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Production of proteolytic enzymes by selected microorganisms
Pala, Martin ; Lichnová, Andrea (referee) ; Márová, Ivana (advisor)
Presented work was focused on study of microbial production of proteolytic enzymes used in many industrial applications. Bacterium Bacillus subtilis was used for laboratory production of microbial proteolytic enzymes. During cultivation production of biomass and proteolytic enzymes were studied influence of substrate type and stress factor application (osmotic shock, hydrogen peroxide and ethanol) was tested too. The highest concentration of biomass was measured in concentrated BM medium after 32 hours of cultivation. Biomass yield was 1.11 g/l. The highest protease activity (88.28 U/ml) was obtained in the same cultivation medium and time of cultivation. According to results of stress experiments it can be concluded that most of used stress factors exhibited a toxic effects to bacterial culture even at low concentrations.
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Secreted proteases of the liver fluke and their interaction with endogenous inhibitor
Buša, Michal ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The liver fluke, Fasciola hepatica, is one of the most important parasites of livestock, and it also infects humans. The proteolytic system of trematodes is critical for their interaction with the host and is a potential target for the development of novel vaccines. This work is focused on proteases secreted by F. hepatica adults and on FheCy2, a new protease inhibitor from the cystatin family. The proteolytic activity of the secreted proteases was analyzed using: (a) chromogenic protein substrates and fluorogenic peptide substrates, (b) selective protease inhibitors, and (c) a fluorescent activity-based probe for visualization of proteases. The results showed that the secreted proteases are cysteine proteases of papain family belonging to cathepsins L and B. These proteases were effectivelly inhibited by FheCy2 as demonstrated by enzymological analysis. It can be assumed that FheCy2 participates in the physiological regulation of endogenous proteases secreted by F. hepatica adults, which makes it attractive candidate protein for vaccination studies. Key words: Fasciola hepatica, cathepsins, proteolytic activity, substrate specificity, protease inhibitors (In Czech)
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Cathepsin L from the hard tick Ixodes ricinus: analysis of proteolytic activity and its regulation
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity...
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Production of proteolytic enzymes by selected microorganisms
Pala, Martin ; Lichnová, Andrea (referee) ; Márová, Ivana (advisor)
Presented work was focused on study of microbial production of proteolytic enzymes used in many industrial applications. Bacterium Bacillus subtilis was used for laboratory production of microbial proteolytic enzymes. During cultivation production of biomass and proteolytic enzymes were studied influence of substrate type and stress factor application (osmotic shock, hydrogen peroxide and ethanol) was tested too. The highest concentration of biomass was measured in concentrated BM medium after 32 hours of cultivation. Biomass yield was 1.11 g/l. The highest protease activity (88.28 U/ml) was obtained in the same cultivation medium and time of cultivation. According to results of stress experiments it can be concluded that most of used stress factors exhibited a toxic effects to bacterial culture even at low concentrations.
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