|
|
Screening of extremozyme production of selected extremophilic PHA producers
Dyagilev, Dmitry ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
This bachelor thesis deals with the screening of the production of extracellular hydrolytic enzymes in thermophilic microorganisms of the genera Aneurinibacillus, Brevibacillus, Chelatococcus, Pseudomonas, Schlegelella, Tepidimonas and Caldimonas. The ability of selected enzymes, namely proteases, lipases, amylases, xylanases, cellulases and pectinases, was tested in the investigated microorganisms. Such testing made it possible to assess in which microorganisms the production of specific enzymes can be observed. Based on the results of the screening, it was found that Schlegelella aquatica LMG 23380, Tepidimonas fonticaldi LMG 26746 and the investigated microorganisms of the genus Chelatococcus did not show the ability to produce any of the tested enzymes extracellularly. In natural isolates of Brevibacillus borstelensis LK 99 and Aneurinibacillus thermoaerophilus LK 102, only the ability to produce lipolytic enzymes was detected. The isolate Brevibacillus borstelensis Bz acts as a universal producer of all selected extremozymes. Enzyme activity was determined for selected producers. The bacterium Brevibacillus borstelensis Bz proved the ability to produce all six selected hydrolytic enzymes and has the highest activity of lipases, xylanases, cellulases and pectinases from the tested microorganisms. The highest proteolytic activity was measured in Thermomonas hydrothermalis DSM 14834 when cultured on skimmed milk powder.
|
|
|
Příprava mutantního serpinu z klíštěte \kur{Ixodes ricinus}
EDEROVÁ, Monika
Point mutation altering arginin for tryptophan amino acid residue in P1 site of tick salivary serpin Iripin-1 was created using specific primers. Recombinant protein with this mutation in nucleotide sequence was then expressed in chemically competent Escherichia coli cells, extracted from them and purified by affinity and size-exclusion chromatography. To see the impact of the mutation on inhibitory function of Iripin-1, its ability to bind trypsin and form covalent complexes was evaluated.
|
|
|
Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
|
|
|
The role of NADPH oxidase and ros in invadopodia formation
Hanušová, Kristýna ; Brábek, Jan (advisor) ; Vrbacký, Marek (referee)
Invadopodia as specific organelles enabling tumour cells movement, spreading over the organism and ultimately formation of metastasis are possible and promising targets of tumour therapy. Recently, many interesting facts about assembly and mechanism of function of invadopodia were discovered. Invadopodia are centres of ECM degradation by extra-cellular proteases facilitating an invasion of tumour cells. For creation of invadopodia a precisely localized increased production of ROS is necessary. ROS work as crucial signalling molecules and participate in many processes resulting in invadopodia formation. ROS in tumour cells are produced by specific extra-mitochondrial NADPH oxidases (Nox). Several regulatory molecules participating in activation and localization of Nox to invadopodia have been discovered recently (Tks organizer proteins). Furthermore, a regulatory role of Src kinase in ROS production and subsequent invadopodia formation was confirmed. Key words: ECM degradation, invadopodia, invasion, proteases, Nox, ROS, Src kinase, Tks proteins
|
|
|
Structural characterization of interaction between transcription factors and DNA
Filandrová, Růžena ; Novák, Petr (advisor) ; Vondrášek, Jiří (referee) ; Wimmerová, Michaela (referee)
Structural characterization of interaction between transcription factors and DNA Mgr. Růžena Filandrová Abstract Transcription factors are proteins that mediate gene expression regulation through interactions with DNA and other factors. They allow a cell to respond to various stimuli and play a crucial role in many biological processes such as control of cell cycle progression, differentiation of cells during development or immune response. To understand these processes, the knowledge of the transcription factors 3D structure together with the mechanism of their interaction with DNA is essential. However, some of the typical features of transcription factors, such as is for example the presence of intrinsically unstructured regions, make the 3D structure determination by the commonly used high resolution methods challenging. Therefore, utilization of complementary methods like structural mass spectrometry (MS), which was used in this thesis, might prove to be beneficial to explore the structural basis of the transcription factor-DNA interaction. In first part of this work, a set of structural mass spectrometry methods with the main focus on hydrogen/deuterium exchange mass spectrometry (HDX-MS) was optimized and tested on two transcription factor-DNA complexes and their DNA binding motifs and proved to be...
|
|
|
Screening of extremozyme production of selected extremophilic PHA producers
Dyagilev, Dmitry ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
This bachelor thesis deals with the screening of the production of extracellular hydrolytic enzymes in thermophilic microorganisms of the genera Aneurinibacillus, Brevibacillus, Chelatococcus, Pseudomonas, Schlegelella, Tepidimonas and Caldimonas. The ability of selected enzymes, namely proteases, lipases, amylases, xylanases, cellulases and pectinases, was tested in the investigated microorganisms. Such testing made it possible to assess in which microorganisms the production of specific enzymes can be observed. Based on the results of the screening, it was found that Schlegelella aquatica LMG 23380, Tepidimonas fonticaldi LMG 26746 and the investigated microorganisms of the genus Chelatococcus did not show the ability to produce any of the tested enzymes extracellularly. In natural isolates of Brevibacillus borstelensis LK 99 and Aneurinibacillus thermoaerophilus LK 102, only the ability to produce lipolytic enzymes was detected. The isolate Brevibacillus borstelensis Bz acts as a universal producer of all selected extremozymes. Enzyme activity was determined for selected producers. The bacterium Brevibacillus borstelensis Bz proved the ability to produce all six selected hydrolytic enzymes and has the highest activity of lipases, xylanases, cellulases and pectinases from the tested microorganisms. The highest proteolytic activity was measured in Thermomonas hydrothermalis DSM 14834 when cultured on skimmed milk powder.
|
|
|
Study of enzymes secreted by oomycete Pythium oligandrum
Hrdinová, Karolína ; Ryšlavá, Helena (advisor) ; Kubíčková, Božena (referee)
Pythium oligandrum is one of the non-pathogenic microorganisms of Pythium species which found its use as biological control agent. The main modes of action of this oomycete lie in inducing the plant immunity and in mycoparasitism of pathogenic fungi and fungus-like organisms. The oomycete attacks the pathogens by secretion of hydrolytic enzymes into the environment. In the first part of this bachelor's thesis, activity of hydrolytic enzymes endo-1,3-β-glucanase, cellulase, chitinase and proteases was observed in a commercial product based on Pythium oligandrum, called Polyversum-Biogarden. It was conclu- ded that the direct hydrolysis of phytopathogens is probably not the main mechanism of this product because the activity of glycosidases increased only after six hour-long incubation of the Polyversum-Biogarden in water and the proteolytic activity was not detected. In the second part of the bachelor's thesis, properties of proteases secreted by orga- nism Pythium oligandrum were studied. The highest proteolytic activity was observed at pH 6,5. Ovomucoid acted as an inhibitor of secreted proteases. The stability of pro- teases was lowered by SDS, detergents present in liquid soaps and by a solution of a solid soap or NaOH. Only the highest concentrations of urea lowered the activity of proteases....
|
|
|
Phenotyping of proteolytic activities enabled by fluorogenic libraries
Pospíšil, Šimon ; Míšek, Jiří (advisor) ; Hocek, Michal (referee)
This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.
|
|
|
Modulation of HIV-1 Protease Activity
Pokorná, Jana ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Ruml, Tomáš (referee)
HIV-1 protease plays a crucial role in the late state of the life cycle of HIV virus when it cleaves the viral polyprotein precursors into the structural and functional proteins. If it is effectively inhibited, HIV particles remain immature and noninfectious. The application of highly active antiretroviral therapy (HAART) including protease inhibitors can reduce plasma HIV-1 levels below the detection limit in adherent patients and thus dramatically change their life expectancy. The clinical utility of the first inhibitors was limited by severe side effects, low bioavailability, high pill burdens, and rapid development of viral resistance under the selection pressure of HIV antiretrovirals. To overcome these difficulties, second-generation inhibitors were developed. Despite an indisputable improvement they brought to antiretroviral therapy, the development of new highly active HIV-1 protease inhibitors with optimal pharmacokinetic properties, higher metabolic stability, little off-target activity, and particularly, more favorable resistance profiles is still of high importance. This thesis provides an overview of anti-HIV- drugs including development of substituted metallacarboranes, a new class of potent, unusual, nonpeptidic HIV protease inhibitors with therapeutic potential. Next, the impact of...
|
|
|
Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
|
guest ::
