Peptidoglycan is a key component of bacterial cell wall and is found in almost all bacteria. It is involved in many important cellular processes for the microorganism and also has an important protective role. Many enzymes that are involved in biosynthesis and in the degradation of peptidoglycan represent an important target in the development of antibiotics, which can induce bacterial cell lysis by inhibiting these enzymes. The following Master’s thesis was written with the intent of evaluating peptidoglycan in terms of phosphate content, which coincides with the content of teichoic acid and free amine groups, which reflect damage on the peptidoglycan. Evaluated peptidoglycan samples were isolated from three different bacterial strains (S. aureus, B. subtilis, ΔSagB S. aureus), then lyophilized, treated and cleaned under different conditions. Phosphate content was determined with a convenient one-step reagent for the determination of free phosphates - BIOMOL® GREEN. The free amine groups were detected by a ninhydrin reaction. Two series of peptidoglycan samples were tested. After evaluation of the first series of samples, we found that those treated with HF contained the least phosphate and free amine groups. Thus, in the second series of samples, all were purified with HF; the difference among them was the length of treatment. Minimal peptidoglycan damage was found in samples treated for 24 h. In the second part of the experimental work, we began to label the peptidoglycan with a fluorescent probe (dye). We selected the most suitable peptidoglycan, i.e. peptidoglycan with the lowest content of phosphate groups and free amine groups for labelling. Labeled peptidoglycan is a substrate for an enzyme test based on fluorescence detection, which evaluates the kinetics of selected enzymes that degrade peptidoglycan. The highest activity was determined for the enzyme AtlA-gl, as it cleaved the substrate most successfully. For the other two enzymes (AtlE and SagB), the activity was barely detectable.
The finding that the most optimal method of peptidoglycan purification for 24 h is HF treatment, represents an important basis for the preparation of peptidoglycan samples, which we would like to use in the future to measure the activity of various enzymes involved in biosynthesis or process of decomposition of this vital component of the bacterial cell wall.
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