|
|
Vacuolar aspartic protease of Candida albicans
Hirko, Dominik ; Heidingsfeld, Olga (advisor) ; Dostál, Jiří (referee)
Aspartic proteases (APs) are crucial for diverse cellular processes. This thesis delves into the complexities of protein expression and characterization of vacuolar aspartic endoprotease Apr1p from Candida albicans, comparing it to its Saccharomyces cerevisiae ortholog, Pep4p. Recombinant expression of Apr1p in Escherichia coli yielded the inactive proenzyme, proApr1p. Extensive refolding efforts failed to produce mature, active Apr1p, suggesting a reliance on intricate cellular machinery or specific post-translational modifications for activation. Attempts to leverage vacuolar enzymes or cell lysates for proApr1p activation were unsuccessful, potentially due to the fragility of isolated vacuoles and the complex mixture of enzymes in cell lysates. Positive results emerged when Apr1p was expressed in S. cerevisiae, where fractionated cell lysates exhibited specific proteolytic activity at acidic pH after inhibiting serine and metalloproteases proteases. The eukaryotic system can probably produce active Apr1p. However, after preliminary small-scale experiments, upscaling of Apr1p expression in S. cerevisiae will be necessary in order to obtain sufficient amount of protein for further characterization. A reciprocal gene swap experiment, exchanging PEP4 in S. cerevisiae with APR1 and vice versa,...
|
|
|
Influence of some medicinal fungi and other prebiotics on growth and metabolism of probiotic bacteria
Husová, Martina ; Bendová, Agáta (referee) ; Márová, Ivana (advisor)
The bachelor thesis deals with the influence of medicinal mushrooms and other prebiotics on the growth and biological activity of probiotic bacteria. Selected genera of lactic acid bacteria Bifidobacterium and Lactobacillus can produce antimicrobial substances bacteriocins and their production depends on cultivation conditions and available nutrient sources. The theoretical review describes and characterizes probiotic bacteria, their metabolism and importance for human health. Similarly, the theoretical section focuses on prebiotics and their selected representatives, which are four species of medicinal fungi, the microscopic algae Chlorella, resistant starch, the cell biomass of the yeast Saccharomyces cerevisiae and dietary supplements such as inulin and psyllium. In the experimental part, extracts were prepared from these prebiotics by three different methods - boiling, macerating and hydrolysis. The obtained extracts were characterized in terms of macronutrient content such as carbohydrates and proteins. Based on the growth of four species of lactic acid bacteria cultured in standard medium and their production of antimicrobial substances, a strain of B. bifidum was selected for cultivation in the presence of prebiotic extracts. After determining the increase in probiotic biomass in the prebiotic extracts, the cultures were centrifuged, and the protein content of the bacterial supernatant was determined. The lactic acid content of the lyophilized probiotic extracts was determined by high-performance liquid chromatography, and finally, the antimicrobial activity was determined by broth dilution method and resazurin viability assay. The results of the antimicrobial tests were compared with the antimicrobial activity of lactic acid at the same concentration as that contained in the probiotic extracts. For the highest macronutrient content of the prebiotic extracts, extraction by boiling was the most suitable method. For insoluble polysaccharides such as resistant starch, hydrolysis might be a better option, but this requires subsequent treatment of the extract to provide suitable conditions for cultivation. The boiled extracts were the best for the growth of probiotic bacteria as well as their production of antimicrobial substances. The highest concentrations of protein and lactic acid were measured in these samples. The likely presence of bacteriocins was confirmed in all culture types. Almost all probiotic extracts showed antimicrobial activity against Staphylococcus epidermidis and Escherichia coli. The likely presence of bacteriocins was confirmed by the broth dilution method, for example in extracts containing chlorella algae or S. cerevisiae yeast, because these extracts were antimicrobial at lower concentrations than the lactic acid present.
|
|
|
Study of the Influence of Faradaic Processes on the Efficiency of Yeast Electroporation
Martinů, Dominik ; Krčma, František (referee) ; Ehlich, Jiří (advisor)
Electroporation is a widely used method in biotechnology and healthcare. It involves the application of short, intense electric pulses, which create pores in the cell membrane. These pores allow the exchange of molecules between the electrolyte and the intracellular environment. Although electroporation is a standard technique, its exact mechanism remains unknown and is the subject of current research. It is assumed that the primary mechanism of electroporation is the effect of the electric field itself. Recent studies, however, suggest that one of the accompanying mechanisms may also be the effect of reactive oxygen species (ROS) and generally the products of faradaic processes. These highly oxidizing molecules can influence both the efficiency of electroporation and cell viability. The hypothesis was that by anodizing electroporation electrodes, i.e., creating a thin dielectric layer on their surface, the production of ROS can be prevented. By subsequently comparing the efficiency of electroporation using prepared capacitive electrodes and classic faradaic electrodes, it would be possible to reveal the effect of ROS on electroporation. The production of H2O2, as a representative of ROS, was mapped using standard electroporation buffers, protocols, and various electrode materials. The concentration of H2O2 was determined spectrophotometrically by its reaction with 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by horseradish peroxidase (HRP). Electrodes were selected to achieve low, medium, and high production of H2O2. These selected electrodes were used for in vitro electroporation of Saccharomyces cerevisiae yeast, with the efficiency of permeabilization determined by electrochemical impedance spectroscopy (EIS) and fluorescence measurement of propidium iodide (PI) intake. It was found that the action of ROS is not the primary mechanism of pore formation in the cell membrane during electroporation and does not significantly affect the efficiency of this method. However, the results do not exclude their role in secondary processes that affect the kinetics of pore closure and cell viability after electroporation.
|
|
|
The enzymatic hydrolysis of waste paper - a source of raw materials for production of liquid biofuels
Lepař, Petr ; Stloukal, Radek (referee) ; Gabriel, Petr (advisor)
In diploma thesis the process of enzymatic hydrolysis of waste paper as a source for the production of liquid biofuels is discused. It follows directly the homonymous diploma thesis from Ing. Brummer, and it is based on the findings, which were solved and decided in previous work. In the theoretical part there is a summarization of basic information on the enzymatic hydrolysis of waste paper and the associated influences of various factors of the rate and degree of hydrolysis. Higher attention is paid to a waste cardboard and its pretreatment methods due to the maximalization of the yield of hydrolysis. The next part summarizes options of the fermentative production of biofuels, focusing on the method of simultaneous saccharification and fermentation, where the further appropriate organism for ethanol fermentation is discussed. The last part is about the technological process from the raw material input to the separation of ethanol. In the experimental section the pre-treatment of waste paper in order to maximize the efficiency of hydrolysis was examined. The best results were achieved using a vibratory mill. In addition, various parameters for simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the yeast Saccharomyces cerevisiae. The conversion rate of waste paper cellulose to reducing sugars was observed by spectrophotometric method by Somogyi - Nelson and the amount of produced ethanol was quantified using HPLC / RI. As a part of this thesis some conditions (amount of enzyme, substrate, nutrients, yeasts, temperature, pH, type of buffer) were optimized to maximize the effectiveness of the overall process. All experiments were carried out on corrugated cardboard, which was chosen as the most promising material for hydrolysis that was among the waste paper pulp in diploma thesis by Ing. Brummer.
|
|
|
Analytical centrifugation as a tool for characterization of microbial cells
Beránková, Barbora ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of centrifugation potential of microorganisms depending on the cultivation conditions The experimental part focuses on the sedimentation velocity and the instability index of the studied microorganisms during growth, under temperature and salt stress, the influence of the medium composition on centrifugation and the effect of osmotic strength on halophilic microbe Haloferax mediterranei. The sedimentation velocity and the instability index are increasing with increasing amount of water present in the bacterial culture of Haloferax mediterranei. The presence and type of polyhydroxyalkanoate (PHA) play a role in the monitoring of growth of bacteria Cupriavidus necator H16, Cupriavidus necator PHB-4, Haloferax mediterranei, Burkholderia cepacia and Burkholderia sacchari at the rate of sedimentation and instability index. Bacteria Cupriavis necator H16 with the highest content of poly(3-hydroxybutyrate) present was the fastest sedimentating and most unstable of the bacteria studied. Bacteria Cupriavidus necator H16 and Cupriavidus necator PHB-4, exposed to temperature and salt stress, exhibited only a slight influence on the rate of sedimentation and instability under temperature stress. The sedimentation velocity and instability index decreased with saline stress with increasing concentration of NaCl solution in culture. When comparing the media composition to polyhydroxyalkanoate (PHA) producing bacteria, Cupriavidus necator H16 cultivated in the mineral medium demonstrated the lowest sedimentation velocity and instability index rates, but Cupriavidus necator PHB-4 reached the lowest sedimentation velocity and instability index when grown in Nutrient Broth medium. For yeast Saccharomyces cerevisiae, the choice of medium had only a slight effect on sedimentation velocity and instability index.
|
|
|
Investigation of yeast properties during the beer fermentation
Kociánová, Lenka ; Vítová, Eva (referee) ; Omelková, Jiřina (advisor)
In this diploma thesis, the properities of brewery’s yeasts during the operation beer fermentation in the selected brewery and dependences of diferent agents and their influence on viability and vitality of the yeasts were studied. Other observed properities were physiological state, pH, temperature, a value of soft and coarse sludges, a value of dissolved oxygen in wort, the level of wort’s fermentation. It were collected the samples of fermenting wort and washing yeasts for determination. The used yeasts were observed from the first time until thein last used (generally 4 times). pH, temperature, apparent fermenting and total numer of yeast cells were measured in the collected samples. The used yeasts were washed before every other application and vitality was determined. The number of death cells were observed by this method. The number of death cells never fell bellow 5 %. The value of dissolved oxygen in wort is also the important agent for the growth of yeasts. The touchs with low number of oxygen showed that the time for fermentation had to be longer (by about 3 or 4 days more). The touchs with higher number of sludges showed that the sludges haven’t any influence on the yeasts and the procces of fermentation. Confirm the accuracy of the repeated used of the yeasts was the object of this thesis.
|
|
|
Emergent properties of the G1/S network
Dražková, Jana ; Tomášek, Petr (referee) ; Palumbo,, Pasquale (advisor)
Tato práce se zabývá buněčným cyklem kvasinky Saccgaromyces cerevisiae. Oblastí našeho zájmu je přechod mezi G1 a S fází, kde je naším cílem identifikovat velikosti buňky v době počátku DNA replikace. Nejprve se věnujeme nedávno publikovanému matematickému modelu, který popisuje mechanismy vedoucí k S fázi. Práce poskytuje detailní popis tohoto modelu, stejně jako časový průběh některých důležitých proteinů či jejich sloučenin. Dále se zabýváme pravděpodobnostním modelem aktivace replikačních počátků DNA. Nově uvažujeme vliv šíření DNA replikace mezi sousedícími počátky a analyzujeme jeho důsledky. Poskytujeme také senzitivní analýzu kritické velikosti buňky vzhledem ke konstantám popisujícím dynamiku reakcí v modelu G1/S přechodu.
|
|
|
A study of the possibility of waste pastries using for the bioproduction of selected metabolites
Hudečková, Helena ; Vránová, Dana (referee) ; Babák, Libor (advisor)
The aim of this diploma thesis was to study the possibility of using waste bread to bioproduction of selected metabolites. As bakery waste was used waste bread that came from coffee-house “Zastávka”. Bread was pre-treated by grinding into small particles and then it was made to form 15% w/v suspension, which was subjected to enzymatic hydrolysis. For the hydrolysis has been used the -amylase for liquefaction of the substrate and that was followed by a glucoamylase which sacharificated the substrate. There have been several methods of hydrolysis from which was chosen the optimal method for pre-treatment of the substrate prior to fermentation. The effectivity and a process of hydrolysis were determined spectrophotometrically by Somogyi-Nelson method. Final yields of glucose from hydrolysis were determined by HPLC method. Enzymatic hydrolysis was followed by fermentation, which was carried out in two ways, namely by adjusting the pH of the hydrolyzate to pH 5, and without pH adjustment. During the fermentation was carried out sampling in which was determined the content of glucose, glycerol and ethanol by HPLC method. The yeasts Saccharomyces cerevisiae were used for the fermentation which was performed at 30 °C. High yield of glucose was achieved by hydrolysis in two steps (70,28 gl-1), but for the fermentation was used mixed hydrolysis (second method of mixed hydrolysis) with yield 67,94 gl-1. High ethanol yield was achieved during fermentation without treatment pH, namely 31,5 gl-1.
|
|
|
Study on potential applications of glutamic acid polymer
Čangelová, Katarína ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.
|
|
|
A bioconversion study of cellulosic waste to ethanol using yeasts systems
Čalová, Iveta ; Vítová, Eva (referee) ; Babák, Libor (advisor)
This diploma thesis deals with the optimization of the production of ethanol from waste paper using yeast. There were used 4 kinds of paper as a substrate - office paper, non-recycled workbook, recycled workbook and newspaper. All papers were pretreated with the following procedures: grinding, microwaves + NaOH, microwave + H2SO4 and microwave + H2SO4 + NaOH. The glucose concentration was determined in enzymatic hydrolysis by HPLC. Saccharomyces cerevisiae were chosen for ethanol production. The production of ethanol was carried out with all the pretreated papers in simultaneous saccharification and fermentation. During hydrolysis, the pretreated papers have reached the highest results in the combination with microwave + H2SO4 + NaOH. Non-recycled workbook was the only exception, where the highest concentration of glucose has been obtained by the pretreatment of microwaves + H2SO4. Following results have been acquired: office paper 24,69 gdm-3, non-recycled workbook 22,47 gdm-3, recycled workbook 16,94 gdm-3 and newspapers 15,36 gdm-3. SSF was carried out again with all the papers and their pretreatments. The highest concentration of ethanol has been achieved in microwave pretreatment + H2SO4 + NaOH. The highest overall concentration has been gained from the office paper, amounted to 16,98 gdm-3. The maximum concentration of ethanol for non-recycled workbook has been 15,25 gdm-3, for recycled workbook 12,2 gdm-3 and for newspapers 12,59 gdm-3.
|
guest ::
