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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Vliv zemědělské techniky na vznik biogenních aminů v silážích
Jandeková, Nela
This diploma thesis is investigating the influence of an agriculture machinery on the quality of protein silage. In the theoretical part, it deals with the production of silages, factors affecting the resulting quality of silages, the formation of undesirable fermentation and proteolytic products and with their impact on a livestock health. In practical part, two different technologies for collection of a clamp silo and their influence on the secondary degradation of the silage were assessed. The assessment was conducted in field conditions in ZS Kosova Hora a.s. and ZOPOS Přestavlky a.s. companies.
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Vliv aditiv na kvalitu a zdravotní bezpečnost siláží jetelovin
Florianová, Viola
The aim of this diploma thesis was to compare quality of protein silages made of lucerne (Medicago sativa L.) (there were used two varieties Holyna and Tereza) and red clover (Trifolium pratense L.) (tetraploid variety Amos and diploid variety Spurt) ensiled with low dry matter content 16–20 %. Experimental parcels were founded in 2013. Plots of lucerne were harvested for three following years, plots of red clover were harvested for two following years. Green feed was ensiled into laboratory microsilos directly after harvesting. The silage quality was compared within species and variety and within different silage additives. There were analyzed contents of organic nutrients, digestibility in green material and organic nutrients, digestibility, quality of silage extracts, losses and amount of silage effluents in silages. Silages made of red clover had significantly (p <0,05) lower pH, ammonia content, content of butyric acid and proteolysis level compared to lucerne silages. Amos variety had also significantly higher content of lactic acid compared to both lucerne silages. The effect of chemical silage additive on silage quality was significant. Silages treated with chemical additive had significantly lower (p <0,05) pH, ammonia content, content of acetic acid, ethanol and level of proteolysis compared to untreated variant and variant treated with biological additive.
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Biogenní aminy v silážích
Jandeková, Nela
Biogenic amines are low-molecular weight nitrogenic substances, which made by microorganisms with decarboxylic activity. These microrganisms contain decarboxylic enzymes, which catalyze decarboxylation of free amino acids. Biogenic amines are essential for organism, because they have their part in metabolism of celullar growth, regulativ of cardiac activity and so on. However, with higher concentrations ( > 100 mg/kg-1) they cause headache, hypotensia, nauzea, dizziness, and laminitiss with cattle. Laminitis is an inflammation of hooves. Goal of this work was so create an overview of origin, elimination and detection of biogenic amines in protein silages. The creation of biogenic amines is mainly caused by bad condition of silage making and its storage. Feeding of this silage can cause serious health issues to cattle and biogenic amines can be transferred to the animal products that could be potentionally harmfull for customers. Creation of these substances could be eliminated by adhering to the correct terms of silage making and its storage and by adding a correct inoculants. Detection of biogenic amides could be achived by chromatography.
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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Kinetic behavior of the NAD(P)H:Quinone oxidoreductase WrbA from Escherichia coli.
KISHKO, Iryna
This Ph.D. thesis addresses the structure-function relationship of the multimeric oxidoreductase WrbA with the principal aim being the explanation of the unusual kinetics of this enzyme in molecular terms, and thus getting an insight about its physiological role in bacteria. WrbA is a multimeric enzyme with FMN as a co-factor, catalyzing the oxidation of NADH by a two electrons transfer. Structure and function analysis of WrbA places this enzyme between bacterial flavodoxins and eukaryotic oxidoreductases in terms of its evolutionary relationship. The kinetic activity of WrbA was studied under varying conditions such as temperature, pH etc, and its kinetic mechanism was evaluated from parameters KM and Vmax and confirmed by product inhibition pattern experiments. Crystallization and proteolytic experiments also underpin the functional importance of the multimeric nature of WrbA and aid the understanding of the physiological role of this enzyme in molecular terms.
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