Complex Study of Haloalkane Dehalogenase by Means of Electrophoretically Mediated Microanalysis
| Title in English | Complex Study of Haloalkane Dehalogenase by Means of Electrophoretlcally Mediated Microanalysis |
|---|---|
| Authors | |
| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Book of abstract 14 th International Symposium on Capillary Electroseparation Techniques ITP 2004 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | microcystins; capillary electrochromatography |
| Description | Halogenated aliphatic hydrocarbons constitute one of the largest groups of environmental pollutants. Haloalkane dehalogenases are a 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. This work aims to develop Electrophoretically Mediated MicroAnalysis (EMMA)-based assays for the complex study of haloalkane dehalogenase. EMMA utilizes the different electrophoretic mobilities of enzyme, substrate and product to initiate reaction inside the capillary and to separate components from each other for final in-capillary quantitation. The Michaelis constants for different substrates, the inhibition constant for inhibitor 1,2-dichloroethane, substrate inhibition of 1,2-dibromoethane 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 partial filling technique. In this set-up part of the capillary is filled with the buffer best for the enzymatic reaction whereas the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The direct and the indirect detection methods have been used in this study. EMMA combines all advantages of capillary electrophoresis in the assay of enzyme kinetics. The minimal sample and reagent requirements as well as high separation efficiency of capillary zone electrophoresis are fully utilized. The absence of manual procedures and complete automation minimize the risk of cross contamination and strongly reduce the assay cost. |
| Related projects: |