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., [2011
], Pavlova et al., [2009
]). Moreover, this parameter can be further extended even towards acceptance of mono-, di-, and trichloro-substituted substrates by enzyme engineering (Banas et al., [2006
], Pavlova et al., [2009
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., [1999
]). 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., [1999
]). 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., [1960
]), allows one to apply the standard procedure described above. Like other methods relying on the determination of halide content (Holloway et al., [1998
], Kurtovic et al., [2007
], [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 ). 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., [2011
]) or DhaA variants that have been optimized by directed evolution (Pavlova et al., [2009
]) 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., [2010
]). 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
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.