| Popis |
Capillary zone electrophoresis (CZE) is a rapidly growing separation technique. One of the greatest advantages is its diverse application range. In addition, CZE offers a simple method development, minimal sample volume requirements, and lack of organic waste. High efficiency and rate of CZE predetermine this method for the analysis of complex mixtures of biological origin, including living bacterial cells. The electrophoretic separation of bacteria is based on the fact that the surface of bacterial cells is covered with charged polymers (sialic acids, polysaccharides, lipopolysaccharides and proteins) that carry a considerable number of ionizable groups (carboxyl, phosphate, sulphate and amino groups). Both positively and negatively charged groups are present on the bacterial surface, which is therefore amphoteric. The net charge is negative at high values of pH and positive at low values of pH. The polymer composition on the bacterial surface depends mainly on the genetic predisposition of the microorganism, but also on the growth conditions. In a liquid medium, the ionizable polymers on the bacterial surface react with the counterions of the surrounding medium, and a electric double layer is formed, which determines the net electrokinetic potential of the cell. The electrokinetic potential may be influenced by many factors, such as type of bacterium and its age, as well as the growth conditions (pH, ionic strength and chemical composition of the surrounding medium). If during the electrophoretic process the composition of the medium is invariable, it may be assumed that on the basis of different electrokinetic potential, those bacteria will be separated that have been cultived in different conditions. Using different experimental conditions, the electrophoretic separations of bacteria Acidithiobacillus ferroxidans, Paracoccus denitrificans, Micrococcus luteus, Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium infantis have been performed. Apart from those types of buffers which are used commonly in CZE (eg. phosphate, borate), a special buffer containing 0,025% of polyethylenoxide (PEO) has been used. This polymer brings about the dynamic deactivation of the capillary, which is an essential requirement for a succesful separation of bacterial cells. The reproducibilities of the retention time and of the peak area are very good: the relative standard deviation does not exceed 5%. A new method for the assay of enzyme activity of lysozyme was being developed. This method is a modification of the classic assay, where the decrease of turbidity in the suspension of cells M. luteus after adding the lysozyme is measured. In the CZE format, the reaction takes place in a reaction vessel, but the decrease of signal is monitored in the capillary. Unfortunately, the procedure optimized for the separation of bacterial cells is not convenient for the assay of enzyme activity. Therefore, the operation procedure will need to be somewhat modified, first of all with regard to the non-enzymatic decrease of turbidity of the bacterial cell suspension.
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