Informace o publikaci
Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary
| Autoři | |
|---|---|
| Rok publikování | 2003 |
| Druh | Článek ve sborníku |
| Konference | Proceeding of 27th Symposium on HPLC and Related Techniques |
| Fakulta / Pracoviště MU | |
| Citace | |
| Obor | Biochemie |
| Klíčová slova | CZE; Enzymes; EMMA; Haloalkane dehalogenase |
| Popis | Halogenated aliphatic hydrocarbons constitute one of the largest groups of environmental pollutants as a result of their widespread use as solvents, pesticides, herbicides, insecticides and chemical intermediates. Because of their toxicity, bioconcentration and persistence, the ubiquitous distribution of halogenated compounds in the biosphere has caused public concern over the possible effects on the quality of life. Haloalkane dehalogenases [EC 3.8.1.5] are group of enzymes involved in the biodegradation of these compounds by catalysing cleavage of the carbon-halogen bond. The study of the biochemistry of dehalogenation processes may help to understand and evaluate the potential for their degradation in nature. Moreover, biotransformation of organic compounds with biocatalysts offers new routes for the synthesis of intermediates. This report describes the application of electrophoretically mediated microanalysis (EMMA) for the study of kinetic parameters of haloalkane dehalogenase. In this technique, substrate(s) and enzyme are introduced in the capillary as distinct plugs, the first analyte injected being the one with the lower electrophoretic mobility. Upon the application of an electric field, the two zones interpenetrate due the differences in their electrophoretic mobilities. Enzymatic reaction takes place and the resultant reaction product(s) and the unreacted substrate(s) are electrophoretically transported towards detector, where they are individually detected. The Michaelis constants for different substrates and the effect of temperature on enzymatic reaction of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this set-up the part of the capillary is filled with the buffer best for the enzymatic reaction whereas the rest of the capillary with the background electrolyte optimal for separation of substrates and products. The basic limitation of EMMA methodology the necessity to have the electrophoretic conditions compatible with both the separation of substrate(s) and product(s) and the enzymatic reaction is thus overcome. |
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