A sensitive method for rapid detection of alkyl halides and dehalogenase activity using a multistep enzyme assay
- Sebastian Fabritz†1,
- Franziska Maaß†1,
- Olga Avrutina1,
- Tim Heiseler1,
- Björn Steinmann1 and
- Harald Kolmar1Email author
© Fabritz et al.; licensee Springer. 2012
Received: 10 August 2012
Accepted: 16 September 2012
Published: 24 September 2012
A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules. Due to its high sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expenditure and the ability of handling a large number of samples in parallel, this method is an attractive alternative to existing procedures for the monitoring of both haloalkanes and dehalogenases.
Haloalkanes are toxic (Akers et al., , [Koch and Strobel 1981], Weber et al., ) and mutagenic (Brem et al., ) environmental contaminants ([Koch and Tunger 1981], Yen et al., ). Many bacterial species that are able to degrade such compounds have been described to date. Some of them can even utilize haloalkanes as a sole carbon source (Manickam et al., , Mattes et al., , Torz et al., ). Bioremediation based on the capability of certain microorganisms to dispose halogenated pollutants is a promising and cost-effective technology ([Beeman and Bleckmann 2002], Marzorati et al., , Megharaj et al., , Vlieg et al., ). Therefore, convenient methods are required to determine the activity of dehalogenases in enrichment cultures and to detect alkyl halides in environmental samples.
Methods for the determination of halogenated content in various samples have been established since the early 1950s (Iwasaki et al., ). A common method for the analysis of haloalkanes is gas chromatography combined with flame-ionization/electron capture or mass spectrometric detection ([Arbon and Grimsrud 1990], Curragh et al., , Phillips et al., , van Wijk et al., ). These methods, although providing detailed information about the nature and composition of haloalkanes present in a sample, are technically demanding. More recently, procedures for haloalkane detection and degradation have been reported which rely on enzyme-catalyzed dehalogenation yielding free protons and halides (van [Pee and Unversucht 2003]). Therefore, the majority of current haloalkane assays is focused on the monitoring of proton or halide release. Several pH dependent detection systems rely on chromatic (Holloway et al., , Phillips et al., ) or fluorescent indicators (Bidmanova et al., ) that require weakly buffered or unbuffered aqueous systems. Alternatively, methods enabling direct estimation of halide concentration have been developed as e.g. the classic colorimetric mercury-iron-thiocyanate method (Cirello-[Egamino and Brindle 1995], Iwasaki et al., , Zall et al., ), iodide detection via starch incorporation (Kurtovic et al., ) or quenching of fluorophores by halides ([Marchesi 2003]).
Materials and methods
Chemicals and enzymes
The chemicals and enzymes were of analytical grade and used without further purification. Alcohol oxidase (AOX) from Pichia pastoris (solution in phosphate-buffered 30% sucrose, 10-40 U/mg) and peroxidase (HRP) from horseradish type VI-A (950-2000 U/mg) were obtained from Sigma-Aldrich (USA). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Fluka. Ampicillin sodium salt, isopropyl-β-D-thiogalactopyranoside (IPTG), potassium dihydrogen phosphate and dipotassium hydrogen phosphate were obtained from Carl Roth (Germany).
Expression and purification of haloalkane dehalogenase
The haloalkane dehalogenase gene (dhaA) from Rhodococcus erythropolis DSM 16550 ([Gray and Thornton 1928]) has been deposited in the GenBank database under accession no. AF060871.1. The gene was isolated by polymerase chain reaction (PCR) and cloned into the pET21d expression vector (Novagen) to yield pET21d-DhaA. The expression vector was transferred into E. coli BL21(DE3). Transformed E. coli cells were cultured in 3 liters of dYT medium (1% yeast extract, 1.6% Bacto tryptone, 0.5% NaCl) supplemented with 100 μg/mL ampicillin at 37°C and 180 rpm. Expression was induced with 1 mM IPTG when bacterial growth reached an A600 of 0.5 and performed for 18 h at 30°C. The cells were harvested by centrifugation, resuspended in potassium phosphate buffer (0.1 M, pH 7.5) and the cell suspension was stored at -80°C for 1 h. Cells were thawed and disrupted using a high-pressure cell disruption system from Constant Systems Limited (United Kingdom). The suspension was centrifuged at 19650 × g for 30 min at 4°C. The enzyme was purified from the supernatant by immobilized metal ion chromatography using Ni-loaded IMAC Sepharose 6 Fast Flow (GE Healthcare) and a step gradient of imidazole as eluent. Purified enzyme was dialyzed against potassium phosphate buffer (0.1 M, pH 7.5) and frozen in aliquots at -80°C until needed.
Detection of haloalkanes
A quartz cuvette with a path length of 5 mm (type: 104B-QS; Hellma Analytics, Germany) was filled with 500 μL potassium phosphate buffer (0.1 M, pH 7.5) containing the corresponding halogenated compound in desired concentration. 5 μL of ABTS solution in aqua bidest (10 mM), 1 μL AOX dissolved in phosphate buffer containing 30% sucrose (1500 U/mL) and 1 μL of a HRP solution in aqua bidest (15 kU/mL) was added successively. After an equilibration time of 15 min, 20 μL of purified DhaA in potassium phosphate buffer (~0.32 U/mL) were added. The absorbance was measured at 405 nm using a Shimadzu UV–vis spectrophotometer UV-1650PC over 5 min at ambient temperature.
To develop appropriate experimental conditions under which the initial reaction velocity of the coupled three-step enzyme reaction directly corresponds to the initial substrate concentration, 1-chlorobutane was used as a model substrate at different concentrations. To obtain reliable values, measurements were performed in triplicate and the initial reaction rates were determined from the reaction progress curve using a time-frame ranging from 2 to 4 minutes. After substrate addition the samples were preincubated (15 min) as an initial delay in reaction progress has been observed.
Haloalkane dehalogenases have been isolated from a number of species and also from uncultivated environmental samples using polymerase chain reaction ([Kotik and Famerova 2012]). Each specific dehalogenase can be expected to have its own characteristic substrate specificity, enantioselectivity and product inhibition properties. For detection of various haloalkanes we have chosen the R. erythropolis DhA since it is well characterized and it has been reported to display broadened substrate specificity (Koudelakova et al., , Pavlova et al., ). Moreover, this parameter can be further extended even towards acceptance of mono-, di-, and trichloro-substituted substrates by enzyme engineering (Banas et al., , Pavlova et al., ).
The coupled triple enzyme reaction described here allows fast simple and sensitive detection of haloalkanes but depending on the nature of the sample to be analysed several potentially limiting conditions have to be carefully considered. It has been reported that certain dehalogenases are inhibited by halides (Schindler et al., ). In the case when R. erythropolis DhaA is used, presence of halide salts in concentrations up to 80 mM should not disturb the reaction (Schindler et al., ). It should also be noted that the signal generation in our method can similarly be triggered by traces of alcohols in the sample. In this case, it may be advisable to preincubate the analyte solution with AOX and a catalase to oxidize the alcohol to the corresponding aldehyde and to remove the hydrogen peroxide generated. Subsequent inactivation of catalase, e.g. by addition of 3-amino-1,2,4-triazole or 4-hydroxypyrazole (Mac[Donald and Pispa 1980], Margoliash et al., ), allows one to apply the standard procedure described above. Like other methods relying on the determination of halide content (Holloway et al., , Kurtovic et al., , [Marchesi 2003]), this assay does not allow to distinguish between individual alkyl halides if a multicomponent mixture of haloalkanes is present in a sample. Since different halogenated alkanes give rise to distinct sensitivity and reaction rates, knowledge of the haloalkane composition of a sample would be required. It can be obtained by using e.g. GC-MS analytics via the generation of an equivalent reference sample for calibration purposes.
It should also be noted that with this triple enzyme assay no linear correlation exists between analyte content and initial velocity of ABTS formation over the range of 0 to 5 mM substrate concentration (Figure 6). Several reasons may account for this finding. At low haloalkane concentration, the accuracy of the measurement may be limited due to the fact that a fraction of the primary aldehydes could react with primary amines of the enzymes present in the reaction mixture via Schiff base formation ([Shan and Hammock 2001]) which would impede oxidation by HRP.
Obviously, besides measurement of haloalkane content in a sample, the coupled assay can also be used for the determination of haloalkane dehalogenase activity, e.g. in an enrichment culture, using halogenated hydrocarbon substrates for which the enzyme of interest displays the highest catalytic efficiency. The detection system we report here is nontoxic, works in a buffered system, is rapid and, due to the enzyme-mediated chromophore formation, highly sensitive. The assay does not require sophisticated machinery (chlorimeter, special electrodes, etc.), and the enzymes apart from haloalkane dehalogenases are inexpensive and commercially available. Furthermore, the established multistage enzyme reaction can be considered as a modular system for haloalkane detection. The usage of different haloalkane dehalogenases ([Janssen 2004], Koudelakova et al., ) or DhaA variants that have been optimized by directed evolution (Pavlova et al., ) should result in an extension of accepted substrates if required.
Recently, an enzyme-based method for the detection of halogenated hydrocarbons that relies on an enzymatic fibre-optic biosensor has been reported with similar detection limits (Bidmanova et al., ). The assembly of such a device needs special equipment and fine-tuned immobilisation chemistry. Nevertheless, it has the inherent capability of continuous in situ measurement. Another interesting approach was developed by Marchesi ([Marchesi 2003]). The assay is based on the fluorescence quenching of 6-methoxy-N-(3-sulfopropyl)-quinolinium by halides. This elegant methodology that allows one to detect halide concentrations in the range of 1-500 mM is restricted to the samples where halide salts are absent since they quench the fluorophore. The approach described here is at least as sensitive as other methods and, depending on the nature of the haloalkane substrate and enzyme, may allow for an even lower detection limit.
In conclusion, we have developed a fast, simple and sensitive detection of haloalkanes and haloalkane dehalogenase activity based on coupled enzymatic reactions. This method may be useful for the detection of halogenated pollutants in environmental samples or for the detection of haloalkane dehalogenase activity e.g. in enrichment cultures or to control dehalogenase activity during bioremediation. Using DhaA from R. erythropolis as a model enzyme, we showed that the rate-determing step of the multistep assay was dehalogenation of a haloalkanes substrate. Detection can be conducted either “on-bench”, with green colour of a sample indicating the enzymatic conversion of haloalkanes, or, more precisely, by photometric monitoring of the formation of an ABTS oxidation product. Our method allows for the detection of enzyme-mediated haloalkane conversion in buffered systems and, depending on the dehalogenase used, in samples that may contain inorganic halides. High sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expedition, and possibilities to vary haloalkane dehalogenase towards broadened substrate tolerance makes this method a versatile alternative to existing procedures.
This work was supported by BMBF through grant Nanokat.
- Akers KS, Sinks GD, Schultz TW: Structure-toxicity relationships for selected halogenated aliphatic chemicals. Environ Toxicol Pharmacol 1999, 7: 33–39. 10.1016/S1382-6689(98)00048-9View ArticlePubMedGoogle Scholar
- Arbon RE, Grimsrud EP: Selective detection of iodinated hydrocarbons by the electron-capture detector with negative-ion hydration and photodetachement. Anal Chem 1990, 62: 1762–1768. 10.1021/ac00216a009View ArticleGoogle Scholar
- Banas P, Otyepka M, Jerabek P, Petrek M, Damborsky J: Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling. J Comput Aided Mol Des 2006, 20: 375–383. 10.1007/s10822-006-9071-1View ArticlePubMedGoogle Scholar
- Beeman RE, Bleckmann CA: Sequential anaerobic-aerobic treatment of an aquifer contaminated by halogenated organics: field results. J Contam Hydrol 2002, 57: 147–159. 10.1016/S0169-7722(02)00008-6View ArticlePubMedGoogle Scholar
- Bidmanova S, Chaloupkova R, Damborsky J, Prokop Z: Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons. Anal Bioanal Chem 2010, 398: 1891–1898. 10.1007/s00216-010-4083-zView ArticlePubMedGoogle Scholar
- Braithwaite A: Unit cell dimensions of crystalline horseradish peroxidase. J Mol Biol 1976, 106: 229–230. 10.1016/0022-2836(76)90309-0View ArticlePubMedGoogle Scholar
- Brem H, Stein AB, Rosenkranz HS: The mutagenicity and DNA-modifying effect of haloalkanes. Cancer Res 1974, 34: 2576–2579.PubMedGoogle Scholar
- Childs RE, Bardsley WG: The steady-state kinetics of peroxidase with 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as a chromogen. Biochem J 1975, 145: 93–103.PubMed CentralView ArticlePubMedGoogle Scholar
- Cirello-Egamino J, Brindle ID: Determination of chloride ions by reaction with mercury thiocyanate in the absence of iron(III) using a UV-photometric, flow injection method. Analyst 1995, 120: 183–186. 10.1039/an9952000183View ArticleGoogle Scholar
- Couderc R, Baratti J: Oxidation of methanol by the yeast. Pichia pastoris. Purification and properties of the alcohol oxidase. Agr Biol Chem 1980, 44: 2279–2289. 10.1271/bbb1961.44.2279View ArticleGoogle Scholar
- Curragh H, Flynn O, Larkin MJ, Stafford TM, Hamilton JTG, Harper DB: Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB 13064. Microbiology-Uk 1994, 140: 1433–1442. 10.1099/00221287-140-6-1433View ArticleGoogle Scholar
- Delincée H, Radola B: Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography. Eur J Biochem 1975, 52: 321–330. 10.1111/j.1432-1033.1975.tb04000.xView ArticlePubMedGoogle Scholar
- Gray PHH, Thornton HG: Soil bacteria that decompose certain aromatic compounds. Zentralbl Bakterio Parasitenkd Infektionskr Hyg Abt I 1928, 73: 74–96.Google Scholar
- Holloway P, Trevors JT, Lee H: A colorimetric assay for detecting haloalkane dehalogenase activity. J Microbiol Methods 1998, 32: 31–36. 10.1016/S0167-7012(98)00008-6View ArticleGoogle Scholar
- Iwasaki I, Utsumi S, Ozawa T: New colorimetric determination of chloride using mercuric thiocyanate and ferric ion. Bull Chem Soc Jpn 1952, 25: 226–226. 10.1246/bcsj.25.226View ArticleGoogle Scholar
- Janssen DB: Evolving haloalkane dehalogenases. Curr Opin Chem Biol 2004, 8: 150–159. 10.1016/j.cbpa.2004.02.012View ArticlePubMedGoogle Scholar
- Koch R, Strobel K: Ökochemische und toxikologische Daten für ausgewählte Halogenalkane und ihre Bewertung. Acta Hydrochim Hydrobiol 1981, 9: 227–246. 10.1002/aheh.19810090302View ArticleGoogle Scholar
- Koch R, Tunger A: Kontamination von Wässern mit Halogenalkanen und -alkenen. Acta Hydrochim Hydrobiol 1981, 9: 471–475. 10.1002/aheh.19810090414View ArticleGoogle Scholar
- Kotik M, Famerova V: Sequence diversity in haloalkane dehalogenases, as revealed by PCR using family-specific primers. J Microbiol Methods 2012, 88: 212–217. 10.1016/j.mimet.2011.11.013View ArticlePubMedGoogle Scholar
- Koudelakova T, Chovancova E, Brezovsky J, Monincova M, Fortova A, Jarkovsky J, Damborsky J: Substrate specificity of haloalkane dehalogenases. Biochem J 2011, 435: 345–354. 10.1042/BJ20101405View ArticlePubMedGoogle Scholar
- Kulakova AN, Larkin MJ, Kulakov LA: The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064. Microbiology-Uk 1997, 143: 109–115. 10.1099/00221287-143-1-109View ArticleGoogle Scholar
- Kurtovic S, Jansson R, Mannervik B: Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates. Arch Biochem Biophys 2007, 464: 284–287. 10.1016/j.abb.2007.04.009View ArticlePubMedGoogle Scholar
- MacDonald E, Pispa JP: Inhibition of catalase in vitro and in vivo by 4-hydroxypyrazole, a metabolite of pyrazole. FEBS Lett 1980, 120: 61–64. 10.1016/0014-5793(80)81046-5View ArticlePubMedGoogle Scholar
- Manickam N, Reddy MK, Saini HS, Shanker R: Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in gamma-HCH degradation. J Appl Microbiol 2008, 104: 952–960. 10.1111/j.1365-2672.2007.03610.xView ArticlePubMedGoogle Scholar
- Marchesi JR: A microplate fluorimetric assay for measuring dehalogenase activity. J Microbiol Methods 2003, 55: 325–329. 10.1016/S0167-7012(03)00132-5View ArticlePubMedGoogle Scholar
- Margoliash E, Novogrodsky A, Schejter A: Irreversible reaction of 3-amino-1:2:4-triazole and related inhibitors with the protein of catalase. Biochem J 1960, 74: 339–348.PubMed CentralView ArticlePubMedGoogle Scholar
- Marzorati M, Borin S, Brusetti L, Daffonchio D, Marsilli C, Carpani G, de Ferra F: Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater. Biodegradation 2006, 17: 41–56. 10.1007/s10532-005-9004-zView ArticleGoogle Scholar
- Mattes TE, Alexander AK, Richardson PM, Munk AC, Han CS, Stothard P, Coleman NV: The genome of Polaromonas sp strain JS666: Insights into the evolution of a hydrocarbon- and xenobiotic-degrading bacterium, and features of relevance to biotechnology. Appl Environ Microbiol 2008, 74: 6405–6416. 10.1128/AEM.00197-08PubMed CentralView ArticlePubMedGoogle Scholar
- Megharaj M, Ramakrishnan B, Venkateswarlu K, Sethunathan N, Naidu R: Bioremediation approaches for organic pollutants: A critical perspective. Environ Int 2011, 37: 1362–1375. 10.1016/j.envint.2011.06.003View ArticlePubMedGoogle Scholar
- Pavlova M, Klvana M, Prokop Z, Chaloupkova R, Banas P, Otyepka M, Wade RC, Tsuda M, Nagata Y, Damborsky J: Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol 2009, 5: 727–733.View ArticlePubMedGoogle Scholar
- Phillips TM, Seech AG, Lee H, Trevors JT: Colorimetric assay for Lindane dechlorination by bacteria. J Microbiol Methods 2001, 47: 181–188. 10.1016/S0167-7012(01)00299-8View ArticlePubMedGoogle Scholar
- Porstmann B, Porstmann T, Nugel E: Comparison of chromogens for the determination of horseradish peroxidase as a marker in enzyme immunoassay. J Clin Chem Clin Biochem 1981, 19: 435–439.PubMedGoogle Scholar
- Sahm H, Schutte H, Kula MR: Alcohol oxidase from Candida boidinii . Methods Enzymol 1982, 89: 424–428.View ArticlePubMedGoogle Scholar
- Schindler JF, Naranjo PA, Honaberger DA, Chang CH, Brainard JR, Vanderberg LA, Unkefer CJ: Haloalkane dehalogenases: steady-state kinetics and halide inhibition. Biochemistry 1999, 38: 5772–5778. 10.1021/bi982853yView ArticlePubMedGoogle Scholar
- Shan G, Hammock BD: Development of sensitive esterase assays based on alpha-cyano-containing esters. Anal Biochem 2001, 299: 54–62. 10.1006/abio.2001.5388View ArticlePubMedGoogle Scholar
- Stsiapanava A, Koudelakova T, Lapkouski M, Pavlova M, Damborsky J, Smatanova IK: Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis. Acta Crystallogr F-Struct Biol Cryst Commun 2008, 64: 137–140. 10.1107/S1744309108002066View ArticleGoogle Scholar
- Torz M, Wietzes P, Beschkov V, Janssen DB: Metabolism of mono- and dihalogenated C1 and C2 compounds by Xanthobacter autotrophicus growing on 1,2-dichloroethane. Biodegradation 2007, 18: 145–157. 10.1007/s10532-006-9050-1View ArticlePubMedGoogle Scholar
- Ukeda H, Ohira M, Sawamura M: Immobilized enzyme-based microtiter plate assay for ethanol in alcoholic beverages. Anal Sci 1999, 15: 447–450. 10.2116/analsci.15.447View ArticleGoogle Scholar
- Van der Klei IJ, Bystrykh LV, Harder W: Alcohol oxidase from Hansenula polymorpha CBS 4732. Methylortophy 1990, 188: 420.Google Scholar
- van Pee KH, Unversucht S: Biological dehalogenation and halogenation reactions. Chemosphere 2003, 52: 299–312. 10.1016/S0045-6535(03)00204-2View ArticlePubMedGoogle Scholar
- van Wijk AM, Beerman B, Niederlander HA, Siebum AH, de Jong GJ: A new approach for generic screening and quantitation of potential genotoxic alkylation compounds by pre-column derivatization and LC-MS/MS analysis. Anal Bioanal Chem 2011, 400: 1375–1385. 10.1007/s00216-011-4901-yView ArticlePubMedGoogle Scholar
- Vlieg J, Poelarends GJ, Mars AE, Janssen DB: Detoxification of reactive intermediates during microbial metabolism of halogenated compounds. Curr Opin Microbiol 2000, 3: 257–262. 10.1016/S1369-5274(00)00086-2View ArticleGoogle Scholar
- Weber LWD, Boll M, Stampfl A: Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003, 33: 105–136. 10.1080/713611034View ArticlePubMedGoogle Scholar
- Welinder KG: Amino acid sequence studies of horseradish peroxidase. Eur. Biochem 1979, 96: 483–502. 10.1111/j.1432-1033.1979.tb13061.xView ArticleGoogle Scholar
- Yen JH, Lin KH, Wang YS: Acute lethal toxicity of environmental pollutants to aquatic organisms. Ecotox Environ Safe 2002, 52: 113–116. 10.1006/eesa.2002.2167View ArticleGoogle Scholar
- Zall DM, Fisher D, Garner MQ: Photometric determination of chlorides in water. Anal Chem 1956, 28: 1665–1668. 10.1021/ac60119a009View ArticleGoogle Scholar
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