Determination of The Kinetic Parameters of Haloalkane
Dehalogenase by EMMA In a Partially Filled Capillary

D 2003

Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary

TELNAROVÁ, Magdalena, Soňa NOVÁKOVÁ and Zdeněk GLATZ

Basic information

Original name

Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary

Authors

TELNAROVÁ, Magdalena (203 Czech Republic, guarantor), Soňa NOVÁKOVÁ (203 Czech Republic) and Zdeněk GLATZ (203 Czech Republic)

Edition

Nice, France, Proceeding of 27th Symposium on HPLC and Related Techniques, p. 133-133, 1 pp. 2003

Publisher

MCI Paris France

Other information

Language

English

Type of outcome

Stať ve sborníku

Field of Study

10600 1.6 Biological sciences

Country of publisher

France

Confidentiality degree

není předmětem státního či obchodního tajemství

RIV identification code

RIV/00216224:14310/03:00008125

Organization unit

Faculty of Science

Keywords in English

CZE; Enzymes; EMMA; Haloalkane dehalogenase

Abstract

V originále

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.

Links

GA203/03/1125, research and development project

Name: Využití kapilární zónové elektroforézy pro studium enzymů

Investor: Czech Science Foundation, Application of capillary zone electrophoresis for the study of enzymes

Citovat

TELNAROVÁ, Magdalena, Soňa NOVÁKOVÁ and Zdeněk GLATZ. Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary. In Proceeding of 27th Symposium on HPLC and Related Techniques. Nice, France: MCI Paris France, 2003, p. 133-133.

@inproceedings{488016,
   author = {Telnarová, Magdalena and Nováková, Soňa and Glatz, Zdeněk},
   address = {Nice, France},
   booktitle = {Proceeding of 27th Symposium on HPLC and Related Techniques},
   keywords = {CZE; Enzymes; EMMA; Haloalkane dehalogenase},
   language = {eng},
   location = {Nice, France},
   pages = {133-133},
   publisher = {MCI Paris France},
   title = {Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary},
   year = {2003}
}

TY  - JOUR
ID  - 488016
AU  - Telnarová, Magdalena - Nováková, Soňa - Glatz, Zdeněk
PY  - 2003
TI  - Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary
PB  - MCI Paris France
CY  - Nice, France
KW  - CZE
KW  - Enzymes
KW  - EMMA
KW  - Haloalkane dehalogenase
N2  - 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.
ER  -

TELNAROVÁ, Magdalena, Soňa NOVÁKOVÁ and Zdeněk GLATZ. Determination of The Kinetic Parameters of Haloalkane Dehalogenase by EMMA In a Partially Filled Capillary. In \textit{Proceeding of 27th Symposium on HPLC and Related Techniques}. Nice, France: MCI Paris France, 2003, p.~133-133.

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