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Stanovení aktivity průmyslově významných enzymů produkovaných mikroorganismy izolovanými z potravinového odpadu
Lymarenko, Sofiia
The microbiome and the produced enzymes play an important role in processing food waste. Bacillus licheniformis is one of the representatives of microflora in composting and producer of industrially important hydrolytic enzymes: protease, lipase and cellulase. As part of the thesis, enzymatic activity using spectrophotometric methods was determined in seven strains of B. licheniformis for a period of 96 to 192 hours. In all seven strains, cellulase, protease and lipase activity was detected, which agrees with previously published literature. The highest cellulase activity determined by two methods was recorded for strain 3A-52 (1 U/ml) and 1A-52 (1.5 U/ml) after 24 hours of cultivation. The pH optimum for cellulase activity was 7, and the temperature optimum 50 °C to 60 °C. The maximum protease activity was detected after 120 and 144 hours of cultivation in two strains: 3A-52 (1.3 U/ml) and 3D-42 (1.4 U/ml). During the optimization, it was found that the pH optimum was 9 to 11 and the temperature optimum was 40 to 45 °C depending on the tested strain. The maximum lipase activity was approximately 0.7 U/ml after 144 hours of cultivation in 4D-41 and 3D-42. The temperature optimum was found at 50 to 60 °C and the range of pH optimum for this activity was very wide depending on the studied strain. For the strains with the highest enzymatic activity, a growth curve was determined, where the highest cellulase activity corresponded to the time of transition of the bacterial culture from the exponential phase to the stationary phase, and the highest lipase and protease activities were found later only at the transition from the stationary phase to the death phase. Strain 3A-52 and 3D-42 showed the highest activity for all three monitored enzymes. Subsequently, these strains isolated during composting can be used to produce enzymes industrially.
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Study of the substrate specificity of the LACTB tumour suppressor enzyme
Baudyšová, Alžběta ; Kečkéšová, Zuzana (advisor) ; Janečková, Lucie (referee)
Serine beta-lactamase-like protein (LACTB) is a tumour suppressor that modulates mitochondrial lipid metabolism and induces differentiation of breast cancer cells. This is achieved by the LACTB-dependent downregulation of phosphatidylserine- decarboxylase (PISD) which subsequently leads to decreases in the amounts of phosphatidylethanolamines and lysophosphatidylethanolamines in mitochondrial membranes. However, PISD was shown to not be a direct substrate of the LACTB enzyme what leaves the identity of the LACTB substrate an open question. To fill this important gap in the mechanism of the LACTB tumour suppressive pathway, this diploma thesis was focused on finding a physiological substrate of LACTB via Proteomic Identification of protease Cleavage Sites (PICS) assay. For this purpose, the other sub-aims of this project were to isolate recombinant wild-type LACTB and its catalytic mutant, to reveal ideal in vitro conditions for LACTB activity and to find out the requirements needed for LACTB multimerization. My results show that in vitro activity of LACTB is increased in the presence of higher pH and calcium ions. I also show that higher LACTB multimeric forms are bound together via disulfide bonds as they disintegrate after treatment with dithiothreitol. Furthermore, and most importantly, I show...
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Protease Sapp3p of the pathogenic yeast Candida parapsilosis
Sochor, Richard ; Heidingsfeld, Olga (advisor) ; Zábranská, Helena (referee)
Pathogenic yeasts of the genus Candida can cause systemic diseases which, in patients suffering from immunosuppression due to a disease such as AIDS, can cause serious pathological conditions, which can lead to the patient's death. One such yeast is the pathogenic yeast Candida parapsilosis. For the colonization and penetration of host tissues this yeast uses various virulence factors. One of these virulence factors are secreted aspartic proteases. The pathogenic yeast C. parapsilosis contains three secreted aspartic proteases Sapp1p, Sapp2p and Sapp3p, which are paralogs. The first two aspartic proteases are responsible for increasing the virulence of C. parapsilosis. They help the yeast survive in the body, by degrading important components of the host's immune system. However, Sapp3p doesn't exhibit these properties, except that it helps the yeast to adhere to abiotic surfaces to some extent. This work is focused on clarification the functions and localization of Sapp3p in the yeast C. parapsilosis. To clarify the function, precursor of Sapp3p (pro-Sapp3p) was recombinantly prepared in E. coli cells. The protein thus prepared was further tested for its autocatalytic activation and assisted activation by trypsin and Kex2p protease, under various conditions. Under the conditions tested, it was not...
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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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Production of recombinant cathepsin C from human blood fluke
Illichová, Hana ; Konvalinka, Jan (advisor) ; Martínková, Markéta (referee)
Blood flukes of the genus Schistosoma cause schistosomiasis, a serious parasitic disease occurring in tropical and subtropical areas. Cathepsin C (EC 3.4.14.1) is a digestive enzyme of the blood flukes which participates in the degradation of hemoglobin through its dipeptidyl aminopeptidase activity. This enzyme is critical for metabolism of the parasite and represents a potential target for the development of antischistosomal drugs. Cathepsin C has not yet been studied in detail. This bachelor thesis is focused on the development of expression systems for production of recombinant cathepsin C of Schistosoma mansoni (SmCC). The yeast Pichia pastoris system was used for the expression of an inactive SmCC precursor (zymogen) whose proteolytic stability was analysed. Furthermore, the expression system for SmCC in the protozoan Leishmania tarentolae was employed, and four different SmCC constructs were prepared to optimize production.
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Non-structural proteins of Zika and Dengue virus with enzyme activity
Krýsová, Eliška ; Konvalinka, Jan (advisor) ; Novotný, Marian (referee)
Zika and Dengue viruses codes their own enzymes which helps them in different stages of the replication cycle. NS3 a NS5 proteins and their cofactors play an essential role in flaviviral life cycle. Although their structure was already solved, many aspects of their function remain unclear. The main subject of this bachelor thesis is the role of these proteins in flaviviral life cycle, polyprotein cleavage, replication and protein-protein interaction. These enzymes keep many particular enzymatic activities such as protease, helicase, methyltrasferase and polymerase. They are both structurally and functionally separated, which is interesting regarding autoactivation and protein-protein interaction. Since Zika and Dengue infections remain a serious health care issue, it is necessary to understand the molecular mechanisms behind their replication. Keywords: protease, polymerase, Zika, Dengue, polyprotein processing, antiviral therapy
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Regulation of cathepsin K activity by reactive inhibitory molecules applicable in biomedicine
Benýšek, Jakub ; Mareš, Michael (advisor) ; Obšilová, Veronika (referee) ; Kutil, Zsófia (referee)
3 Abstract Human cathepsin K (KatK) is a lysosomal cysteine protease expressed predominantly in osteoclasts. It is the most effective enzyme for collagen breakdown and its physiological function lies in the degradation of the extracellular bone matrix. Increased enzymatic activity of KatK is associated with osteoporosis, rheumatoid arthritis, and osteoarthritis. This makes KatK a target for the treatment of pathologies, and chemotherapeutics based on protease inhibitors are being developed for its regulation. This work deals with reactive peptidomimetic and low molecular weight inhibitors of KatK namely thiadiazoles, vinyl ketones, and cyanohydrazides. It focuses mainly on the determination of the binding mode of selective inhibitors and characterization of their key interactions with the active site of KatK. Crystallographic structural analysis was combined with approaches of computational chemistry, enzymological analysis, and cell-based assays to elucidate the relationship between structure and biochemical activity of the investigated inhibitors. The obtained results provide important information for the design and optimization of new highly effective and selective KatK inhibitors as potential drugs and diagnostic probes. Keywords: cathepsin K, protease, inhibitor, 3D structure, osteoporosis
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Proteolytic enzymes of the blood fluke Schistosoma mansoni: pathobiochemistry and their use in biomedicine
Leontovyč, Adrian ; Mareš, Michael (advisor) ; Kašný, Martin (referee) ; Mikeš, Libor (referee)
Blood flukes of the genus Schistosoma are causative agents of the disease schistosomiasis, which affects more than 250 million people worldwide and together with malaria represents the most important parasitic infection. There is a high risk of resistance development against the only drug in use, therefore novel therapeutic approaches for schistosomiasis are intensively researched. Proteolytic enzymes of schistosomes are crucial for their survival in the host and thus are promising drug and vaccine targets. This thesis is focused on two proteases of the human blood fluke Schistosoma mansoni, which were produced as recombinant proteins and functionally characterized. The first one is serine protease SmSP2, which is localized at the surface of the adult worms and secreted into the blood of the host. It was identified as a vasodilatory and fibrinolytic agent, and its modulatory role in host-parasite interactions was proposed. The second one is cysteine cathepsin SmCL3, which is involved in the digestion of host blood proteins serving schistosomes as nutrients. Potent peptidomimetic inhibitors of SmCL3 were identified, and their antischistosomal activity was demonstrated in an assay with live parasites. The thesis provides new important information about S. mansoni proteases, their pathobiochemistry...
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BIOPHYSICAL AND FUNCTIONAL CHARACTERIZATION OF DDI1-LIKE ASPARTIC PROTEASES INVOLVED IN REPLICATION STRESS RESPONSE
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Čermák, Lukáš (referee)
Accurate, timely replication of a DNA molecule is a pivotal moment in the life cycle of every living organism. Any temporal or spatial defect putting the fine-tuned replication machinery off balance causes the so-called replication stress. As the replication machinery consists mainly of enzymes and other proteins, it is not surprising that many of the obstacles most severely blocking the replication machinery progress are of protein origin. Therefore, specialized proteases responsible for relieving replication stress matured during evolution. However, neither the full repertoire of proteolytic enzymes and their particular substrates taking place in countering the DNA replication stress nor detailed molecular mechanisms involved remain unknown. This thesis describes how conserved putative aspartic proteases of the Ddi1-like family engage in countering DNA replication stress via a proteolysis dependent mechanism. We structurally and biophysically characterized yeast and human members of the Ddi1-like family, explored their interactions with ubiquitin and polyubiquitin chains, and identified hypersensitivity to DNA replication inhibitor hydroxyurea in a yeast strain double deleted for DDI1 gene together with a DNA dependent metalloprotease WSS1. Detailed analysis of the DDI1 role in hydroxyurea...
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Study of the substrate specificity of the LACTB tumour suppressor enzyme
Baudyšová, Alžběta ; Kečkéšová, Zuzana (advisor) ; Janečková, Lucie (referee)
Serine beta-lactamase-like protein (LACTB) is a tumour suppressor that modulates mitochondrial lipid metabolism and induces differentiation of breast cancer cells. This is achieved by the LACTB-dependent downregulation of phosphatidylserine- decarboxylase (PISD) which subsequently leads to decreases in the amounts of phosphatidylethanolamines and lysophosphatidylethanolamines in mitochondrial membranes. However, PISD was shown to not be a direct substrate of the LACTB enzyme what leaves the identity of the LACTB substrate an open question. To fill this important gap in the mechanism of the LACTB tumour suppressive pathway, this diploma thesis was focused on finding a physiological substrate of LACTB via Proteomic Identification of protease Cleavage Sites (PICS) assay. For this purpose, the other sub-aims of this project were to isolate recombinant wild-type LACTB and its catalytic mutant, to reveal ideal in vitro conditions for LACTB activity and to find out the requirements needed for LACTB multimerization. My results show that in vitro activity of LACTB is increased in the presence of higher pH and calcium ions. I also show that higher LACTB multimeric forms are bound together via disulfide bonds as they disintegrate after treatment with dithiothreitol. Furthermore, and most importantly, I show...
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