) fermentation. Strain WCP931 exhibited a significant survival rate of 51 to 96% under artificial gastric acid conditions at pH 2 to 3 after 3 h. The opdC gene, consisting of 831 bp encoding 276 amino acids, was cloned from strain WCP907. Recombinant Escherischia coli har- boring the opdC gene depleted 77% chlorpyrifos (CP) in M9 minimal medium after 6 days of incubation. The OpdC enzyme represents a novel member of the GHSQG family of esterolytic enzymes or a new Opd group. The OpdC molecular mass was estimated to be approximately 31 kDa by SDS-PAGE and showed maximum activity at pH 6 and 35 °C. The mutated OpdC (Ser116 → Ala116) enzyme had no activity towards OP insecticides and ρ-nitrophenol-β- butyrate. Importantly, the relative activity of OpdC protein against chlorpyrifos, coumafos, diazinon, fenamifos, methyl parathion, and parathion was higher than that against cadosafos, dyfonate, and ethoprofos insecticides. These results suggested the involvement of OpdC in the biodegradation of OP insecticide-contaminated cabbage during fermen- tation. The new OpdC enzyme expands the heterogeneity of the lactic acid bacterial Opd enzyme group in nature. Keywords: Organophosphorus insecticides, Kimchi, Lactobacillus plantarum WCP931, OpdC gene, Biodegradation Introduction various pesticides, including organophosphates, are com- Pesticide-contaminated vegetables are more frequently monly used in agriculture for crop cultivation and pro- ingested by humans in developing countries. Vegetables tection worldwide. Organophosphate insecticides are are the most common foodstuff, and people consume preferred over organochlorine insecticides due to their 150–250 g of vegetables daily in Asian countries. In fact, extensive efficacy and longer persistence in the environ - ment and on crops. As a consequence, organochlorine has been replaced with organophosphate insecticides in *Correspondence: helalbmb2016@hstu.ac.bd; kmcho@gntech.ac.kr recent decades. Some preliminary work carried out sev- Jin Hwan Lee and Hee Yul Lee made equal contributions to this work eral years ago reported that extensively applied organo- (co-first author) Department of Food Science, Gyeongnam National University phosphorus (OP) insecticide residues not only persist of Science and Technology, Jinju 52725, Republic of Korea in the environment but also enter vegetables cultivated Department of Biochemistry and Molecular Biology, Hajee Mohammad on polluted sites, consequently posing a great threat to Danesh Science & Technology University, Dinajpur 5200, Bangladesh Full list of author information is available at the end of the article human health [1]. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Appl Biol Chem (2021) 64:62 Page 2 of 12 In a major advance in 2018, Hwang and Moon [2] [12–14]. In fact, the existence of opd genes is widely surveyed the levels of chlorpyrifos (CP) in Korean cab- distributed; as a consequence, opd genes have been bage crops and detected 0.12–0.75  mg/kg of CP after increasingly described [15–19]. Interestingly, a new 32–35  days of treatment. This research outcome led group of organophosphorus hydrolase (opd) genes was to anxiety among Korean citizens about consuming isolated from lactic acid bacteria that drive fermenta- fermented food kimchi made from Chinese cabbage. tion in Korean kimchi [18–20]. The OP hydrolase genes Therefore, considerable attention must be paid to using opdA and opdE, opdD, and opdB were isolated from Leu. organophosphate pesticides when cultivating Chinese mesenteriodes WCP907, L. sakei WCP904, and L. brevis cabbage plants, and pesticide residue avails in fermented WCP902 strains, respectively, of kimchi origin. kimchi should also be monitored. Interestingly, a neg- This study will unravel a new organophosphorus hydro - ligible concentration of CP residues in CP-impregnated lase gene named opdC from Lactobacillus plantarum fermented kimchi was estimated due to the catalytic WCP931 of kimchi origin. The essential amino acid in the strengths of fermentation driving bacteria such as L. catalytic site that played a vital role in the biodegradation mesenteroides WCP907, L. sakei WCP904, L. brevis of organophosphate insecticides was predicted by the WCP902, and L. plantarum WCP931 [3]. The most strik - site-directed mutagenesis and bioinformatics analysis. ing result emerged that the LAB strains could use OP The biochemical and genetic properties of the opd gene insecticides as a source of carbon and phosphorus in a also deviated from those of the opdA, opdE, opdD, and defined medium and decontaminate vegetable insecti - opdB genes [18–20]. The new opdC gene has boosted the cides used in mulkimchi fermentation [3]. diversity of opd gene in nature. The fermented mulkimchi character is developed by the influence of lactic acid bacteria in fermentation. The Materials and methods acidity of kimchi varies markedly at the initial, immature, Materials, chemicals and instruments optimum-ripening, overripening, and rancid stages [4] of Analytical grade OP insecticides, including cadusafos fermentation. Several studies [4–6] have been performed (CS), chlorpyrifos (CP), coumaphos (CM), diazinon (DZ), on the dynamics of LAB in kimchi fermentation and con- dyfonate (DF), ethoprophos (EP), fenamiphos (FA), meth- cluded that Lactobacillus sp. were dominated by Leucon- ylparathion (MPT), parathion (PT), and their residues, ostoc sp. A recent review of the literature on this topic such as 3, 5, 6-trichloro-2-pyridinol (TCP), and diethyl- found that the cell growth of L. mesenteroides reached thiophosphate (DEPT), were purchased from ChemSer- highest at the ripening period of kimchi and later reduced vice (West Chester, PA, USA) and Sigma-Aldrich Inc. as the pH of kimchi decreased, whereas that of acid-tol- (St. Louis, MO, USA). The esterase enzyme assay sub - erant L. plantarum continuously augmented until the strates tributyrin, ρ-nitrophenol-β-butyrate (ρ-NPB), and completion of fermentation [4]. Moreover, Cho et  al. [3] ρ-nitrophenol (ρ-NP) were also purchased from Sigma- concluded that L. plantarum plays a vital role of organo- Aldrich Inc. (St. Louis, MO, USA). The ultrapure deion - phosphorus insecticides degradation in kimchi fermen- ized water, acetic acid, methanol, and hydrochloric acid tation. It has also been reported that the phosphatase used were of analytical grade. Microbial growth and of L. plantarum might degrade OP insecticides during enzyme assays were performed using ultraviolet (UV)- skimmed milk fermentation [7]. Like all probiotics, L. visible absorption spectra on a Shimadzu Scientific Korea plantarum is gastric acid-tolerant and bile salt-tolerant, Corp. spectrometer (UV-1800 240  V, Seoul, Korea). which provides it to survive in the harsh environment The analysis of OP insecticides was performed by using of gastrointestinal tract [8]. In addition, it inhibits the a high-performance liquid chromatography (HPLC) growth of harmful pathogens and preserves critical nutri- (PerkinElmer Inc., Norwalk, CT, USA) including a Perki- ents, vitamins, and antioxidants [9, 10]. Moreover, L. nElmer UV detector, qutermary pump, autosampler, and plantarum provides beneficial immunomodulatory func - Phenomenx C18-RP column (250 × 4.6  mm, 5  µm, Phe- tion by increasing the synthesis of interleukin-10 and nomenx Inc., Torrance, CA, USA). secretion of macrophage and T-cell in the affected colon [11]. Therefore, L. plantarum-enriched kimchi ingestion Experimental bacterial strains, plasmids, and culture media has health-beneficial probiotic activities. For opdC gene cloning, subcloning, and high expression, However, there is still uncertainty concerning the the competent Escherichia coli DH5α strain and BL21 degradation of OP insecticides by organophosphorus (DE3) cells were purchased from Novagen (Washigton, hydrolases of L. plantarum strains. Multiple OP hydro- DC, USA). The culture medium such as MRS (de Man, lase genes, such as opd, opdA, opdB, mpd, ophc2, OPAA, Rogosa & Sharpe), LB (Luria–Bertani), and M9 minimal hocA, and adpB NC ADPase Oph, have been isolated medium (standard) were procured from Becton Difico from a wide range of bacteria in the last few decades Co. (Sparks, MD, USA). The cloning vector pGEM-T, L ee et al. Appl Biol Chem (2021) 64:62 Page 3 of 12 which was utilized for cloning and sequencing the opdC for insecticide quantification was adopted from our pre - gene, was collected from Promega Co. (Madison, WI, viously described methods [3, 18–20]. In an attempt, a (+) (+) USA). The vectors pBluescript II SK and pET32a 50 μL recombinant OpdC enzyme solution was mixed to used for the overexpression and purification of the gene a 700 μL phosphate buffer (200  mM, pH 6.5) and added product were procured from Stratagene (La Jolla, CA, separately with 250 μL OP insecticides (200 mg/L). After USA) and Novagen. Genomic DNA and plasmid isola- that, it was kept at 30 °C for 12 h in an incubation cham- tion were conducted using commercial DNA extraction ber. Next, the solution filtrate (10 μL) was separated and kits (iNtRON Biotechnology, South Korea). The restric - added with methanol (1:1). Then, it was went across 0.45- tion enzymes BamHI and HindIII were purchased from µm PVDF filter and injected into HPLC column (C18, Promega Co. (USA), respectively. 250 × 4.6 mm, 5 μm, Phenomenex, CA, USA). The 10 μL filtered sample was injected in to the HPLC column, and Acid and artificial gastric acid tolerance of L. plantarum the 0.5% acetic acid and methanol (1:4 v/v) were used as WCP931 eluent at 1 mL/min flow rate. The strain WCP931, capable of degrading OP insecti - cides, was isolated from mulkimchi samples and was Cloning of the opdC gene identified as described by Cho et  al. [3]. The 16S rRNA The genomic databases of the L. plantarum strains were gene sequence of the strain was submitted to the NCBI screened to design suitable primers for cloning the opdC and its accession number was provided as FJ480209. gene. For cloning the complete open reading frame of Moreover, the concentrated acid and gastric acid toler- opdC from L. plantarum, WCP931 genomic DNA was ance capability of the strain L. plantarum WCP931 was amplified using 5’-AAA GGA TCC TGA TTG ATC determined according to Lee et al. [21]. TGA CAA TGG G-3’ (sense, BamHI sites are indicated by underline), and 5’-AAA GAA TTC C TT GCT ATA Biodegradation of OP insecticides by L. plantarum WCP931 CTG ATT CGC TAG CC-3’ (antisense, HindIII sites and recombinant E. coli are indicated by underline) primer sequences based on One hundred microliters of the WCP931 strains in MRS the carboxylesterase sequence available in the database. broth culture suspension containing 10   CFU/mL was The purified opdC gene was amplified and ligated with inoculated into 50  mL of 1/25 MRS medium containing pGEM-T easy vector (Promega, USA), and after cloning, 100  mg/L CP. Likewise, the E. coli DH5α was cultured the plasmid (opdC- pGEM-T) was amplified by E. coli under similar conditions as control. E. coli DH5α harbor- DH5α culture and isolated as instructed by the manu- ing the opdC gene (pGCY300) was grown in M9 medium facturer. Afterthat, the plasmid was cut with BamHI supplemented with 100 mg/L CP. Likewise, other organ- and HindIII enzyme, next to the isolated opdC gene was ophosphate such as CP, CS, DF, DZ, EP, MPT, FA, and cloned into the pBluscript II SK vector. The nucleotide PT insecticide mineralizing capabilities of L. plantarum sequence of the opdC was analyzed according to Haque WCP931, E. coli DH5α, and E. coli DH5α carrying opdC et  al. [19]. The GenBank accession number of the opdC gene were evaluated using the above mentioned condi- gene was obtained MT472461. The phylogenetic tree and tions. Culture flasks containing specific insecticides were conserved regions of the OpdC enzyme with related Opd sacrificed after periodic intervals. Thereafter, the insecti - and esterase enzyme sequences were accomplished using cide concentrations and strain growth were determined. DNAMAN10.0 [19]. To ensure the degradation accuracy, the cultures of these strains were run in triplicate [19]. Expression and purification of the OpdC enzyme To overexpress the opdC gene, the PCR product gener- OP degradation assay for L. plantarum WCP931, ated with primers 5’-AAA GGA TCC T GA TTG ATC recombinant E. coli, and OpdC protein TGA CAA TGG G-3’ and 5’-AAAA GA ATT CCT TGC The concentrations of insecticides and their residues in TAT ACT GAT TCG CTA GCC-3’ was cloned into the (+) strain WCP931, recombinant E. coli, and OpdC protein expression vector pET-32a (Novagen, USA), which were determined using thin layer chromatography (TLC) encodes a C-terminal (His) tag within the recombinant (+) and HPLC as described by Cho et  al. [3]. In brief, 4-mL protein. BL21 (DE3) cells harboring pET-32a /OpdC of filtrate was extracted with ethyl acetate from 5-mL ali - were grown at 37  °C to mid-log phase in LB medium quot of culture supernatant. The TLC plate was set up supplemented with 50  μg/mL ampicillin antibiotic. The to analyze the degradation of CP and TCP according to recombinant E. coli cells were centrifuged at 6,000  rpm Islam et al. [19]. All experimental OP insecticide concen- for 10  min to get pellet and later washed with 10  mM trations were determined at 214  nm by HPLC (Perkin- Tris–HCl buffer (pH 7.0). Next, the pellet was resus - Elmer 200 series, CT, USA). The HPLC analysis protocol pended in the same buffer and kept at −20 °C for 30 min. Lee et al. Appl Biol Chem (2021) 64:62 Page 4 of 12 Thereafter, it was mixed with 1 mg of bovine DNase I and Pfu DNA polymerase buffer (20 mM MgSO ), and 2.5 U incubated at 37  °C for 30  min. Triton X-100 was added of Pfu DNA polymerase (Stratagene, CA, USA). The to the suspension at a final concentration of 2.5%. The PCR products were incubated on ice for 5  min, and 1 supernatant of the suspension was collected and stored μL of DpnI restriction enzyme (10 U/μL) was added. immediately at 4  °C. The overexpressed His -tagged Then, the mixture was incubated for 1  h at 37  °C. The OpdC protein was purified using a HisTrap kit (Amer - DpnI-treated plasmids were then transformed into E. shan Pharmacia Biotech). The elution of OpdC protein coli DH5α according to the manufacturer’s specifica - was conducted using 100  mM imidazole with 0.1% Tri- tions. The site-directed mutagenesis procedure was ton X-100. The purity and molecular weight of the OpdC adapted as described by Haque et al. [20]. protein were evaluated by sodium dodecyl sulfate–poly- acrylamide gel electrophoresis. The quantity of OpdC protein in the solution was adjusted to 50  μg/mL and Homology modeling, molecular docking, and visualization used for the activity assay towards OP insecticides. of OpdC enzyme The 3D structure of insecticides degrading model OpdC protein was built in the I-TASSER server [22– pH and temperature effects on OpdC enzymatic activity 24]. The model OpdC protein structure was later sub - The pH and temperature effects of the OpdC enzyme jected to energy minimization using the Swiss-PDB were examined by considering esterase activity. The Viewer. Next, the energy minimization followed by effect of pH (range 3.0–11.0) on the esterase activity of structure validation was conducted using "SAVESv OpdC protein was determined according to the above- 6.0”, which verify 3D models based on several param- mentioned protocol at 30 ± 0.5  °C. While, the tempera- eters such as non-bonded interactions of atoms and the ture effect of the OpdC enzyme was determined at 10 compatibility of the model amino acid sequence, ste- to 70 °C for 1 h. The degree of OP hydrolysis was meas - reochemical properties of the model, etc. Additionally, ured using HPLC. Fifty microliters of enzyme solution “Ramachandran” plot analyses were done for the model were poured into a solution containing 250 μL insecti- OpdC protein as well. The 3D structures of organo - cide (200  mg/L) and 700 μL phosphate buffer (200  mM, phosphate insecticides were traced and assembled from pH 6.5). To calculate the experimental error, assays were the “Pubchem" website. The energy minimization and performed three times. The classical spectrophotomet - optimization of the ligands were conducted using the ric method was used to measure the activity of esterase mmff94 force field and the steepest descent algorithm. provided by the native and mutant OpdC enzymes. The Multiple docking of OpdC protein was performed for rate of hydrolysis of the ρ-NPB (100 mg/L) substrate was tracing out the active sites and catalytic interactions measured in 50  mM sodium phosphate buffer (pH 7.0) using PyRx in Autodoc vina. at 35 ± 0.5 °C using a spectrophotometer at 420 nm. One unit of esterase activity was defined as the amount of enzyme required to release 1 μmol of ρ-NPB per minute under the assay conditions. The assay was conducted in Results triplicate [19]. Identification and gastric juice tolerance ability of L. plantarum WCP931 The 16S rRNA gene similarity of the WCP931 strain with Site‑directed mutation of opdC gene reference LAB was 85.4 to 99.5%. The phylogenetic tree The organophosphorus hydrolase enzyme OpdA, showed that the strain WCP931 was related to Lactoba- OpdE, OpdB, OpdD contains conserved domain G-X- cillus sp. (Additional file  1: Fig. S1). As a consequence, S-X-G [18–20]. According to this database of organo- the chlorpyrifos-degrading WCP931 strain was named as phosphorus hydrolase, the conserved domain of the Lactobacillus plantarum WCP931. The strain’s 16S rRNA OpdC protein was analyzed and identified. In addition, gene sequence was deposited in the NCBI database. The to confirm the location of the catalytic sites in OpdC, accession number of the strain is FJ480209. a site-directed mutagenesis technique was employed to The survival rates of L. plantarum WCP931 under introduce amino acid changes at position 116 (serine acidic and artificial gastric acidic conditions are shown in to alanine) using oligonucleotide primers: 5’-TCT TGC Fig. 1. The CP-degrading strain WCP931 showed moder - CGG GTT TTCG GCTGG CGG CCACG-3’ (sense) and ate survival rates of 86% (acidic condition) and 51% (arti- 5’-CGT GGC CGC CAGC CGA AA ACC CGG CAAGA- ficial gastric acidic condition) at pH 2.0, 95% (acidic) and 3’ (antisense) 5’. The underlined codons were mutated. 84% (artificial gastric acidic) at pH 2.5, and 99% (acidic) The PCR mixture (50  μL) composed by 1 μL pET- and 96% (artificial gastric acidic) at pH 2.5 after 3  h, 32a( +)/opdC DNA (80  ng/μL), 4  μL (10  ρmol) of each respectively. primer, 5  μL (2  mM) dNTP mixture, and 5 μL (10 ×) L ee et al. Appl Biol Chem (2021) 64:62 Page 5 of 12 (A) (A) ab b ab ab cd cd d d cd pH : Incubaon me : 3 h 6 h Incubaon me (day) (B) ab (B) ab c b ab pH : Incubaon me : 3 h 6 h Fig. 1 Survival rates of L. plantarum WCP931 under acidic conditions (A) and survival rates of L. plantarum WCP931 under artificial gastric acidic conditions (B) at pH 2.0, 2.5, and 3.0 after 3 and 6 h Incubaon me (day) of incubation. L. plantarum WCP931 was tested in triplicate for its tolerance in acidified and artificial gastric acidified MRS. Means (C) with different lowercase letters (a–d) indicate significant (p < 0.05) ab differences of survival rate by Duncan’s multiple range test Degradation of OP insecticides by L. plantarum WCP931 The cell growth response and degradation pattern f f are shown in Fig.  2. The L. plantarum WCP931 grew markedly until the 1st day (OD 0.85), slightly declined at 2  days, and gradually decreased at 6  days (OD 0.94) OP inseccides during incubation. However, E. coli DH5α did not grow Fig. 2 Cell growth response of L. plantarum WCP931 and E. coli DH5α in the presence of CP 100 mg/L (Fig.  2A). The L. plan - in 1/25 MRS and M9 medium, respectively, containing 100 mg/L tarum WCP931 exhibited an initial rapid degradation CP after 9 days (A), change of CP concentrations of L. plantarum of CP of approximately 66 mg/L during the first 3 days WCP931 and E. coli DH5α in 1/25 MRS and M9 medium, respectively, of incubation and then exhibited a maximum degrada- containing 100 mg/L CP after 9 days (B), insecticide degradation tion of 86  mg/L at 9  days of incubation. On the other pattern of L. plantarum WCP931 in 1/25 MRS containing 100 mg/L insecticides after 9 days (C). Means with different lowercase letters hand, in the case of E. coli DH5α, it slightly decreased (a–f ) indicate significant (p < 0.05) differences in survival rate by to 83  mg/L on the 9  days (Fig.  2B). The L. plantarum Duncan’s multiple range test. Names of insecticides on the X-axis WCP931 was able to degrade CP to DEPT and TCP and of the figure are abbreviated as follows: OP, organophosphorus; utilized DETP as the sole source of carbon and phos- CP, chlorpyrifos; CS, cadusafos; CM, comnaphos; DZ, diazinon; DF, phorus. All OP insecticides tested in the cross-feeding dyfonate; EP, ethoprophos; FA, fenamiphos; MPT, methylparathion; and PT, parathion experiment were degraded by L. plantarum WCP931. All OP insecticides tested, such as CP, CM, DZ, MPT, and PT, had DEPT side chains, while CS, DF, EP, and from 72 to 88% for the CP, CM, DZ, MPT, and PT FA had no DEPT. Except for DF, eight other OP insecti- insecticides, respectively. cides (including CS, CP, CM, DZ, EP, FA, MPT, and PT) were hydrolyzed at a phosphoester bond by L. plan- tarum WCP931. However, a decreased degradation rate Sequence analysis of the opdC gene and the OpdC protein of OP insecticides was observed, as shown in Fig.  2C. PCR amplification of the total DNA from L. plantarum In particular, on the 9 days, degradation was enhanced WCP931 with specific primers produced an amplification Survival rate (%) Survival rate (%) OP inseccides Microbial growth CP inseccide concentraon (mg/L) concentraon (mg/L) (OD ) 600 nm Lee et al. Appl Biol Chem (2021) 64:62 Page 6 of 12 product of approximately 1.5  kb. After sequencing, a (opdC) are shown in Fig.  4. CP and TCP with R values nucleotide sequence 1500  bp in length was found in the of 0.57 and 0.66, respectively, were detected in sam- open reading frame (ORF) of opdC. Its ORF started with ples drawn at 0, 1, 3, 6, and 9  days (Fig.  4A). The clone an ATG start codon and ended with a TAA Ochre stop decomposed CP markedly until 2  days (78  mg/L), then codon (Additional file  2: Fig. S2). The opdC gene product decreased rapidly at 6  days (24  mg/L), and subsequently is predicted to contain 276 amino acids with a molecu- grew slowly until 9  days during incubation. At 3  days, lar mass of 31  kDa (http:// web. expasy. org/ compu te_ the clone exhibited a gradual increase in TCP concentra- pi/). Analysis of the amino acid sequence with the pro- tion to approximately 32  mg/L at 3  days. After that, the gram PSORT (http:// www. cbs. dtu. dk/ servi ces/ Signa lP/) TCP concentration was increased to 68  mg/L at 6  days revealed no potential signal sequences. The calculated pI (Fig. 4B). Nine OP insecticides (CS, CP, CM, DZ, EP, FA, of OpdC was 5.18. MPT, and PT) were mineralized by recombinant E. coli The amino acid sequence GFSAG, starting at residue with the opdC gene. The recombinant cells exhibited 46 116 for OpdC (Additional file  2: Fig. S2 and Fig.  3), fits to 90% degradation of CP, CM, DZ, FA, MPT, and PT at the Gly-X-Ser-X-Gly motif found in most bacterial and 37 °C for 9 days (Fig. 4C). eukaryotic serine hydrolases, such as lipase, esterase, and serine proteinase, as well as in β-lactamase [25–27]. A Purification and characterization of the OpdC protein phylogenetic tree containing the esterolytic and lipolytic The OpdC protein was purified from E. coli BL21 (DE3) proteins showed that the OpdC enzyme did not belong overproducing OpdC using column filtration techniques. to groups I, II, III or IV (Fig. 3). This separation of OpdC Protein fractions were analyzed by SDS-PAGE, and one suggested a new type of esterase. protein band (31  kDa) was present after the final purifi - cation step (Fig.  5A). The ability of OpdC to hydrolyze Degradation of CP in liquid culture by E. coli harboring ρ-NPB was determined at 30 ± 0.5  °C with various buff - opdC gene ers ranging from pH 3 to 11. The maximum activity was To confirm the insecticide degradation function of the observed at pH 6 (Fig.  5B). The optimal hydrolysis tem - opdC gene, the gene was cloned into E. coli DH5α cells. perature was determined at pH 6 by measuring the activ- The degradation patterns of CP by the clone pGCY300 ity across a temperature range. The maximum activity 0.05 Lbr -OpdB Lpl -OpdC LAB- Opd Lsa -OpdD group Lme -OpdA Lme -OpdE A. thaliana (AAB84335) A. azollae (AF035558) Est/Lip H. sapiens (NP_001975) Group I E. coli K12 (AAC73458) S. cerevisiae S288C (CAA84054) L. laccs MG1363 (AAF02201) Est/Lip Group II L. laccs MG1363 (AF157601) S. albus (AAA53485) New Est/Lip S. colicolor (NP_625018) group Uncultured bacterium (AF223645) S. aureus (AAA26633) Est/Lip S. epidermidis (AF090142) Group IV L. casei CL96 (AY251019) G. stearothermophilus P1 (AF237623) Est/Lip G. thermocatenulatus DSM730 (CAA64621) Group III D. radiodurans R1 (AAF09912) Fig. 3 Phylogenetic tree showing the evolutionary relatedness and levels of homology between the esterolytic and lipolytic enzyme amino acid sequences and the alignment of the conserved regions found in the primary esterolytic and lipolytic enzymes. The esterolytic groups are classified according to the catalytic conserved domain G-X-S-X-G-G of the protein sequence L ee et al. Appl Biol Chem (2021) 64:62 Page 7 of 12 G-F-S116-A-G of OpdC. To determine whether Ser116 S 0 1 3 6 9 (A) was involved in catalytic esterase action, it was replaced CP by site-direct mutagenesis, and the mutant proteins were expressed in E. coli and purified. The purified OpdC TCP enzyme showed 78% degradation, while the mutant OpdC had no enzymatic activity towards ρ-NPB and CP (B) (Table 1). ab Five different 3D models of the OpdC protein were b a built and provided by the Iterative Threading ASSEmbly Refinement (I-TASSER) server. According to the best scoring value, model 1 of OpdC was chosen for analysis, as shown in Fig.  6. The OpdC 3D model protein showed a C-score of 0.87, a TM-score of 0.83 ± 0.08, an RMSD of 4.2 ± 2.8, and a cluster density of 0.7167. The 3D model of OpdC showed eight α-helices, eight β-sheets, two ran- Incubaon me (day) dom coils (η), and six different hydrogen-bonded turns (T) in the whole structure (Fig.  6A). In particular, the (C) G-F-S-A-G motif for OpdC was found in the β5 and α3 helices of the predicted structure from the N-terminus (Fig.  6A).  In ERRAT server, a model is evaluated based on non-bonded interactions between different types of atom to assess error rate with the standard optimized model, while in Verify 3D the 3D to 1D comparisons are made based on surrounding environment and locations of the α-helix, β-sheets, loops, etc. The protein model was fine-tuned using loop refining and energy minimi - OP inseccides zation. The loop refined and energy minimized OpdC Fig. 4 TLC profile (A) and changes of CP and TCP concentration model protein showed an overall quality factor 80.22% (B) and changes of OP insecticides concentrations (C) by the and verify 3D score 93.84%. The Ramachandran plot recombinant E. coli with opdC gene growing in the M9 medium analysis for the OpdC protein showed that 92.1%, 7.5%, containing 100 mg/L of CP and OP insecticides after 9 days, respectively. Means with different lowercase letters (a–f ) indicate 0.0%, and 0.4% amino acid residues are centered in the significant (p < 0.05) differences of survival rate by Duncan’s multiple most favorable regions, additional allowed regions, gen- range test. Names of insecticides as the X-axis of the figure shows erously allowed region, and in the disallowed regions, the abbreviations as follow: OP, organophosphorus; CS, cadusafos; respectively (Fig. 6B). Thus, the Ramachandran plot anal - CP, chlorpyrifos; CM, comnaphos; DZ, diazinon; DF, dyfonate; EP, ysis results for the OpdC protein substantiate the quality ethoprophos; FA, fenamiphos; MPT, methylparathion; PT, parathion; and TCP, 3,5,6-trichloro-2-pyridinol of the model. Using the COACH Meta server, the highest poten- tial ligand-binding sites were identified and observed at Gly42, Gly43, Gly44, Phe115, Ser116, Ala117, and was observed at 35  °C (Fig.  5C). Nine OP insecticides Val156 for cluster 1, but they were recorded at Asp201, were decomposed by the OpdC enzyme (Fig. 5D). Except Glu202, Ser203, Ile232, and His233 for cluster 2. Based for DF, all OP insecticides are hydrolyzed at a phosphoe- on the molecular docking of the OpdC protein with ster bond by the OpdC protein. In particular, the relative chlorpyrifos (Fig.  6C), the critical amino acids (Ser116, activity of the enzyme was higher towards CP, CM, DZ, Asp201, His233, Glu52) of the catalytic triad were pre- FA, MPT, and PT insecticides than towards CS, DF, and sent in an area of 5  Å from chlorpyrifos. Interestingly, EP insecticides. the distance of the P-atom of the phosphodiester of CH and the O-atom of the hydroxyl group was meas- ured to be 3.3  Å, which may initiate the nucleophilic Identification of residues essential for enzymatic activity attack on the P-atom by the O-atom and might liber- of the OpdC protein ate TCP. Consequently, the P-tom of the phosphodi- Most lipases and carboxyl esterases have the consensus ester of CP might be attacked by the O-atom of water, sequence motif Gly-X-Ser-X-Gly with the serine active resulting in further degradation of the nontoxic residue site. Analysis of the deduced amino acid sequences DEPT and the return of Ser116 to its original state. In showed a potential serine hydrolase motif, such as CP concentraon OP inseccides (mg/L) concentraon (mg/L) TCP concentraon (mg/L) Lee et al. Appl Biol Chem (2021) 64:62 Page 8 of 12 (A) 1 2 3 4 (B) 116.0 66.2 ab 45.0 35.0 31 kDa 25.0 d 18.4 14.4 (kDa) pH (C) (D) ab bc f f Temperature ( C) OP inseccides Fig. 5 Electrophoretic analysis of the purified OpdC protein (A). Separation was performed on a 12.5% (w/v) SDS polyacrylamide gel and after was stained with 0.025% Coomassie blue R-250. Lane 1, standard marker; lane 2, crude extract from E. coli BL21 (DE3) containing pET-32( +)/opdC; lane 3, crude extract from IPTG-induced E. coli BL21 (DE3) containing pET-32( +)/opdC; lane 4, purified OpdC protein from Hi-Trap kit (Amersham). pH effect on the relative activity of OpdC (B). The esterase activity of OpdC was assayed using ρ- NPB as substrate at different pH values at 30 ± 0.5 °C for 1 h. Eec ff t of temperature on the relative activity of OpdC (C). The esterase activity of OpdC was assayed using ρ- NPB as substrate at different temperature values at pH 6 for 1 h. Substrate relative activities of OpdC on the various OP insecticides (D). The OP hydrolase activity of OpdC was assayed using as substrate with 200 mg/L OP in insecticides at 35 ± 0.5 °C and pH 6.0 for 12 h. Names of insecticides as the X-axis of the figure shows the abbreviations as follow: OP, organophosphorus; CS, cadusafos; CP, chlorpyrifos; CM, comnaphos; DZ, diazinon; DF, dyfonate; EP, ethoprophos; FA, fenamiphos; MPT, methylparathion; and PT, parathion. Means with different lowercase letters (a-f ) indicate significant (p < 0.05) differences of survival rate by Duncan’s multiple range test this circumstance, the OpdC showing higher relative Table 1 Esterase and orangophosphorus (OP) hydrolase activity towards MPT and PT, which docked complex, activities for the hydrolysis of ρ-nitrophenyl butyrate (ρ-NPB) and was visualized to get the catalytic insights, as seen in chlorpyrifos (CP) by the OpdC and mutant OpdC enzyme Fig. 6D and E ProteinsEsterase activity (U/mg)/ b Figures  6D and E demonstrate the direct interac- CP degradation degree (%) tion of Ser116 and His233 with MPT and PT. Besides, Glu52 is closely positioned near the Ligands (MPT, OpdC 397 ± 15.88 /78 PT). Therefore, the predicted homology model of OpdCM < 0.01/2 OpdC revealed that the active site of this enzyme a / Esterase activity is indicated the micromoles of ρ-NPB hydrolyzed min mg. The was located in the known architecture of the hydro- OpdC and OpdCM activities were assayed with ρ-NPB as substrate at pH 6 and lases. As seen in Fig. 6D, the OpdC protein abundantly 35 ± 0.5 °C for 1 h in constant temperature incubator, respectively interacts with methyl-parathion through multiple The OpdC and OpdCM activities were assayed with CP as substrate at pH 6 and 35 ± 0.5 °C for 12 h in constant temperature incubator, respectively amino acid residues. In fact, the O-atom of methyl- Values indicate the means of three replications. The standard errors were within parathion is attacked by Ser116, His233, Gly44, and 5% of the mean Ser203, Glu52 attacks P-atom via attractive charge. Relave acvity (%) Relave acvity (%) Relave acvity (%) L ee et al. Appl Biol Chem (2021) 64:62 Page 9 of 12 (B) (C) (A) (D) (E) Fig. 6 3D modeling of OpdC protein. The model was built using I-TASSER server (A). The α-helix, β-sheet, and random coil (η) are marked with red, yellow, and light blue color. The critical amino acid Ser116 side chains are marked with magenta color, while His233 and Glu52 are marked with yellow color. Ramacahndran plot analysis of the OpdC protein (B). Molecular docking model of the OpdC protein with chlorpyrifos (CP) (C). The critical amino acid (Ser116, His233, Glu 52) side chains within 5 Å of chlorpyrifos are marked in yellow. Visualization of ligand binding sites of OpdC within 5 Å of methyl parathion (D) and parathion (E) The Ser116, His233, Arg51, and Gly44 provide a con- Discussion ventional hydrogen bond with the O-atom of the To date, three different classes of organophosphorus ligand molecule. Notably, the Ser203 residues provided hydrolase genes namely, opd, mpd, and ophc2, had been carbon-hydrogen bond interaction with O-atom, while discovered in the last decades. Among these organo- Ser49 provide unfavorable acceptor-acceptor interac- phosphorus hydrolases, the opd gene is extensively dis- tion with O-atom of the ligand molecule, respectively. tributed, especially it is sourced from different bacterial Dramatically, the OpdC protein formed a possible cat- species [15–20]. The reported opd genes belonged to alytic triad in the binding pocket region with residues the chromosome [12, 15] or the plasmid [26] of the iso- Ser116, His233, Glu52. lated strains. Yet several classes of esterase are revealed; The OpdC protein-parathion docked complex among them, some are capable of degrading OP insecti- showed multiple residues interactions (Fig. 6E). In par- cides. However, the classes of organophosphorus hydro- ticular, the conventional hydrogen bond interaction lase are not much reported. Recently, we have reported was observed for Ser116 and Arg51 with the O-atom of organophosphorus hydrolase gene opdA, opdB, opdD, parathion. At the same time, His233 formed π- π-alkyl opdE from Kimchi originated Lactobacillus species bonds with a benzene ring and O- atom of parathion strains, which are deviated from the common ester- and Glu52, which contribute an attractive charge to ase groups. Exploring new esterase having evolutionary the parathion. Consequently, the possible catalytic history with OP insecticides degrading activity might triad was supposed to be made as Ser116, His233, increase the diversity of organophosphorus hydrolase Glu52. According to docking analyses, these two pro- in nature. Therefore, the present study was focused on teins, catalytically crucial residues, are placed within another new specific organophosphorus hydrolase that 1.5–5.5 Å that might participate in the biodegradation could degrade a range of OP insecticide, which might of organophosphates like MPT, PT, CP, and others. derive the strain L. plantarum WCP931 in insecticides Lee et al. Appl Biol Chem (2021) 64:62 Page 10 of 12 bioremediation during kimchi fermentation. Therefore, neutral and alkaline soils [30, 31]. Importantly, the opti- the cloned and functionally expressed chromosome- mum temperature (35 °C) for OpdC varied slightly from based opdC gene increases the diversity of the hosts of that observed for the OpdB protein of L. brevis WCP902 OP hydrolases. Because of the lack of a signal sequence (40 °C) [18] but was higher than that recorded for OpdD in the N-terminal region of OpdC, it is assumed that the of L. sakei WCP904 (30 °C) [19]. E. coli cells expressing OpdC protein might degrade CP The OpdC protein contains Gly-X-Ser-X-Gly conserved in the intracellular environment. Therefore, hydrolysis domain and a catalytic site comprised of serine residues, could take place inside the cell, followed by the release which are routinely appeared in bacterial and eukaryotic of the hydrolysis product into the culture medium. How- serine hydrolases, e.g., serine proteinases, lipases, ester- ever, products in the culture medium do not rule out the ases as well as in β-lactamases [25, 35, 36]. However, the possibility that hydrolysis takes place inside the cell. The phylogenetic tree analysis of the OpdC protein showed nonclassical secreted proteins often seem to have both that it did not belong to the known families of esterolytic cytoplasmic and extracellular functions [27]. Moreover, and lipolytic proteins (groups I, II, III, IV or even a new several carbohydrate- and protein-degrading enzymes group of soil metagenomes), indicating the existence of were identified as extracellular despite the lack of extra - a new LAB esterase/opdase group, represented by OpdC cellular signal peptides [28, 29]. Organophosphate insec- (Fig.  3). Importantly, our previously reported OpdB and ticides with residues were extracted from recombinant OpdD enzymes showed the Gly-X-Ser-X-Gly motif and E. coli harboring OpdC culture medium, indicating that catalytic active site of serine residues. Therefore, a con - the OpdC enzymes use a nonclassical pathway to exhibit temporary LAB-opd esterase can be brought forward in extracellular activities. Generally, OP is hydrophobic in the present study, consisting of OP hydrolase genes from nature; thus, compounds in the culture medium are in LAB strains isolated during kimchi fermentation. equilibrium with compounds inside bacterial cells. As seen in Fig.  3, α-helices, β sheets, random coils, Interestingly, the OpdC enzyme hydrolyzed a range of and β turns were observed in both structures of OpdC OP insecticides containing a P–O bond and a P–S bond, enzymes and were matched with the catalytic motif G-X- indicating that recombinant OpdC has broad substrate S-X-G of OpdC and OpdD enzymes [18, 19]. When the specificity. This finding showed similarities with some Ser116 residue was replaced by Ala, the mutant OpdC previous reports [19, 27, 30, 31]. However, the enzyme enzymes had no enzymatic activity towards ρ-NPB and relative activity against the P–O bond consisting of CP. In 3D modeling, the quality of the 3D model made insecticides was much higher than that against the P–S in I-TASSER is predicted by the confidence score, i.e., bond, which is consistent with the previously reported C-score [22–24]. The C-score is generated according to OpdB and OpdD enzymes [18, 19]. However, minor vari- the significance of threading template alignments and ations in relative substrate activities were observed for the convergence parameters of the structure assembly OpdC compared with those reported for OpdB, OpdA, simulations. In fact, it should be ranged of −5 maxi- and OpdE enzymes. Thus, the OpdC hydrolysis activity mum, where a higher value of C-score indicates a model depends on the molecular structure of insecticides used with increased confidence and vice versa [22–24]. In this in this study. Temperature influenced the OpdC activ - study, the CS scores ranged between −1.64 and −5.0 for ity. The optimum pH values of OpdD (6.0) from L. sakei all other four models except 3D model 1. Since model 1 WCP904 [19] and OpdB from L. brevis WCP902 (6.0) has a positive (+ 0.87) CS score, we chose model 1 for [18] were less than that of OpdB from Pesudomonas sp. analyses and docking. BP3 (8.0) [32]. The L. plantarum is known to be adapted The nearest homologs carboxylesterase Cest-2923 to stressful environments such as those in the gastro- (PDB ID: 4BZW) of Lactobacillus plantarum WCFS- intestinal tract with a low pH or a high salt content. To 1’s nucleophile Ser116 was located in the nucleophile survive in acidic environments, this bacterium uses elbow, with its backbone angles residing in an unfavora- F F -ATPase and sodium-proton pumps to regulate and ble region in Ramachandran plot (ɸ = 52º, ψ = −180º) o 1 maintain the intracellular pH [33]. Kimchi fermenta- [37]. Alike, the residue Ser116 of OpdC protein is found tion involving LAB is conducted at acidic pH, in which in the unfavorable in Ramachandran plot. In fact, its L. plantarum is quite predominant and is responsible for catalytic triad (Ser116-His233-Asp201/Glu52) was made acidifying kimchi. Therefore, the OpdC highest activity in a canonical site of the OpdC protein sequence, which observed at pH 5–6 (acidic) is quite logical. In fact, OP also consistent with the homologs 4BZW protein. Thus, insecticides are immovable in pH 5–7, but it is easily Ramachandran plot analysis validated the model OpdC decomposed in alkaline pH [34]. Therefore, acidic soils protein structure. are more preferable to slower CP degradation than the L ee et al. Appl Biol Chem (2021) 64:62 Page 11 of 12 Similar to CP, MPT, PT, very similar docking results Additional file 1: Figure S1. Phylogenetic relationships of L. plantarum and catalytic interactions were observed for the other WCP931 and other LAB closely related bacterial based on 16S rRNA sequence. Number above each node is confidence levels (%) generated insecticides and ρ-NPB evaluated in this study (data from 1000 bootrap trees. The scale bar is in fixed nucleotide substitutions not shown). These results suggested that Ser116 might per sequences position. be the crucial amino acid for the degradation of CP and Additional file 2: Figure S2. Nucleotide and deduced amino acid other insecticides evaluated in this study. Our previous sequences of opdC gene from L. plantarum WCP931. Bold letters and underlines the start codon and serine residue. The stop codon is indicated studies have reported the role of serine in insecticide by asterisk. The consensus sequences region is indicated by yellow box. degradation [19, 20]. In addition, His233 and Glu52 of the catalytic triad were located within 5  Å of the CP Acknowledgements molecule, indicating that His233 and Glu52 might also Not applicable. be involved in the degradation of CP. The amino acid 10 154 157 residues Ser, Asp , and His of thioesterase I/pro- Authors’ contributions KMC conceived and designed the experiments. KMC, MAH, and JHL tease of E. coli are appeared in the catalytic site [38]. interpreted the data and wrote the manuscript. HYL, DYC, MJK, JGJ, and EHJ 156 281 Moreover, Ser and His residues of a novel chlor- performed the experiments and analyzed the data. MAH performed and pyrifos hydrolase was reported to be participated in interpreted Bioinformatics analyses. All authors read and approved the final manuscript. the chlorpyrifos degradation [39]. Notably, the nucleo- phile Ser, a general bases His/Arg, and an acid Glu/Asp Funding residues are apparent in the OpdC catalytic site, which This work was supported by the research invigoration program of 2020 Gyeo- ngnam National University of Science and Technology. might forming an oxyanion hole. The predicted struc - ture of OpdC protein partially shares esterase along Availability of data and materials with a new LAB-Opd hydrolase structure. As a result, The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. the classification of esterase is expanded into the LAB- Opd group [18, 19], where OpdCs are included in this Declarations study. To date, this study reports a new organophos- phorus hydrolase (OpdC) enzyme from the kimchi Consent for publication originated L. plantarum strain that can degrade nine This research article entitled as “Biodegradable properties of organophospho- rus insecticides by the potential probiotic Lactobacillus plantarum WCP931 insecticides containing P–O and P–S bonds as well as with a degrading gene (opdC)” an original work was carried out by authors: All unmask its potential catalytic insights by site-directed authors approve of its submission to as Applied Biological Chemistry. It is not mutation and molecular docking. under consideration by another journal at the same time as Applied Biological Chemistry. I am the author responsible for the submission of this article and In conclusions, the recombinant OpdC enzyme dem- I accept the conditions of submission and the Springer Open Copyright and onstrated robust degradation of OP insecticides such as License Agreement. MT, CP, CM, DZ, and PT. To date, the OpdC enzyme Competing interests sequence deviates from the common families of ester- As a corresponding author, I confirm that I have read Springer Open’s guid- ase and lipase proteins available. As a result, OpdC is ance on competing interests and have included a statement indicating that suggested to be a novel protein. Importantly, the cata- none of the authors have any competing interests in the paper. lytic action of the OpdC enzyme seemed to be per- Author details formed by the serine (116) amino acid. With regard Department of Life Resources Industry, Dong-A University, Busan 49315, to the safety of insecticides in kimchi, it is assumed Republic of Korea. Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea. Depar t- that fermented kimchi meets the minimal residue cri- ment of Biochemistry and Molecular Biology, Hajee Mohammad Danesh teria for food safety due to OP degradation by kimchi Science & Technology University, Dinajpur 5200, Bangladesh. fermentation driving lactic acid bacteria, including L. Received: 17 June 2021 Accepted: 8 August 2021 plantarum. The present study suggested that the opdC gene in L. plantarum WCP931 along with opdB in L. bevis WCP902 and opdD in L. sakei WCP904 play roles in the degradation of OP insecticides during kimchi fer- References mentation. Hence, L. plantarum WCP931 establish as 1. Huete-Soto A, Castillo-González H, Masís-Mora M, Chin-Pampillo JS, Rod- a worthy candidate for the potential bioremediation of ríguez-Rodríguez CE (2017) Eec ff ts of oxytetracycline on the performance and activity of biomixtures: removal of herbicides and mineralization of environments contaminated with organophosphorus chlorpyrifos. J Hazard Mater 321:1–8 insecticides. 2. 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Biodegradable properties of organophosphorus insecticides by the potential probiotic Lactobacillus plantarum WCP931 with a degrading gene (opdC)

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Abstract

An organophosphorus (OP) insecticide-mineralizing strain, Lactobacillus plantarum WCP931, harboring a new OP hydrolase (opdC) gene, was isolated during kimchi (Korean traditional food) fermentation. Strain WCP931 exhibited a significant survival rate of 51 to 96% under artificial gastric acid conditions at pH 2 to 3 after 3 h. The opdC gene, consisting of 831 bp encoding 276 amino acids, was cloned from strain WCP907. Recombinant Escherischia coli har- boring the opdC gene depleted 77% chlorpyrifos (CP) in M9 minimal medium after 6 days of incubation. The OpdC enzyme represents a novel member of the GHSQG family of esterolytic enzymes or a new Opd group. The OpdC molecular mass was estimated to be approximately 31 kDa by SDS-PAGE and showed maximum activity at pH 6 and 35 °C. The mutated OpdC (Ser116 → Ala116) enzyme had no activity towards OP insecticides and ρ-nitrophenol-β- butyrate. Importantly, the relative activity of OpdC protein against chlorpyrifos, coumafos, diazinon, fenamifos, methyl parathion, and parathion was higher than that against cadosafos, dyfonate, and ethoprofos insecticides. These results suggested the involvement of OpdC in the biodegradation of OP insecticide-contaminated cabbage during fermen- tation. The new OpdC enzyme expands the heterogeneity of the lactic acid bacterial Opd enzyme group in nature. Keywords: Organophosphorus insecticides, Kimchi, Lactobacillus plantarum WCP931, OpdC gene, Biodegradation Introduction various pesticides, including organophosphates, are com- Pesticide-contaminated vegetables are more frequently monly used in agriculture for crop cultivation and pro- ingested by humans in developing countries. Vegetables tection worldwide. Organophosphate insecticides are are the most common foodstuff, and people consume preferred over organochlorine insecticides due to their 150–250 g of vegetables daily in Asian countries. In fact, extensive efficacy and longer persistence in the environ - ment and on crops. As a consequence, organochlorine has been replaced with organophosphate insecticides in *Correspondence: helalbmb2016@hstu.ac.bd; kmcho@gntech.ac.kr recent decades. Some preliminary work carried out sev- Jin Hwan Lee and Hee Yul Lee made equal contributions to this work eral years ago reported that extensively applied organo- (co-first author) Department of Food Science, Gyeongnam National University phosphorus (OP) insecticide residues not only persist of Science and Technology, Jinju 52725, Republic of Korea in the environment but also enter vegetables cultivated Department of Biochemistry and Molecular Biology, Hajee Mohammad on polluted sites, consequently posing a great threat to Danesh Science & Technology University, Dinajpur 5200, Bangladesh Full list of author information is available at the end of the article human health [1]. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Appl Biol Chem (2021) 64:62 Page 2 of 12 In a major advance in 2018, Hwang and Moon [2] [12–14]. In fact, the existence of opd genes is widely surveyed the levels of chlorpyrifos (CP) in Korean cab- distributed; as a consequence, opd genes have been bage crops and detected 0.12–0.75  mg/kg of CP after increasingly described [15–19]. Interestingly, a new 32–35  days of treatment. This research outcome led group of organophosphorus hydrolase (opd) genes was to anxiety among Korean citizens about consuming isolated from lactic acid bacteria that drive fermenta- fermented food kimchi made from Chinese cabbage. tion in Korean kimchi [18–20]. The OP hydrolase genes Therefore, considerable attention must be paid to using opdA and opdE, opdD, and opdB were isolated from Leu. organophosphate pesticides when cultivating Chinese mesenteriodes WCP907, L. sakei WCP904, and L. brevis cabbage plants, and pesticide residue avails in fermented WCP902 strains, respectively, of kimchi origin. kimchi should also be monitored. Interestingly, a neg- This study will unravel a new organophosphorus hydro - ligible concentration of CP residues in CP-impregnated lase gene named opdC from Lactobacillus plantarum fermented kimchi was estimated due to the catalytic WCP931 of kimchi origin. The essential amino acid in the strengths of fermentation driving bacteria such as L. catalytic site that played a vital role in the biodegradation mesenteroides WCP907, L. sakei WCP904, L. brevis of organophosphate insecticides was predicted by the WCP902, and L. plantarum WCP931 [3]. The most strik - site-directed mutagenesis and bioinformatics analysis. ing result emerged that the LAB strains could use OP The biochemical and genetic properties of the opd gene insecticides as a source of carbon and phosphorus in a also deviated from those of the opdA, opdE, opdD, and defined medium and decontaminate vegetable insecti - opdB genes [18–20]. The new opdC gene has boosted the cides used in mulkimchi fermentation [3]. diversity of opd gene in nature. The fermented mulkimchi character is developed by the influence of lactic acid bacteria in fermentation. The Materials and methods acidity of kimchi varies markedly at the initial, immature, Materials, chemicals and instruments optimum-ripening, overripening, and rancid stages [4] of Analytical grade OP insecticides, including cadusafos fermentation. Several studies [4–6] have been performed (CS), chlorpyrifos (CP), coumaphos (CM), diazinon (DZ), on the dynamics of LAB in kimchi fermentation and con- dyfonate (DF), ethoprophos (EP), fenamiphos (FA), meth- cluded that Lactobacillus sp. were dominated by Leucon- ylparathion (MPT), parathion (PT), and their residues, ostoc sp. A recent review of the literature on this topic such as 3, 5, 6-trichloro-2-pyridinol (TCP), and diethyl- found that the cell growth of L. mesenteroides reached thiophosphate (DEPT), were purchased from ChemSer- highest at the ripening period of kimchi and later reduced vice (West Chester, PA, USA) and Sigma-Aldrich Inc. as the pH of kimchi decreased, whereas that of acid-tol- (St. Louis, MO, USA). The esterase enzyme assay sub - erant L. plantarum continuously augmented until the strates tributyrin, ρ-nitrophenol-β-butyrate (ρ-NPB), and completion of fermentation [4]. Moreover, Cho et  al. [3] ρ-nitrophenol (ρ-NP) were also purchased from Sigma- concluded that L. plantarum plays a vital role of organo- Aldrich Inc. (St. Louis, MO, USA). The ultrapure deion - phosphorus insecticides degradation in kimchi fermen- ized water, acetic acid, methanol, and hydrochloric acid tation. It has also been reported that the phosphatase used were of analytical grade. Microbial growth and of L. plantarum might degrade OP insecticides during enzyme assays were performed using ultraviolet (UV)- skimmed milk fermentation [7]. Like all probiotics, L. visible absorption spectra on a Shimadzu Scientific Korea plantarum is gastric acid-tolerant and bile salt-tolerant, Corp. spectrometer (UV-1800 240  V, Seoul, Korea). which provides it to survive in the harsh environment The analysis of OP insecticides was performed by using of gastrointestinal tract [8]. In addition, it inhibits the a high-performance liquid chromatography (HPLC) growth of harmful pathogens and preserves critical nutri- (PerkinElmer Inc., Norwalk, CT, USA) including a Perki- ents, vitamins, and antioxidants [9, 10]. Moreover, L. nElmer UV detector, qutermary pump, autosampler, and plantarum provides beneficial immunomodulatory func - Phenomenx C18-RP column (250 × 4.6  mm, 5  µm, Phe- tion by increasing the synthesis of interleukin-10 and nomenx Inc., Torrance, CA, USA). secretion of macrophage and T-cell in the affected colon [11]. Therefore, L. plantarum-enriched kimchi ingestion Experimental bacterial strains, plasmids, and culture media has health-beneficial probiotic activities. For opdC gene cloning, subcloning, and high expression, However, there is still uncertainty concerning the the competent Escherichia coli DH5α strain and BL21 degradation of OP insecticides by organophosphorus (DE3) cells were purchased from Novagen (Washigton, hydrolases of L. plantarum strains. Multiple OP hydro- DC, USA). The culture medium such as MRS (de Man, lase genes, such as opd, opdA, opdB, mpd, ophc2, OPAA, Rogosa & Sharpe), LB (Luria–Bertani), and M9 minimal hocA, and adpB NC ADPase Oph, have been isolated medium (standard) were procured from Becton Difico from a wide range of bacteria in the last few decades Co. (Sparks, MD, USA). The cloning vector pGEM-T, L ee et al. Appl Biol Chem (2021) 64:62 Page 3 of 12 which was utilized for cloning and sequencing the opdC for insecticide quantification was adopted from our pre - gene, was collected from Promega Co. (Madison, WI, viously described methods [3, 18–20]. In an attempt, a (+) (+) USA). The vectors pBluescript II SK and pET32a 50 μL recombinant OpdC enzyme solution was mixed to used for the overexpression and purification of the gene a 700 μL phosphate buffer (200  mM, pH 6.5) and added product were procured from Stratagene (La Jolla, CA, separately with 250 μL OP insecticides (200 mg/L). After USA) and Novagen. Genomic DNA and plasmid isola- that, it was kept at 30 °C for 12 h in an incubation cham- tion were conducted using commercial DNA extraction ber. Next, the solution filtrate (10 μL) was separated and kits (iNtRON Biotechnology, South Korea). The restric - added with methanol (1:1). Then, it was went across 0.45- tion enzymes BamHI and HindIII were purchased from µm PVDF filter and injected into HPLC column (C18, Promega Co. (USA), respectively. 250 × 4.6 mm, 5 μm, Phenomenex, CA, USA). The 10 μL filtered sample was injected in to the HPLC column, and Acid and artificial gastric acid tolerance of L. plantarum the 0.5% acetic acid and methanol (1:4 v/v) were used as WCP931 eluent at 1 mL/min flow rate. The strain WCP931, capable of degrading OP insecti - cides, was isolated from mulkimchi samples and was Cloning of the opdC gene identified as described by Cho et  al. [3]. The 16S rRNA The genomic databases of the L. plantarum strains were gene sequence of the strain was submitted to the NCBI screened to design suitable primers for cloning the opdC and its accession number was provided as FJ480209. gene. For cloning the complete open reading frame of Moreover, the concentrated acid and gastric acid toler- opdC from L. plantarum, WCP931 genomic DNA was ance capability of the strain L. plantarum WCP931 was amplified using 5’-AAA GGA TCC TGA TTG ATC determined according to Lee et al. [21]. TGA CAA TGG G-3’ (sense, BamHI sites are indicated by underline), and 5’-AAA GAA TTC C TT GCT ATA Biodegradation of OP insecticides by L. plantarum WCP931 CTG ATT CGC TAG CC-3’ (antisense, HindIII sites and recombinant E. coli are indicated by underline) primer sequences based on One hundred microliters of the WCP931 strains in MRS the carboxylesterase sequence available in the database. broth culture suspension containing 10   CFU/mL was The purified opdC gene was amplified and ligated with inoculated into 50  mL of 1/25 MRS medium containing pGEM-T easy vector (Promega, USA), and after cloning, 100  mg/L CP. Likewise, the E. coli DH5α was cultured the plasmid (opdC- pGEM-T) was amplified by E. coli under similar conditions as control. E. coli DH5α harbor- DH5α culture and isolated as instructed by the manu- ing the opdC gene (pGCY300) was grown in M9 medium facturer. Afterthat, the plasmid was cut with BamHI supplemented with 100 mg/L CP. Likewise, other organ- and HindIII enzyme, next to the isolated opdC gene was ophosphate such as CP, CS, DF, DZ, EP, MPT, FA, and cloned into the pBluscript II SK vector. The nucleotide PT insecticide mineralizing capabilities of L. plantarum sequence of the opdC was analyzed according to Haque WCP931, E. coli DH5α, and E. coli DH5α carrying opdC et  al. [19]. The GenBank accession number of the opdC gene were evaluated using the above mentioned condi- gene was obtained MT472461. The phylogenetic tree and tions. Culture flasks containing specific insecticides were conserved regions of the OpdC enzyme with related Opd sacrificed after periodic intervals. Thereafter, the insecti - and esterase enzyme sequences were accomplished using cide concentrations and strain growth were determined. DNAMAN10.0 [19]. To ensure the degradation accuracy, the cultures of these strains were run in triplicate [19]. Expression and purification of the OpdC enzyme To overexpress the opdC gene, the PCR product gener- OP degradation assay for L. plantarum WCP931, ated with primers 5’-AAA GGA TCC T GA TTG ATC recombinant E. coli, and OpdC protein TGA CAA TGG G-3’ and 5’-AAAA GA ATT CCT TGC The concentrations of insecticides and their residues in TAT ACT GAT TCG CTA GCC-3’ was cloned into the (+) strain WCP931, recombinant E. coli, and OpdC protein expression vector pET-32a (Novagen, USA), which were determined using thin layer chromatography (TLC) encodes a C-terminal (His) tag within the recombinant (+) and HPLC as described by Cho et  al. [3]. In brief, 4-mL protein. BL21 (DE3) cells harboring pET-32a /OpdC of filtrate was extracted with ethyl acetate from 5-mL ali - were grown at 37  °C to mid-log phase in LB medium quot of culture supernatant. The TLC plate was set up supplemented with 50  μg/mL ampicillin antibiotic. The to analyze the degradation of CP and TCP according to recombinant E. coli cells were centrifuged at 6,000  rpm Islam et al. [19]. All experimental OP insecticide concen- for 10  min to get pellet and later washed with 10  mM trations were determined at 214  nm by HPLC (Perkin- Tris–HCl buffer (pH 7.0). Next, the pellet was resus - Elmer 200 series, CT, USA). The HPLC analysis protocol pended in the same buffer and kept at −20 °C for 30 min. Lee et al. Appl Biol Chem (2021) 64:62 Page 4 of 12 Thereafter, it was mixed with 1 mg of bovine DNase I and Pfu DNA polymerase buffer (20 mM MgSO ), and 2.5 U incubated at 37  °C for 30  min. Triton X-100 was added of Pfu DNA polymerase (Stratagene, CA, USA). The to the suspension at a final concentration of 2.5%. The PCR products were incubated on ice for 5  min, and 1 supernatant of the suspension was collected and stored μL of DpnI restriction enzyme (10 U/μL) was added. immediately at 4  °C. The overexpressed His -tagged Then, the mixture was incubated for 1  h at 37  °C. The OpdC protein was purified using a HisTrap kit (Amer - DpnI-treated plasmids were then transformed into E. shan Pharmacia Biotech). The elution of OpdC protein coli DH5α according to the manufacturer’s specifica - was conducted using 100  mM imidazole with 0.1% Tri- tions. The site-directed mutagenesis procedure was ton X-100. The purity and molecular weight of the OpdC adapted as described by Haque et al. [20]. protein were evaluated by sodium dodecyl sulfate–poly- acrylamide gel electrophoresis. The quantity of OpdC protein in the solution was adjusted to 50  μg/mL and Homology modeling, molecular docking, and visualization used for the activity assay towards OP insecticides. of OpdC enzyme The 3D structure of insecticides degrading model OpdC protein was built in the I-TASSER server [22– pH and temperature effects on OpdC enzymatic activity 24]. The model OpdC protein structure was later sub - The pH and temperature effects of the OpdC enzyme jected to energy minimization using the Swiss-PDB were examined by considering esterase activity. The Viewer. Next, the energy minimization followed by effect of pH (range 3.0–11.0) on the esterase activity of structure validation was conducted using "SAVESv OpdC protein was determined according to the above- 6.0”, which verify 3D models based on several param- mentioned protocol at 30 ± 0.5  °C. While, the tempera- eters such as non-bonded interactions of atoms and the ture effect of the OpdC enzyme was determined at 10 compatibility of the model amino acid sequence, ste- to 70 °C for 1 h. The degree of OP hydrolysis was meas - reochemical properties of the model, etc. Additionally, ured using HPLC. Fifty microliters of enzyme solution “Ramachandran” plot analyses were done for the model were poured into a solution containing 250 μL insecti- OpdC protein as well. The 3D structures of organo - cide (200  mg/L) and 700 μL phosphate buffer (200  mM, phosphate insecticides were traced and assembled from pH 6.5). To calculate the experimental error, assays were the “Pubchem" website. The energy minimization and performed three times. The classical spectrophotomet - optimization of the ligands were conducted using the ric method was used to measure the activity of esterase mmff94 force field and the steepest descent algorithm. provided by the native and mutant OpdC enzymes. The Multiple docking of OpdC protein was performed for rate of hydrolysis of the ρ-NPB (100 mg/L) substrate was tracing out the active sites and catalytic interactions measured in 50  mM sodium phosphate buffer (pH 7.0) using PyRx in Autodoc vina. at 35 ± 0.5 °C using a spectrophotometer at 420 nm. One unit of esterase activity was defined as the amount of enzyme required to release 1 μmol of ρ-NPB per minute under the assay conditions. The assay was conducted in Results triplicate [19]. Identification and gastric juice tolerance ability of L. plantarum WCP931 The 16S rRNA gene similarity of the WCP931 strain with Site‑directed mutation of opdC gene reference LAB was 85.4 to 99.5%. The phylogenetic tree The organophosphorus hydrolase enzyme OpdA, showed that the strain WCP931 was related to Lactoba- OpdE, OpdB, OpdD contains conserved domain G-X- cillus sp. (Additional file  1: Fig. S1). As a consequence, S-X-G [18–20]. According to this database of organo- the chlorpyrifos-degrading WCP931 strain was named as phosphorus hydrolase, the conserved domain of the Lactobacillus plantarum WCP931. The strain’s 16S rRNA OpdC protein was analyzed and identified. In addition, gene sequence was deposited in the NCBI database. The to confirm the location of the catalytic sites in OpdC, accession number of the strain is FJ480209. a site-directed mutagenesis technique was employed to The survival rates of L. plantarum WCP931 under introduce amino acid changes at position 116 (serine acidic and artificial gastric acidic conditions are shown in to alanine) using oligonucleotide primers: 5’-TCT TGC Fig. 1. The CP-degrading strain WCP931 showed moder - CGG GTT TTCG GCTGG CGG CCACG-3’ (sense) and ate survival rates of 86% (acidic condition) and 51% (arti- 5’-CGT GGC CGC CAGC CGA AA ACC CGG CAAGA- ficial gastric acidic condition) at pH 2.0, 95% (acidic) and 3’ (antisense) 5’. The underlined codons were mutated. 84% (artificial gastric acidic) at pH 2.5, and 99% (acidic) The PCR mixture (50  μL) composed by 1 μL pET- and 96% (artificial gastric acidic) at pH 2.5 after 3  h, 32a( +)/opdC DNA (80  ng/μL), 4  μL (10  ρmol) of each respectively. primer, 5  μL (2  mM) dNTP mixture, and 5 μL (10 ×) L ee et al. Appl Biol Chem (2021) 64:62 Page 5 of 12 (A) (A) ab b ab ab cd cd d d cd pH : Incubaon me : 3 h 6 h Incubaon me (day) (B) ab (B) ab c b ab pH : Incubaon me : 3 h 6 h Fig. 1 Survival rates of L. plantarum WCP931 under acidic conditions (A) and survival rates of L. plantarum WCP931 under artificial gastric acidic conditions (B) at pH 2.0, 2.5, and 3.0 after 3 and 6 h Incubaon me (day) of incubation. L. plantarum WCP931 was tested in triplicate for its tolerance in acidified and artificial gastric acidified MRS. Means (C) with different lowercase letters (a–d) indicate significant (p < 0.05) ab differences of survival rate by Duncan’s multiple range test Degradation of OP insecticides by L. plantarum WCP931 The cell growth response and degradation pattern f f are shown in Fig.  2. The L. plantarum WCP931 grew markedly until the 1st day (OD 0.85), slightly declined at 2  days, and gradually decreased at 6  days (OD 0.94) OP inseccides during incubation. However, E. coli DH5α did not grow Fig. 2 Cell growth response of L. plantarum WCP931 and E. coli DH5α in the presence of CP 100 mg/L (Fig.  2A). The L. plan - in 1/25 MRS and M9 medium, respectively, containing 100 mg/L tarum WCP931 exhibited an initial rapid degradation CP after 9 days (A), change of CP concentrations of L. plantarum of CP of approximately 66 mg/L during the first 3 days WCP931 and E. coli DH5α in 1/25 MRS and M9 medium, respectively, of incubation and then exhibited a maximum degrada- containing 100 mg/L CP after 9 days (B), insecticide degradation tion of 86  mg/L at 9  days of incubation. On the other pattern of L. plantarum WCP931 in 1/25 MRS containing 100 mg/L insecticides after 9 days (C). Means with different lowercase letters hand, in the case of E. coli DH5α, it slightly decreased (a–f ) indicate significant (p < 0.05) differences in survival rate by to 83  mg/L on the 9  days (Fig.  2B). The L. plantarum Duncan’s multiple range test. Names of insecticides on the X-axis WCP931 was able to degrade CP to DEPT and TCP and of the figure are abbreviated as follows: OP, organophosphorus; utilized DETP as the sole source of carbon and phos- CP, chlorpyrifos; CS, cadusafos; CM, comnaphos; DZ, diazinon; DF, phorus. All OP insecticides tested in the cross-feeding dyfonate; EP, ethoprophos; FA, fenamiphos; MPT, methylparathion; and PT, parathion experiment were degraded by L. plantarum WCP931. All OP insecticides tested, such as CP, CM, DZ, MPT, and PT, had DEPT side chains, while CS, DF, EP, and from 72 to 88% for the CP, CM, DZ, MPT, and PT FA had no DEPT. Except for DF, eight other OP insecti- insecticides, respectively. cides (including CS, CP, CM, DZ, EP, FA, MPT, and PT) were hydrolyzed at a phosphoester bond by L. plan- tarum WCP931. However, a decreased degradation rate Sequence analysis of the opdC gene and the OpdC protein of OP insecticides was observed, as shown in Fig.  2C. PCR amplification of the total DNA from L. plantarum In particular, on the 9 days, degradation was enhanced WCP931 with specific primers produced an amplification Survival rate (%) Survival rate (%) OP inseccides Microbial growth CP inseccide concentraon (mg/L) concentraon (mg/L) (OD ) 600 nm Lee et al. Appl Biol Chem (2021) 64:62 Page 6 of 12 product of approximately 1.5  kb. After sequencing, a (opdC) are shown in Fig.  4. CP and TCP with R values nucleotide sequence 1500  bp in length was found in the of 0.57 and 0.66, respectively, were detected in sam- open reading frame (ORF) of opdC. Its ORF started with ples drawn at 0, 1, 3, 6, and 9  days (Fig.  4A). The clone an ATG start codon and ended with a TAA Ochre stop decomposed CP markedly until 2  days (78  mg/L), then codon (Additional file  2: Fig. S2). The opdC gene product decreased rapidly at 6  days (24  mg/L), and subsequently is predicted to contain 276 amino acids with a molecu- grew slowly until 9  days during incubation. At 3  days, lar mass of 31  kDa (http:// web. expasy. org/ compu te_ the clone exhibited a gradual increase in TCP concentra- pi/). Analysis of the amino acid sequence with the pro- tion to approximately 32  mg/L at 3  days. After that, the gram PSORT (http:// www. cbs. dtu. dk/ servi ces/ Signa lP/) TCP concentration was increased to 68  mg/L at 6  days revealed no potential signal sequences. The calculated pI (Fig. 4B). Nine OP insecticides (CS, CP, CM, DZ, EP, FA, of OpdC was 5.18. MPT, and PT) were mineralized by recombinant E. coli The amino acid sequence GFSAG, starting at residue with the opdC gene. The recombinant cells exhibited 46 116 for OpdC (Additional file  2: Fig. S2 and Fig.  3), fits to 90% degradation of CP, CM, DZ, FA, MPT, and PT at the Gly-X-Ser-X-Gly motif found in most bacterial and 37 °C for 9 days (Fig. 4C). eukaryotic serine hydrolases, such as lipase, esterase, and serine proteinase, as well as in β-lactamase [25–27]. A Purification and characterization of the OpdC protein phylogenetic tree containing the esterolytic and lipolytic The OpdC protein was purified from E. coli BL21 (DE3) proteins showed that the OpdC enzyme did not belong overproducing OpdC using column filtration techniques. to groups I, II, III or IV (Fig. 3). This separation of OpdC Protein fractions were analyzed by SDS-PAGE, and one suggested a new type of esterase. protein band (31  kDa) was present after the final purifi - cation step (Fig.  5A). The ability of OpdC to hydrolyze Degradation of CP in liquid culture by E. coli harboring ρ-NPB was determined at 30 ± 0.5  °C with various buff - opdC gene ers ranging from pH 3 to 11. The maximum activity was To confirm the insecticide degradation function of the observed at pH 6 (Fig.  5B). The optimal hydrolysis tem - opdC gene, the gene was cloned into E. coli DH5α cells. perature was determined at pH 6 by measuring the activ- The degradation patterns of CP by the clone pGCY300 ity across a temperature range. The maximum activity 0.05 Lbr -OpdB Lpl -OpdC LAB- Opd Lsa -OpdD group Lme -OpdA Lme -OpdE A. thaliana (AAB84335) A. azollae (AF035558) Est/Lip H. sapiens (NP_001975) Group I E. coli K12 (AAC73458) S. cerevisiae S288C (CAA84054) L. laccs MG1363 (AAF02201) Est/Lip Group II L. laccs MG1363 (AF157601) S. albus (AAA53485) New Est/Lip S. colicolor (NP_625018) group Uncultured bacterium (AF223645) S. aureus (AAA26633) Est/Lip S. epidermidis (AF090142) Group IV L. casei CL96 (AY251019) G. stearothermophilus P1 (AF237623) Est/Lip G. thermocatenulatus DSM730 (CAA64621) Group III D. radiodurans R1 (AAF09912) Fig. 3 Phylogenetic tree showing the evolutionary relatedness and levels of homology between the esterolytic and lipolytic enzyme amino acid sequences and the alignment of the conserved regions found in the primary esterolytic and lipolytic enzymes. The esterolytic groups are classified according to the catalytic conserved domain G-X-S-X-G-G of the protein sequence L ee et al. Appl Biol Chem (2021) 64:62 Page 7 of 12 G-F-S116-A-G of OpdC. To determine whether Ser116 S 0 1 3 6 9 (A) was involved in catalytic esterase action, it was replaced CP by site-direct mutagenesis, and the mutant proteins were expressed in E. coli and purified. The purified OpdC TCP enzyme showed 78% degradation, while the mutant OpdC had no enzymatic activity towards ρ-NPB and CP (B) (Table 1). ab Five different 3D models of the OpdC protein were b a built and provided by the Iterative Threading ASSEmbly Refinement (I-TASSER) server. According to the best scoring value, model 1 of OpdC was chosen for analysis, as shown in Fig.  6. The OpdC 3D model protein showed a C-score of 0.87, a TM-score of 0.83 ± 0.08, an RMSD of 4.2 ± 2.8, and a cluster density of 0.7167. The 3D model of OpdC showed eight α-helices, eight β-sheets, two ran- Incubaon me (day) dom coils (η), and six different hydrogen-bonded turns (T) in the whole structure (Fig.  6A). In particular, the (C) G-F-S-A-G motif for OpdC was found in the β5 and α3 helices of the predicted structure from the N-terminus (Fig.  6A).  In ERRAT server, a model is evaluated based on non-bonded interactions between different types of atom to assess error rate with the standard optimized model, while in Verify 3D the 3D to 1D comparisons are made based on surrounding environment and locations of the α-helix, β-sheets, loops, etc. The protein model was fine-tuned using loop refining and energy minimi - OP inseccides zation. The loop refined and energy minimized OpdC Fig. 4 TLC profile (A) and changes of CP and TCP concentration model protein showed an overall quality factor 80.22% (B) and changes of OP insecticides concentrations (C) by the and verify 3D score 93.84%. The Ramachandran plot recombinant E. coli with opdC gene growing in the M9 medium analysis for the OpdC protein showed that 92.1%, 7.5%, containing 100 mg/L of CP and OP insecticides after 9 days, respectively. Means with different lowercase letters (a–f ) indicate 0.0%, and 0.4% amino acid residues are centered in the significant (p < 0.05) differences of survival rate by Duncan’s multiple most favorable regions, additional allowed regions, gen- range test. Names of insecticides as the X-axis of the figure shows erously allowed region, and in the disallowed regions, the abbreviations as follow: OP, organophosphorus; CS, cadusafos; respectively (Fig. 6B). Thus, the Ramachandran plot anal - CP, chlorpyrifos; CM, comnaphos; DZ, diazinon; DF, dyfonate; EP, ysis results for the OpdC protein substantiate the quality ethoprophos; FA, fenamiphos; MPT, methylparathion; PT, parathion; and TCP, 3,5,6-trichloro-2-pyridinol of the model. Using the COACH Meta server, the highest poten- tial ligand-binding sites were identified and observed at Gly42, Gly43, Gly44, Phe115, Ser116, Ala117, and was observed at 35  °C (Fig.  5C). Nine OP insecticides Val156 for cluster 1, but they were recorded at Asp201, were decomposed by the OpdC enzyme (Fig. 5D). Except Glu202, Ser203, Ile232, and His233 for cluster 2. Based for DF, all OP insecticides are hydrolyzed at a phosphoe- on the molecular docking of the OpdC protein with ster bond by the OpdC protein. In particular, the relative chlorpyrifos (Fig.  6C), the critical amino acids (Ser116, activity of the enzyme was higher towards CP, CM, DZ, Asp201, His233, Glu52) of the catalytic triad were pre- FA, MPT, and PT insecticides than towards CS, DF, and sent in an area of 5  Å from chlorpyrifos. Interestingly, EP insecticides. the distance of the P-atom of the phosphodiester of CH and the O-atom of the hydroxyl group was meas- ured to be 3.3  Å, which may initiate the nucleophilic Identification of residues essential for enzymatic activity attack on the P-atom by the O-atom and might liber- of the OpdC protein ate TCP. Consequently, the P-tom of the phosphodi- Most lipases and carboxyl esterases have the consensus ester of CP might be attacked by the O-atom of water, sequence motif Gly-X-Ser-X-Gly with the serine active resulting in further degradation of the nontoxic residue site. Analysis of the deduced amino acid sequences DEPT and the return of Ser116 to its original state. In showed a potential serine hydrolase motif, such as CP concentraon OP inseccides (mg/L) concentraon (mg/L) TCP concentraon (mg/L) Lee et al. Appl Biol Chem (2021) 64:62 Page 8 of 12 (A) 1 2 3 4 (B) 116.0 66.2 ab 45.0 35.0 31 kDa 25.0 d 18.4 14.4 (kDa) pH (C) (D) ab bc f f Temperature ( C) OP inseccides Fig. 5 Electrophoretic analysis of the purified OpdC protein (A). Separation was performed on a 12.5% (w/v) SDS polyacrylamide gel and after was stained with 0.025% Coomassie blue R-250. Lane 1, standard marker; lane 2, crude extract from E. coli BL21 (DE3) containing pET-32( +)/opdC; lane 3, crude extract from IPTG-induced E. coli BL21 (DE3) containing pET-32( +)/opdC; lane 4, purified OpdC protein from Hi-Trap kit (Amersham). pH effect on the relative activity of OpdC (B). The esterase activity of OpdC was assayed using ρ- NPB as substrate at different pH values at 30 ± 0.5 °C for 1 h. Eec ff t of temperature on the relative activity of OpdC (C). The esterase activity of OpdC was assayed using ρ- NPB as substrate at different temperature values at pH 6 for 1 h. Substrate relative activities of OpdC on the various OP insecticides (D). The OP hydrolase activity of OpdC was assayed using as substrate with 200 mg/L OP in insecticides at 35 ± 0.5 °C and pH 6.0 for 12 h. Names of insecticides as the X-axis of the figure shows the abbreviations as follow: OP, organophosphorus; CS, cadusafos; CP, chlorpyrifos; CM, comnaphos; DZ, diazinon; DF, dyfonate; EP, ethoprophos; FA, fenamiphos; MPT, methylparathion; and PT, parathion. Means with different lowercase letters (a-f ) indicate significant (p < 0.05) differences of survival rate by Duncan’s multiple range test this circumstance, the OpdC showing higher relative Table 1 Esterase and orangophosphorus (OP) hydrolase activity towards MPT and PT, which docked complex, activities for the hydrolysis of ρ-nitrophenyl butyrate (ρ-NPB) and was visualized to get the catalytic insights, as seen in chlorpyrifos (CP) by the OpdC and mutant OpdC enzyme Fig. 6D and E ProteinsEsterase activity (U/mg)/ b Figures  6D and E demonstrate the direct interac- CP degradation degree (%) tion of Ser116 and His233 with MPT and PT. Besides, Glu52 is closely positioned near the Ligands (MPT, OpdC 397 ± 15.88 /78 PT). Therefore, the predicted homology model of OpdCM < 0.01/2 OpdC revealed that the active site of this enzyme a / Esterase activity is indicated the micromoles of ρ-NPB hydrolyzed min mg. The was located in the known architecture of the hydro- OpdC and OpdCM activities were assayed with ρ-NPB as substrate at pH 6 and lases. As seen in Fig. 6D, the OpdC protein abundantly 35 ± 0.5 °C for 1 h in constant temperature incubator, respectively interacts with methyl-parathion through multiple The OpdC and OpdCM activities were assayed with CP as substrate at pH 6 and 35 ± 0.5 °C for 12 h in constant temperature incubator, respectively amino acid residues. In fact, the O-atom of methyl- Values indicate the means of three replications. The standard errors were within parathion is attacked by Ser116, His233, Gly44, and 5% of the mean Ser203, Glu52 attacks P-atom via attractive charge. Relave acvity (%) Relave acvity (%) Relave acvity (%) L ee et al. Appl Biol Chem (2021) 64:62 Page 9 of 12 (B) (C) (A) (D) (E) Fig. 6 3D modeling of OpdC protein. The model was built using I-TASSER server (A). The α-helix, β-sheet, and random coil (η) are marked with red, yellow, and light blue color. The critical amino acid Ser116 side chains are marked with magenta color, while His233 and Glu52 are marked with yellow color. Ramacahndran plot analysis of the OpdC protein (B). Molecular docking model of the OpdC protein with chlorpyrifos (CP) (C). The critical amino acid (Ser116, His233, Glu 52) side chains within 5 Å of chlorpyrifos are marked in yellow. Visualization of ligand binding sites of OpdC within 5 Å of methyl parathion (D) and parathion (E) The Ser116, His233, Arg51, and Gly44 provide a con- Discussion ventional hydrogen bond with the O-atom of the To date, three different classes of organophosphorus ligand molecule. Notably, the Ser203 residues provided hydrolase genes namely, opd, mpd, and ophc2, had been carbon-hydrogen bond interaction with O-atom, while discovered in the last decades. Among these organo- Ser49 provide unfavorable acceptor-acceptor interac- phosphorus hydrolases, the opd gene is extensively dis- tion with O-atom of the ligand molecule, respectively. tributed, especially it is sourced from different bacterial Dramatically, the OpdC protein formed a possible cat- species [15–20]. The reported opd genes belonged to alytic triad in the binding pocket region with residues the chromosome [12, 15] or the plasmid [26] of the iso- Ser116, His233, Glu52. lated strains. Yet several classes of esterase are revealed; The OpdC protein-parathion docked complex among them, some are capable of degrading OP insecti- showed multiple residues interactions (Fig. 6E). In par- cides. However, the classes of organophosphorus hydro- ticular, the conventional hydrogen bond interaction lase are not much reported. Recently, we have reported was observed for Ser116 and Arg51 with the O-atom of organophosphorus hydrolase gene opdA, opdB, opdD, parathion. At the same time, His233 formed π- π-alkyl opdE from Kimchi originated Lactobacillus species bonds with a benzene ring and O- atom of parathion strains, which are deviated from the common ester- and Glu52, which contribute an attractive charge to ase groups. Exploring new esterase having evolutionary the parathion. Consequently, the possible catalytic history with OP insecticides degrading activity might triad was supposed to be made as Ser116, His233, increase the diversity of organophosphorus hydrolase Glu52. According to docking analyses, these two pro- in nature. Therefore, the present study was focused on teins, catalytically crucial residues, are placed within another new specific organophosphorus hydrolase that 1.5–5.5 Å that might participate in the biodegradation could degrade a range of OP insecticide, which might of organophosphates like MPT, PT, CP, and others. derive the strain L. plantarum WCP931 in insecticides Lee et al. Appl Biol Chem (2021) 64:62 Page 10 of 12 bioremediation during kimchi fermentation. Therefore, neutral and alkaline soils [30, 31]. Importantly, the opti- the cloned and functionally expressed chromosome- mum temperature (35 °C) for OpdC varied slightly from based opdC gene increases the diversity of the hosts of that observed for the OpdB protein of L. brevis WCP902 OP hydrolases. Because of the lack of a signal sequence (40 °C) [18] but was higher than that recorded for OpdD in the N-terminal region of OpdC, it is assumed that the of L. sakei WCP904 (30 °C) [19]. E. coli cells expressing OpdC protein might degrade CP The OpdC protein contains Gly-X-Ser-X-Gly conserved in the intracellular environment. Therefore, hydrolysis domain and a catalytic site comprised of serine residues, could take place inside the cell, followed by the release which are routinely appeared in bacterial and eukaryotic of the hydrolysis product into the culture medium. How- serine hydrolases, e.g., serine proteinases, lipases, ester- ever, products in the culture medium do not rule out the ases as well as in β-lactamases [25, 35, 36]. However, the possibility that hydrolysis takes place inside the cell. The phylogenetic tree analysis of the OpdC protein showed nonclassical secreted proteins often seem to have both that it did not belong to the known families of esterolytic cytoplasmic and extracellular functions [27]. Moreover, and lipolytic proteins (groups I, II, III, IV or even a new several carbohydrate- and protein-degrading enzymes group of soil metagenomes), indicating the existence of were identified as extracellular despite the lack of extra - a new LAB esterase/opdase group, represented by OpdC cellular signal peptides [28, 29]. Organophosphate insec- (Fig.  3). Importantly, our previously reported OpdB and ticides with residues were extracted from recombinant OpdD enzymes showed the Gly-X-Ser-X-Gly motif and E. coli harboring OpdC culture medium, indicating that catalytic active site of serine residues. Therefore, a con - the OpdC enzymes use a nonclassical pathway to exhibit temporary LAB-opd esterase can be brought forward in extracellular activities. Generally, OP is hydrophobic in the present study, consisting of OP hydrolase genes from nature; thus, compounds in the culture medium are in LAB strains isolated during kimchi fermentation. equilibrium with compounds inside bacterial cells. As seen in Fig.  3, α-helices, β sheets, random coils, Interestingly, the OpdC enzyme hydrolyzed a range of and β turns were observed in both structures of OpdC OP insecticides containing a P–O bond and a P–S bond, enzymes and were matched with the catalytic motif G-X- indicating that recombinant OpdC has broad substrate S-X-G of OpdC and OpdD enzymes [18, 19]. When the specificity. This finding showed similarities with some Ser116 residue was replaced by Ala, the mutant OpdC previous reports [19, 27, 30, 31]. However, the enzyme enzymes had no enzymatic activity towards ρ-NPB and relative activity against the P–O bond consisting of CP. In 3D modeling, the quality of the 3D model made insecticides was much higher than that against the P–S in I-TASSER is predicted by the confidence score, i.e., bond, which is consistent with the previously reported C-score [22–24]. The C-score is generated according to OpdB and OpdD enzymes [18, 19]. However, minor vari- the significance of threading template alignments and ations in relative substrate activities were observed for the convergence parameters of the structure assembly OpdC compared with those reported for OpdB, OpdA, simulations. In fact, it should be ranged of −5 maxi- and OpdE enzymes. Thus, the OpdC hydrolysis activity mum, where a higher value of C-score indicates a model depends on the molecular structure of insecticides used with increased confidence and vice versa [22–24]. In this in this study. Temperature influenced the OpdC activ - study, the CS scores ranged between −1.64 and −5.0 for ity. The optimum pH values of OpdD (6.0) from L. sakei all other four models except 3D model 1. Since model 1 WCP904 [19] and OpdB from L. brevis WCP902 (6.0) has a positive (+ 0.87) CS score, we chose model 1 for [18] were less than that of OpdB from Pesudomonas sp. analyses and docking. BP3 (8.0) [32]. The L. plantarum is known to be adapted The nearest homologs carboxylesterase Cest-2923 to stressful environments such as those in the gastro- (PDB ID: 4BZW) of Lactobacillus plantarum WCFS- intestinal tract with a low pH or a high salt content. To 1’s nucleophile Ser116 was located in the nucleophile survive in acidic environments, this bacterium uses elbow, with its backbone angles residing in an unfavora- F F -ATPase and sodium-proton pumps to regulate and ble region in Ramachandran plot (ɸ = 52º, ψ = −180º) o 1 maintain the intracellular pH [33]. Kimchi fermenta- [37]. Alike, the residue Ser116 of OpdC protein is found tion involving LAB is conducted at acidic pH, in which in the unfavorable in Ramachandran plot. In fact, its L. plantarum is quite predominant and is responsible for catalytic triad (Ser116-His233-Asp201/Glu52) was made acidifying kimchi. Therefore, the OpdC highest activity in a canonical site of the OpdC protein sequence, which observed at pH 5–6 (acidic) is quite logical. In fact, OP also consistent with the homologs 4BZW protein. Thus, insecticides are immovable in pH 5–7, but it is easily Ramachandran plot analysis validated the model OpdC decomposed in alkaline pH [34]. Therefore, acidic soils protein structure. are more preferable to slower CP degradation than the L ee et al. Appl Biol Chem (2021) 64:62 Page 11 of 12 Similar to CP, MPT, PT, very similar docking results Additional file 1: Figure S1. Phylogenetic relationships of L. plantarum and catalytic interactions were observed for the other WCP931 and other LAB closely related bacterial based on 16S rRNA sequence. Number above each node is confidence levels (%) generated insecticides and ρ-NPB evaluated in this study (data from 1000 bootrap trees. The scale bar is in fixed nucleotide substitutions not shown). These results suggested that Ser116 might per sequences position. be the crucial amino acid for the degradation of CP and Additional file 2: Figure S2. Nucleotide and deduced amino acid other insecticides evaluated in this study. Our previous sequences of opdC gene from L. plantarum WCP931. Bold letters and underlines the start codon and serine residue. The stop codon is indicated studies have reported the role of serine in insecticide by asterisk. The consensus sequences region is indicated by yellow box. degradation [19, 20]. In addition, His233 and Glu52 of the catalytic triad were located within 5  Å of the CP Acknowledgements molecule, indicating that His233 and Glu52 might also Not applicable. be involved in the degradation of CP. The amino acid 10 154 157 residues Ser, Asp , and His of thioesterase I/pro- Authors’ contributions KMC conceived and designed the experiments. KMC, MAH, and JHL tease of E. coli are appeared in the catalytic site [38]. interpreted the data and wrote the manuscript. HYL, DYC, MJK, JGJ, and EHJ 156 281 Moreover, Ser and His residues of a novel chlor- performed the experiments and analyzed the data. MAH performed and pyrifos hydrolase was reported to be participated in interpreted Bioinformatics analyses. All authors read and approved the final manuscript. the chlorpyrifos degradation [39]. Notably, the nucleo- phile Ser, a general bases His/Arg, and an acid Glu/Asp Funding residues are apparent in the OpdC catalytic site, which This work was supported by the research invigoration program of 2020 Gyeo- ngnam National University of Science and Technology. might forming an oxyanion hole. The predicted struc - ture of OpdC protein partially shares esterase along Availability of data and materials with a new LAB-Opd hydrolase structure. As a result, The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. the classification of esterase is expanded into the LAB- Opd group [18, 19], where OpdCs are included in this Declarations study. To date, this study reports a new organophos- phorus hydrolase (OpdC) enzyme from the kimchi Consent for publication originated L. plantarum strain that can degrade nine This research article entitled as “Biodegradable properties of organophospho- rus insecticides by the potential probiotic Lactobacillus plantarum WCP931 insecticides containing P–O and P–S bonds as well as with a degrading gene (opdC)” an original work was carried out by authors: All unmask its potential catalytic insights by site-directed authors approve of its submission to as Applied Biological Chemistry. It is not mutation and molecular docking. under consideration by another journal at the same time as Applied Biological Chemistry. I am the author responsible for the submission of this article and In conclusions, the recombinant OpdC enzyme dem- I accept the conditions of submission and the Springer Open Copyright and onstrated robust degradation of OP insecticides such as License Agreement. MT, CP, CM, DZ, and PT. To date, the OpdC enzyme Competing interests sequence deviates from the common families of ester- As a corresponding author, I confirm that I have read Springer Open’s guid- ase and lipase proteins available. As a result, OpdC is ance on competing interests and have included a statement indicating that suggested to be a novel protein. Importantly, the cata- none of the authors have any competing interests in the paper. lytic action of the OpdC enzyme seemed to be per- Author details formed by the serine (116) amino acid. With regard Department of Life Resources Industry, Dong-A University, Busan 49315, to the safety of insecticides in kimchi, it is assumed Republic of Korea. Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea. Depar t- that fermented kimchi meets the minimal residue cri- ment of Biochemistry and Molecular Biology, Hajee Mohammad Danesh teria for food safety due to OP degradation by kimchi Science & Technology University, Dinajpur 5200, Bangladesh. fermentation driving lactic acid bacteria, including L. Received: 17 June 2021 Accepted: 8 August 2021 plantarum. The present study suggested that the opdC gene in L. plantarum WCP931 along with opdB in L. bevis WCP902 and opdD in L. sakei WCP904 play roles in the degradation of OP insecticides during kimchi fer- References mentation. Hence, L. plantarum WCP931 establish as 1. Huete-Soto A, Castillo-González H, Masís-Mora M, Chin-Pampillo JS, Rod- a worthy candidate for the potential bioremediation of ríguez-Rodríguez CE (2017) Eec ff ts of oxytetracycline on the performance and activity of biomixtures: removal of herbicides and mineralization of environments contaminated with organophosphorus chlorpyrifos. J Hazard Mater 321:1–8 insecticides. 2. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Organophosphorus insecticides; Kimchi; Lactobacillus plantarum WCP931; OpdC gene; Biodegradation

References