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Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
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Passive microrheology of colloidal systems based on biopolymers.
Bjalončíková, Petra ; Burgert, Ladislav (referee) ; Mravec, Filip (advisor)
Diploma thesis was aimed to deal with evaluation of microrheology method in the research of biopolymer-protein. Used biopolymer was sodium hyaluronate and proteins were trypsin and chymotrypsin. For measuring of microrheology were used particles with different radius (0,5 m and 1 m). It was found, that both substances have viscous charakter. Passive microrheology is suitable for measuring the viscoelastic properties of biopolymers.
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Effects of trypsin to HEK293 cell membrane
Lipnická, Soňa ; Provazník, Ivo (referee) ; Čmiel, Vratislav (advisor)
This Bachelor’s thesis examines the effect of trypsin on the membranes of cell cultures. HEK293 cell lines nontransfected and cell lines stably transfected through the CaV3.1 membrane channels were selected for the optimization. They were gradually exposed to 0.25, 0.375 and 0.5 % trypsin. The concentration of trypsin influences the separation of the adherent cells on structure, as well as the degradation of cell membranes, which influences the cell cultures viability. Part of this thesis is an attempt to establish a methodology for the research of the effect of trypsin on membranes of HEK293 cell lines.
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Biodegradable Nanogels from Biocompatible Polymer Precursors
Oleshchuk, Diana ; Proks, Vladimír (advisor) ; Uchman, Mariusz Marcin (referee) ; Trachtová, Štěpánka (referee)
This doctoral thesis focuses on the development and application of hydrophilic, biocompatible, and biodegradable poly(amino acids)-based nanogels as a new type of delivery system. Within this Ph.D. thesis, nanogelation via horseradish peroxidase (HRP)/H2O2-mediated crosslinking in an inverse miniemulsion was investigated and optimized. The effect of three different surfactants (SPAN 80, TWEEN 85, and AOT) with various concentrations (5 wt% - 25 wt% relative to the dispersed phase with step 5 wt%) on the nanogelation of polyglutamine-based polymer precursor modified with tyramine units (PHEG-Tyr) was investigated. The results indicated that the stabilization of nanogel was most effectively achieved using 20 wt% of the nonionic surfactant SPAN 80. The hydrodynamic diameter (Dh) determined by dynamic light scattering (DLS) was 230 nm, and this size range was further confirmed to be between 200 and 300 nm using (nanoparticle tracking analysis) NTA. Analysis using transmission (TEM) and cryogenic transmission electron microscopy (cryo-TEM) techniques revealed that the nanogel in its dry state exhibited almost spherical shape with a number-average diameter (Dn) of 26 nm and dispersity (Ɖ) of 1.91, and in the frozen hydrate state, the nanogel displayed a larger size with Dn = 182 nm and Ɖ = 1.52,...
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Production of enzyme hydrolysates from flaxseed proteins and evaluation of their properties
HAŠKOVÁ, Kateřina
This thesis focuses on effect on antioxidant properties and chosen functional properties: solubility, water and fat holding capacity, emulsification activity and stability caused by hydrolysis of protein isolates (PI) made from three different flax varieties (Libra, Agriol and Raciol). Part of this work was production of PI from flax seed. For hydrolysis trypsin, alcalase and papain were used - hydrolysis by trypsin was the mildest, while hydrolysis by papain was the most intensive one. Dynamics and degree of hydrolysis were also observed. Solubility of PI and hydrolysates was discovered to be very high (almost 100%). On the contrary water holding capacity was lower (3,93 g/g and less) than fat holding capacity (4,34 g/g), which increased upon hydrolysis for most enzymes, while water holding capacity decreased. Emulsification activity rised (1,47×) under the effect of hydrolysis, however stability dropped (1,3×). The best emulsifiction properties were found out in trypsin hydrolysates. Hydrolysis caused great impact on content of polyphenols which were in alcalase hydrolysates up to 3× higher and on antioxidant activity which grew in papain hydrolysates up to 2,5×.
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Study of peptide digestion kinetics by trypsin and chiral separations of biologically active compounds by HPLC
Šlechtová, Tereza ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Mikšík, Ivan (referee)
This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....
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Study of peptide digestion kinetics by trypsin and chiral separations of biologically active compounds by HPLC
Šlechtová, Tereza ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Mikšík, Ivan (referee)
This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....
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Serine protease SmSP2 of Schistosoma mansoni
Leontovyč, Adrian ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (In Czech)
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Bloodmeal digestion of phlebotomine sand flies and its effect on Leishmania development
Pružinová, Kateřina ; Volf, Petr (advisor) ; Šobotník, Jan (referee)
Leishmania development in their vectors is closely connected with bloodmeal digestion. This thesis focuses on factors affecting bloodmeal digestion, egg development and Leishmania infection within the sand fly gut. First, we compared the effect of mammalian (rabbit) and avian (chicken) blood on digestion and eggs development in Phlebotomus duboscqi. Sand flies fed on chickens had twice lower protein concentrations in the midgut and significantly lower trypsin activity compared to those fed on rabbits. The highest differences in the trypsin activity were observed during first 24 hours post bloodmeal. In addition, females fed on chickens had slower eggs development and their eggs were 10 % smaller compared to those fed on rabbits. In the second part of the thesis we tested the effect of mosquito hormone TMOF on the trypsin activity and eggs development of Lutzomyia longipalpis. Rabbit blood with TMOF (28 mg/ml) was presented to the females via a membrane feeding system. Sand flies fed on blood with TMOF had 15 - 35 % less trypsin activity than control females fed on only rabbit blood. In addition, females fed on blood with TMOF had developed 30 % less eggs and their eggs were 12 - 24 % smaller compared to control group. However the effect of TMOF we observed was lower than that described previously...
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