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Keywords:

  • Dioxins;
  • Microbial degradation;
  • Fluorescent assay;
  • Reductive cleavage;
  • Diaryl ether bonds

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The degradation of 2-chloro-4,5-O-(4′-methyl-7′, 8′-diphenyl)ether (CMDPE), an analog of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), mediated by Geobacillus sp. UZO 3 cell-free extract was monitored. Ethyl acetate extracts of a complete reaction mixture incubated at 65°C for 18 h were analyzed either by thin layer chromatography (TLC) fractionation coupled with spectrometric detection or by gas chromatography–mass spectrometry (GC-MS). The reaction product 4-methylumbelliferone (4MU) was successfully isolated by TLC and visualized by a transilluminator at 450 nm. The 4MU, 4-chlorophenol, and reaction intermediate 6-chlorophenoxy-4-methylumbelliferone were all successfully detected by GC-MS. The presence of these compounds suggest that Geobacillus sp. UZO 3 cell-free extract also catalyzes the reductive cleavage of the diaryl ether bonds of CMDPE in a similar mechanism previously reported in 2,7-DCDD. In the present study, the authors describe a simple and highly sensitive fluorescent assay for a new dioxin degrading enzyme(s). Environ. Toxicol. Chem. 2012; 31: 1072–1075. © 2012 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans continue to pose a serious threat to the environment. To date, manufacturing herbicides and pesticides and incinerating chlorinated compounds have produced a total of 75 and 135 isomers of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, respectively. These chemically stable pollutants also present health hazards as they have been found to taint the food chain 1, 2.

In recent years, attention has been directed toward developing technology to clean up these environmental pollutants by bioremediation 3–12. Rapid advances have also been made in the analysis of dioxins in the field of environmental biotechnology. However, these analytical methods have found limited applicability in more thorough studies on dioxin degradation because of the complex extraction operations they involve, which often results in low recovery rates.

We have previously demonstrated that a cell-free extract prepared from cultured cells of Geobacillus sp. UZO 3 reductively cleaves the diaryl ether bonds of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), producing 4′,5-dichloro-2-hydroxydiphenyl ether (DCDE) as an intermediate reaction product and 4-chlorophenol (4CP) as the final reaction product (Fig. 1) 13. The detection of DCDE implicated the discovery of an unprecedented dioxin degradation enzyme that reductively cleaves the diaryl ether bonds of 2,7-DCDD similar to glutathione S-transferase, a reduction cleavage enzyme 14–17. The aim of the present study is to develop a highly sensitive fluorescent assay for the new dioxin degrading enzyme(s) based on the synthesis of a fluorescent analog of 2,7-DCDD.

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Figure 1. Proposed degradation pathway for 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) and 2-chloro-4,5-O-(4′-methyl-7′, 8′-diphenyl)ether (CMDPE) by the Geobacillus sp. UZO 3 cell-free extract. DCDE = 4′,5-dichloro-2-hydroxydiphenyl ether; CPMU = 6-chlorophenoxy-4-methylumbelliferone; 4CP = 4-chlorophenol; 4MU = 4-methylumbelliferone.

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In the present study, 2-chloro-4,5-O-(4′-methyl-7′, 8′-diphenyl)ether (CMDPE), an analog of 2,7-DCDD, was synthesized and used as the substrate. The CMDPE consists of a monochlorophenol and a 4-methylumbelliferone (4MU) structural component. We hypothesize that the enzyme(s) from the cell-free extract of Geobacillus sp. UZO 3 reductively cleaves the diaryl ether bond of CMDPE to produce the intermediate 6-chlorophenoxy-4-methylumbelliferone (CPMU) and the final product 4MU, which strongly fluoresces at 450 nm (Fig. 1). Using 4MU as analyte will allow for the detection of compounds at concentrations as low as 10 ppb in a conventional spectrofluorometer, and even at a lower concentration of 1 ppt with thin layer chromatography (TLC) 14–18. Current techniques that use chlorophenol as analyte in a conventional gas chromatography–mass spectrometry (GC-MS) setup only has a limit of detection of 100 ppt.

This study presents evidence that the enzyme(s) that reductively cleave the diaryl ether bond of chlorinated dioxin in the cell-free extract of Geobacillus sp. UZO 3 also reductively cleave the diaryl ether bonds of CMDPE. A highly sensitive and simple new assay of chlorinated dioxin degradation activity is hereby described.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Chemicals

Mono-chlorophenol (2-,3-,4-chlorophenol), Cu(OAc)2, CH2Cl2, CDCl3, tetramethylsilane, and pyridine were purchased from Wako Pure Chemical. N,O-Bis(trimethyl silyl)trifluoroacetamide, 4MU, 4-methylesculetin, and 2,4-dichloronitrobenzene were purchased from Tokyo Kasei. Potassium hydride, 4-chlorophenylboronic acid, and hexamethylphosphoric triamide were purchased from Sigma-Aldrich. Silica gel 60 F254 (20 × 20 cm, thickness 0.25 mm) TLC plates were purchased from Merck. Purities of these chemicals ranged from 96.6 to 100%. All other chemicals used were of analytical grade and of the highest purity available.

Synthesis of CMDPE

The CMDPE was synthesized from 2,4-dichloronitrobenzene and 4-methylescretin according to the procedure of Lee and Denny 19. Briefly, a flask was charged with 2,4-dichloronitrobenzene (3.13 mmol), 4-methylescretin (6.26 mmol), and potassium hydride (220 mmol) in dry hexamethylphosphoric triamide (50 ml) under a slow stream of dry Ar. After stirring (2 h, 135°C), the cooled mixture was poured over ice to decompose the residual potassium hydride and then acidified with 0.5 M HCl. The mixture was extracted with ethyl acetate (50 ml), and the organic layer was washed sequentially with 0.5 M HCl and water. The diaryl ether was purified by silica gel chromatography (chloroform:ethyl acetate:formic acid, 9:8:1, v/v) and analyzed by GC-MS and nuclear magnetic resonance (NMR). The yield of this reaction was 30%.

Synthesis of CPMU

The CPMU was synthesized from 4-methylescretin and 4-chlorophenylboronic acid according to the procedure of Evans et al. 20. Briefly, a flask was charged with 4-methylescretin (1.0 equiv), Cu(OAc)2 (1.0 equiv), 4-chlorophenylbolonic acid (1.0–3.0 equiv), and powdered molecular sieves. The reaction mixture was diluted with CH2Cl2, and pyridine as the amino base (5.0 equiv) was added. After stirring (24 h, 25°C) the colored heterogeneous reaction mixture under ambient atmosphere, the resulting slurry was filtered and washed with distilled water, and the organic layer was collected. The diaryl ether was purified by silica gel chromatography (chloroform:hexane:ethyl acetate, 8:4:1, v/v) and analyzed by GC-MS and NMR. The yield of this reaction was only 25%.

Culture condition and extracts

The preparation for cell-free extract of Geobacillus sp. UZO 3 was prepared as previously described by Suzuki et al. 13.

Enzymatic reaction assays

The assay was performed as described previously by Suzuki et al. 13. Ethyl acetate extracts (10 µl) from the reaction mixture were fractionated by TLC on the silica gel 60 F254 using chloroform:ethyl acetate:formic acid (10:8:1,v/v) as the developing solvent.

Analytical methods

Gas chromatography–mass spectrometry and NMR analyses were performed as described previously by Suzuki et al. 13. Fluorescent spots on TLC were visualized by ultraviolet irradiation (excitation at 360 nm) using a transilluminator. Authentic CMDPE, CPMU, and 4MU were used as standards.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Synthesis of CMDPE and CPMU

Both CMDPE and CPMU were synthesized using the methods of Lee and Denny 19 and Evans et al. 20, respectively. Analysis by GC-MS shows that the synthesized compound has a molecular ion peak of m/z = 300 (Fig. 2A,G) and m/z = 374 (Fig. 2B,H) that corresponds to the estimated molecular weights of CMDPE and CPMU, respectively. Both 1H-NMR and 13C-NMR analyses have confirmed consistently the identity of these compounds (data not shown).

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Figure 2. Gas chromatography–mass spectrometry analysis of the reaction milieu for 2-chloro-4,5-O-(4′-methyl-7′, 8′-diphenyl)ether (CMDPE) degradation mediated by Geobacillus sp. UZO 3 cell-free extract. The selective ion monitoring (SIM) chromatogram of the detected substrate CMDPE at m/z = 300 (C) was compared to that of the authentic compound (A) and its corresponding MS spectrum (G). The SIM chromatograms of the detected intermediate 6-chlorophenoxy-4-methylumbelliferone (CPMU) at m/z = 374 (D), 4-methylumbelliferone (4MU) at m/z = 248 (E), and 4-chlorophe (4CP) at m/z = 200 (F) and their corresponding MS spectrum (Trimethysilyl derivative) (I, J, and K, respectively). D and I were compared to authentic, CPMU (Trimethysilyl derivative) (B and H).

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Reductive degradation of CMDPE

The ethyl acetate extract of the complete reaction mixture containing CMDPE and Geobacillus sp. UZO 3 cell-free extract showed strong fluorescence at 450 nm. Based on the authentic preparation results, the compound was speculated to be 4MU (Fig. 3). Consistently, 4MU and 4CP were detected by GC-MS from the same extract (Fig. 2E,F,J,K). These compounds were not detected in reaction mixtures that contained the cell-free extract (control 1) or CMDPE (control 2) alone. Apparently, the enzyme(s) contained in Geobacillus sp. UZO 3 cell-free extract mediated the reductive cleavage of the diaryl ether bonds of CMDPE to produce 4MU in a similar mechanism, as we have previously described for 2,7-DCDD.

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Figure 3. Thin layer chromatography analysis of the reaction milieu for 2-chloro-4,5-O-(4′-methyl-7′, 8′-diphenyl)ether (CMDPE) degradation mediated by Geobacillus sp. UZO 3 cell-free extract. Lanes 1, 2, and 3 are authentic compound, CMDPE, 6-chlorophenoxy-4-methylumbelliferone (CPMU), and 4-methylumbelliferone (4MU), respectively; lane 4 is the control culture containing no cell-free extract; lane 5 is the reaction milieu.

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Conclusive CPMU identification

Thin layer chromatography and GC-MS were used to detect the production of CPMU as intermediate in the degradation of CMDPE in Geobacillus sp. UZO 3 cell-free extract (Figs. 2 and 3). In the conditions employed in the present experiment, TLC alone could not detect CPMU production, which may be attributed to the nearly similar behavior of this compound that has a partially cleaved diaryl ether bond with CMDPE.

These results provide strong evidence that Geobacillus sp. UZO 3 cell-free extract reductively cleaves the diaryl ether bonds of CMDPE in a stepwise fashion to produce 4MU through the intermediate CPMU. Because this stepwise mechanism is reminiscent of the two-step degradation reaction we previously reported 13, the degradation of both 2,7-DCDD and CMDPE may be catalyzed by the same enzyme(s) in the cell-free extract. This, however, requires further investigation because other enzymes in the extract may possibly be involved in the degradation of chlorinated dioxin in the cell-free extract.

We hereby described a simple and sensitive quantitative assay to detect and monitor enzymatic activity for the degradation of chlorinated dioxin. This method presents a powerful tool in our current efforts to isolate and purify enzyme(s) in the cell-free extract of Geobacillus sp. UZO 3 involved in the reductive cleavage of chlorinated dioxins. Furthermore, this method may find potential application in the following: (1) uncovering the intracellular locus of the enzyme(s) in Geobacillus sp. UZO 3 that reductively cleave the diaryl ether bonds of 2,7-DCDD; (2) analyzing their substrate specificity to polychlorinated dibenzo-p-dioxins; and (3) measuring these enzymatic activities in the contaminated environment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank S. Hoshina (School of Medicine, Jikei University) for helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research (A), 21248037, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a fund from the Ministry of Environment of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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