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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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Synthesis of peptides as potential ligands of aspartate protease HIV and MAY1 and confirmation of their inhibitory activity
Klikarová, Ivana ; Šácha, Pavel (advisor) ; Žáková, Lenka (referee)
The lentivirus known as the human immunodeficiency virus (HIV) is transmitted through blood and body fluids, causing the destruction of CD4 lymphocytes and leading to opportunistic infections that define acquired immunodeficiency syndrome (AIDS). The urgent need for new antiretroviral drugs stems from concerns about the long-term toxicity of existing drugs, HIV-1 variants resistant to treatment, and frequent changes in patient treatment. Drug development is focused on inhibitors of two viral enzymes, reverse transcriptase and protease. Antiretroviral therapy uses protease inhibitors in combination with nucleoside analogs to effectively suppress viral replication, prolonging the lives of HIV-infected patients and reducing morbidity. Cryptococcus neoformans and cryptococcus gattii infections primarily affect the immunocompromised population and have high morbidity and mortality rates. Resistance to commonly used antifungals has been emerging, making it more difficult to treat these infections. Protease inhibitor components used in antiretroviral therapies have shown some clinical efficacy in these opportunistic infections, particularly in Major Aspartyl peptidase 1, an aspartate protease belonging to the same family of proteases as the HIV protease. To search for low molecular weight peptide ligands,...
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The development, constitution and functions of the gland apparatus in schistosome cercariae and endopeptidases in its contents
Titlová, Lucie ; Mikeš, Libor (advisor) ; Konečný, Lukáš (referee)
Flukes of the family Schistosomatidae are blood parasites with two-host life cycles involving aquatic snails as intermediate hosts and avian or mammalian definitive hosts. Cercariae, as invasive aquatic stages of schistosomes, enter the lumen of vessels by penetrating the skin of the definitive host. During their short life, cercariae possess a glandular apparatus consisting of three types of glands with external secretion: penetration glands, escape glands, and a head gland. These glands secrete granules containing, among other things, proteolytic enzymes, which play an important role in the process of penetrating the host's skin, but at the same time they occupy many other functions during the life of the cercaria and other life stages of schistosomes. This thesis summarizes basic knowledge about the anatomy, function and development of the gland apparatus of schistosome cercariae and further focuses on the representation of proteolytic enzymes in these glands, specifically endopeptidases. At the end, it briefly compares the different representation of endopeptidases in some members of the family.
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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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Cathepsins L of Diplostomum pseudospathaceum cercariae
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Hartmann, David (referee)
This study is focused on cercarial cysteine peptidases of the trematode Diplostomum pseudospathaceum. It follows previous research which confirmed the presence of a 24kDa cysteine peptidase in cercariae biochemically and by mass spectrometry. It was postulated, that the function of this peptidase is histolytic, when cercariae penetrate the tissues. During an attempt to purify this peptidase and characterize its peptidolytic activity, it was found out that the cercarial homogenate containsmore different peptidases varying in their pI. Tests of peptidolytic activity and inhibition have shown that these peptidases are cathepsin L-like. They are active over a broad spectrum of pH with optima of activities in weakly acidicor neutral pH. Using degenerate primers based on conserved motifs of cysteine pepridases, partial sequences of three genes for cathepsin L of D. pseudospataceum (DpCL1, 2 a 3) were obtained. Then the complete sequences of DpCL2 and 3 genes and partial sequence (without 5'end) of DpCL1 were obtained by RACE PCR. To confirm function of these peptidases we tried to immunolocalize them. We assumed that they are localized in penetration glands. Preliminary results suggested that some of the cathepsins could be also localized in the gut of cercariae. For more detailed biochemical...
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Cathepsin L by parasites - occurrence, features, functions
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Cathepsines L are lysosomal cysteine endopeptidases with an universal function in protein catabolism. This work discusses present knowledge about their characteristics in the context of their specific function in parasites. Features and function differences are described in detail on molecular level. The emphasis is on the biochemical properties with resultant use of these enzymes. Cathepsines L of kinetoplastida, aplikomplexa, entamoeba and helmints (focused on Fasciola spp and Schistosoma spp) are each discussed in appropriate chapters. Key words: hydrolase, protease, cysteine peptidase, cathepsin L, lysosome, parasite
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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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