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Enzyme content monitoring in wheat malt
Šubertová, Hana ; Benešová, Karolína (referee) ; Hartman,, Ivo (advisor)
The aim Master thesis was to evaluate the influence of malting technology on the content of hydrolytic enzymes -amylase and -amylase. Another aspect of this thesis was to select a wheat variety with the highest activity of enzymes -amylase and -amylase. The first part of the practical part of my work is shown measuring enzyme malt model varieties of wheat set (Triticum aestivum), where individual samples differ technological processing of malting and the second part is focused on the application of efficient malting conditions on other varieties from different locations in the Czech Republic in 2016 . All malt samples were analysed to -amylase and -amylase activity by assay Ceralpha method and Betamyl-3 method made by Megazyme company. After measuring the first part, the most effective malting condition was evaluated, such as a 7 day malting time, a 45% water content, a germination temperature of 18 ° C, and a drying temperature of 50 ° C. In the second part, the varieties are so malted and the highest content of enzyme -amylase was Rumor, it had averafe contanin -amylase 277 U/g, Bonanza – 255 U/g and Izzy –254 U/g. The biggest contain of enzymes -amylasehad varieties Matchball – 37,9 U/g , Fakir – 35,6 U/g, and Bernstein – 35,3 U/g.
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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.
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Incorporation of glucose oxidase into hydrogel structures
Suchá, Klára ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the incorporation of the enzyme glucose oxidase into hydrogel structures, while the activity of the enzyme after the incorporation into the hydrogel structure was monitored. Glucose oxidase was incorporated into the agarose hydrogel at various concentrations (1 and 2 wt. %). Glucose oxidase activity was determined using the resulting hydrogen peroxide, the concentration of which was measured by fluorescence spectroscopy using an Amplex Red fluorescence probe. The enzyme was found to be active even after incorporation into the hydrogel, but the concentrations of hydrogen peroxide formed were lower compared to the free enzyme. Furthermore, it was found that the enzyme reacts with agarose itself, but this reaction did not significantly affect the rheological properties of the hydrogel.
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Incorporation of glucose oxidase into hydrogel structures
Suchá, Klára ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the incorporation of the enzyme glucose oxidase into hydrogel structures, while the activity of the enzyme after the incorporation into the hydrogel structure was monitored. Glucose oxidase was incorporated into the agarose hydrogel at various concentrations (1 and 2 wt. %). Glucose oxidase activity was determined using the resulting hydrogen peroxide, the concentration of which was measured by fluorescence spectroscopy using an Amplex Red fluorescence probe. The enzyme was found to be active even after incorporation into the hydrogel, but the concentrations of hydrogen peroxide formed were lower compared to the free enzyme. Furthermore, it was found that the enzyme reacts with agarose itself, but this reaction did not significantly affect the rheological properties of the hydrogel.
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Study of phosphorylation of inorganic pyrophosphatase from Streptococcus pneumoniae
Štechová, Michaela ; Doubravová, Linda (advisor) ; Svobodová, Jaroslava (referee)
The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...
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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.
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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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Enzyme content monitoring in wheat malt
Šubertová, Hana ; Benešová, Karolína (referee) ; Hartman,, Ivo (advisor)
The aim Master thesis was to evaluate the influence of malting technology on the content of hydrolytic enzymes -amylase and -amylase. Another aspect of this thesis was to select a wheat variety with the highest activity of enzymes -amylase and -amylase. The first part of the practical part of my work is shown measuring enzyme malt model varieties of wheat set (Triticum aestivum), where individual samples differ technological processing of malting and the second part is focused on the application of efficient malting conditions on other varieties from different locations in the Czech Republic in 2016 . All malt samples were analysed to -amylase and -amylase activity by assay Ceralpha method and Betamyl-3 method made by Megazyme company. After measuring the first part, the most effective malting condition was evaluated, such as a 7 day malting time, a 45% water content, a germination temperature of 18 ° C, and a drying temperature of 50 ° C. In the second part, the varieties are so malted and the highest content of enzyme -amylase was Rumor, it had averafe contanin -amylase 277 U/g, Bonanza – 255 U/g and Izzy –254 U/g. The biggest contain of enzymes -amylasehad varieties Matchball – 37,9 U/g , Fakir – 35,6 U/g, and Bernstein – 35,3 U/g.
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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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Study of phosphorylation of inorganic pyrophosphatase from Streptococcus pneumoniae
Štechová, Michaela ; Svobodová, Jaroslava (referee) ; Doubravová, Linda (advisor)
The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...
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