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Characterization of specific proteins form selected animal products.
Janhuba, Filip ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.
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The changes of Rubisco amount in Norway spruce grown at gradient of altitudes
Tomasz, Teresa ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
The study deals with differences of Rubisco enzyme content in Norway spruce needles (Picea abies L.) growing at different altitudes, thus at different concentrations of carbon dioxide. The collection of samples was performed in Jeseníky by Global Change Research Centre, Academy of Sciences of the Czech Republic. The samples of exposed and shaded needles were collected from 39 spruce trees in three altitudes – low (L), medium (M) and high (H). To separate Rubisco enzyme, the SDS-PAGE method was used, the content od Rubisco was determined by computer densitometry. The results of exposed needles confirmed that Rubisco content decreases with increasing altitude, however, in the case of shaded needles, there was no proof of a significant influence of altitudes on Rubisco enzyme amount. At low altitude, Rubisco content was significantly higher in exposed needles in comparison with shaded needles. At medium and high altitude no significant difference was detected between exposed and shaded needles.
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Impact of elevated carbon dioxide concentration on Rubisco content in Fagus sylvatica and Picea abies
Hamříková, Dominika ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
In this bachelor’s thesis content of Rubisco in beech (Fagus sylvatica) and Norway spruce (Picea abies) was studied. The plants were cultivated in conditions with ambient carbon dioxide concentration (350 mol·mol-1) and elevated carbon dioxide concentration (700 mol·mol-1). The content of Rubisco in exposed and shaded leaves or needles was compared. The content of Rubisco was determined by SDS-PAGE method. A significant decrease in Rubisco content was demonstrated in conditions E versus A in shaded beech leaves. In other cases, the decrease was not so significant. A comparison of exposed and shaded needles also did not show a statistically significant difference, but in shaded beech leaves against exposed leaves Rubisco content was significantly lower, especially in conditions E.
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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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Patatin proteins relative abundance in correlation to antioxidative activity of tubers of cultivated and wild potato species
MEJZLÍK, Petr
The aim of the thesis was to determine the content of pure tuber proteins, patatin relative abundance in total tuber protein, and subsequently its antioxidant potential. The theoretical part dealt with general potato characteristics, potato tuber chemical composition and description of analyzed cultivated and wild potato species. In the practical part results of laboratory analysis were presented. It was determined statistically significant genotype variability of antioxidant activity for potato tuber protein as well as for potato tuber dry matter here ranging from 0.221 (Solanum berthaultii 07S0300031) to 0.0066 mg of ascorbic acid/g dry matter (Solanum tuberosum cultivar Kuras). Any direct relationship between potato tuber protein content or patatin relative abundance and antioxidant activity was found. The findings are compared with the literature.
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Evaluation of storage protein profiles modifications in barley (\kur{Hordeum vulgare} L.) grains during malting procedure using gel electrophoresis
BINDER, Richard
This bachelor thesis is focused on evaluation of storage protein profiles modifications in barley (Hordeum vulgare L.) grains during malting procedure using gel electrophoresis. It has been analyzed three varieties of spring barley. One nonmalting variety Herris and two malting varieties Radegast and Sebastien. There are significant proteolysis changes during the malting procedure. Theese changes of storage proteins were captured and evaluated by gel electrophoresis (SDS-PAGE) on the basis of changes in the molecular weight in the different phases of malting procedure. Next observation which has been done was the measurement of the thousand grain weight (TGW) before and after the malting procedure. Theese results have been evaluated by statistic program.
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