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The influence of alkali activator on immobilization of metals in alkali activated blast furnace slag
Bystrianska, Emília ; Kalina, Lukáš (referee) ; Koplík, Jan (advisor)
In this work the influence of alkaline activator on immobilization of lead and copper in alkali-activated blast furnace slag was investigated. A total of five activators were used; sodium water glass, potassium water glass, sodium hydroxide, potassium hydroxide and sodium carbonate. The leaching test according to ČSN EN 12457-4 was used to evaluate the level of immobilization of heavy metals, the leached solutions were analyzed by ICP-OES. For a better understanding of immobilization, the selected samples were characterized by analytical methods (FTIR, XRD, SEM, XPS). It was found that the degree of immobilization Pb2+ and Cu2+ in AAS was very high, regardless of the type of alkaline activator used.
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Primer Extension Reaction at the Gold Electrode Surface
Vidláková, Pavlína ; Havran, Luděk ; Fojta, Miroslav
One option of covalent DNA labeling is incorporation of chemically modified nucleotides using corresponding deoxynucleotide triphosphates into DNA molecules using polymerase-catalyzed primer extension. Primer extension is usually performed in solution and then the product is analyzed, often after a suitable separation. In our study we tested the possibility to prepare oligonucleotides with enzymatically incorporated anthraquinone labeled nucleotides using primer extension on templates immobilize at a gold electrode surface.
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Electrochemical Analysis of Chemically Modified DNA at Gold Electrodes
Vidláková, Pavlína ; Havran, Luděk ; Fojta, Miroslav
DNA labeling is used to increase sensitivity of its electrochemical detection. Covalently bound markers based on a complex of osmium tetroxide with 22-bipyridine(Os,bipy) is one of those frequently applied. Os bipy reacts with pyrimidines (mainly thymines) in single stranded regions of DNA, therefore it represents a useful marker for the analysis of DNA structure or labeling of hybridization probes . Another possibility of DNA labeling is the incorporation of chemically modified dNTPs into DNA molecules with using primer extension. DNA labeling in connection with voltammetry at gold electrodes has application potential in development of sensors for studying of DNA structure and interaction.
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Immobilization of protein macromolecules onto polymer carriers: An overview
Badalcová, Helena ; Holas, Ondřej (advisor) ; Svačinová, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Helena Badalcová Title of Thesis: Immobilization of protein macromolecules onto polymer carriers: An overview Since the 70s, the immobilised enzymes have been getting the attention of not only scientific and laboratory workers, but also industrial companies. Enzymes are unique biocatalysts, which are distinguished by their specificity, environment-friendliness and the ability to react under mild conditions can be easily subject of denaturation or inhibition. With regard to the usually high cost of purchase, the use of these enzymes could often be disadvantageous. Immobilization techniques offer an efficient solution to this problem and greatly simplify the use of enzymes in industry and research. Compared to the free forms, immobilized enzymes show greater activity, stability and allow repeated use as well as easier separation from products. This thesis contains an overview of the basic methods of immobilization - physical absorption and covalent bonds to the carrier, entrapment, encapsulation and carrier- free techniques using cross-linking. Finally, we outline possible biomedical applications as well as the use of immobilised enzymes in biosensors.
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The influence of different anions on fixation of Cu in alkali-activated blast furnace slag
Böhm, Petr ; Kotrla, Jan (referee) ; Koplík, Jan (advisor)
The work deals with the fixation of copper in alkali activated blast furnace slag and with the influence of anionts (sulphate, nitrate, chloride and oxide) to its immobilization. This is tested according to leaching tests ČSN EN – 12457 4. The concentration of copper in leachates was determined by inductively coupled plasma optical emission spectrometry. The aim of this work is to check the influence of addition (copper) to the resulting mechanical properties and also specify, how the heavy metal is bonded in the matrix. The structure was examined by scanning electron microscopy.
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Protein macromolecules immobilization onto polymer carriers
Šitnerová, Michaela ; Holas, Ondřej (advisor) ; Ondrejček, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Michaela Šitnerová Title of Thesis: Protein macromolecules immobilization onto polymer carriers Enzymes are unique biocatalysts because of their properties. They are highly specific, selective and functional even under mild reaction conditions. The method of immobilization is used to increase their operational stability, activity and possible reuse. This process allows the wide use of enzymes in industry, for example in the food industry, analytical chemistry, chemical synthesis and in the pharmaceutical industry. The aim of my thesis was immobilized enzyme acetylcholinesterase (AChE) on the surface pellets of microcrystalline cellulose (MCC). Used method was simple sorption, immobilization using glutaraldehyde, and TEMPO oxidation using MCC. Well known Ellman's method served to measure the activity of AChE. The absorbance of the solution with the immobilized AChE was measured spectrophotometrically at 412 nm.
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Chemo-enzymatic synthesis of antiviral prodrugs
Tupec, Michal ; Stiborová, Marie (advisor) ; Indra, Radek (referee)
Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it...
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav ; Ledvinová, Jana (advisor) ; Stiborová, Marie (referee) ; Holčapek, Michal (referee)
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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Preparation of Microbial Metabolites from Waste Materials
Zichová, Miroslava ; Márová, Ivana (referee) ; Stloukal, Radek (referee) ; Rosenberg, Michal (advisor)
In this thesis the use of waste materials for the microbial production of important metabolites is reported. The first part is focused on the use of waste paper (a lignocellulosic material) as a non-traditional source for the production of bioethanol. The second part is focused on the immobilization of cellulolytic enzymes, which are used for the hydrolysis of lignocellulosic materials. First, the waste paper (cardboard) was pre-treated using a blender and a vibratory mill. The pre-treated cardboard was used for the production of ethanol by the method of simultaneous saccharification and fermentation. This method was optimized with free cells of Saccharomyces cerevisiae. Then strains suitable for the immobilization were selected. Strains of S. cerevisiae and Pichia kudriavzevii were immobilized by encapsulation into the polyvinyl alcohol carrier and tested again for the ethanol production by simultaneous saccharification and fermentation. In the second part of the work a carrier from waste polyethylene terephthalate bottles was prepared and used for the immobilization of the cellulolytic complex. The basic characteristics were determined, such as optimal pH and optimal temperature, storage, operational and thermal stability, enzyme kinetics and the mode of action of the enzyme. Compared to two other commercial carriers this carrier showed to be suitable for the immobilization of the cellulolytic complex.
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