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Biotransformation of phenols by enzymatic systems of Candida tropicalis yeast and Comamonas testosteroni bacteria
Vilímková, Lenka ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Mareš, Jaroslav (referee)
Candida tropicalis yeast and bacteria Comamonas testosteroni have been considered to be able to metabolize phenol and utilize it as the only source of carbon and energy. In our laboratory we investigated the cytoplasmic enzymes responsible for the first and second step of phenol degradation, NADPH-dependent phenol hydroxylase of both C. tropicalis and C. testosteroni and catechol 1,2-dioxygenase of C. tropicalis. The aim of our study was to isolate and partially characterize those enzymes. Phenol hydroxylase purification consisted of preparation of cytosol from C. tropicalis yeast by fraction centrifugation, chromatography and re-chromatography on a column of DEAE Sepharose, fractionation by precipitation of the enzyme with polyethylene glycol 6000 and gel permeation chromatography on a column of Sephacryl S-300. Extracellular phenol hydroxylase of C. testosteroni was purified by fraction precipitation with polyethylene glycol 6000 and by gel permeation chromatography on 4B Sepharose and Sephacryl S-300. Catechol 1,2-dioxygenase was purified using the procedure consisting of: chromatography and re- chromatography on a column of DEAE Sepharose, lyophilization of the enzyme and gel permeation chromatography on a column of Sephadex G-100. The enzyme activity was determined by two methods: use of HPLC...
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Asymmetric biotransformation of certain tetralones to tetralols
Andrejch, Jan ; Poljaková, Jitka (advisor) ; Martínek, Václav (referee)
Preparation of pure enantiomers is very important part of chemical synthesis of industrially and pharmaceutically useful compounds. In recent years traditional chemical synthesis is replaced by biotransformations. Biotransformation means the use of isolated enzymes, enzymatic systems or whole cells in catalysis of chemical reactions. In comparison with chemical synthesis it has certain advantages. In this work the activity of yeast strains Saccharomyces uvarum K8, Saccharomyces cerevisiae K12, Torulopsis azyma K43, Torulopsis sphaerica K46 and Candida tropicalis K49, K51 a K52 in catalysis of reduction 6- bromo-2-tetralone, 7-hydroxy-2-tetralone and 7-methoxy-2-tetralone was studied. Products of these reductions are used in preparation of pharmaceutical drugs. Suitable conditions for biotransformations were found and it was proved that the yeast strains Saccharomyces uvarum K8 and Candida tropicalis K52 are able to produce 2- tetralones with ee greater than 90% with conversion 100% after four-hour biotransformation. This thesis is written in Czech.
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Se-Metabolism inside the mammalian organism fed Se-supplemented Brassica napus forage
Žíla, Ondřej ; Čadková, Zuzana (advisor) ; Václav, Václav (referee)
The aim of the thesis was to determine whether the individual Se-speciation in the mammalian organism are affected by the form of received selenium. Selenium is an essential micronutrient important for humans and animals. It plays an important role in the antioxidant protection of the organism and in the conversion of thyroid hormones. In our experiment the laboratory Wistar rats were divided into three groups. Each group had a different diet. The rats were fed with selenium in the form of soy, sodium selenite and extracted rapeseed meal. Urine samples were regularly collected during the four-week experiment and in the end of the feeding study, the blood serum was also collected. The total selenium content was measured by ICP-MS, while the individual Se-speciation in urine and serum by HPLC coupled with ICP-MS. In the urine the identified speciation were methylselenocystein (MeSeCys), trimethylselenium (TMSe) and selenosugar 1 and 3. In the blood serum the measured speciation were TMSe, selenite, selenate and selenosugar 1. For the group fed with sodium selenite the measured values in the urine were generally higher, this might be due to a higher overall intake and also an inorganic form of selenium with a lower absorbency. Groups that received selenium from plant sources took in several Se-compounds and the total measured content of Se-speciation and secretion dynamics were not significantly different. Additionally speciation of selenosugar 2 was measured for the group fed with rapeseed meals, which in the other groups did not appear. When receiving selenium from plant sources the biotransformation in the mammalian organism differs in comparison to receiving selenium from mineral salts. The initial hypothesis that Se-speciation is influenced by the form of selenium administered in the diet was confirm by our results. Since the group fed rapeseed showed similar results as the group fed a standard feed with soy, the extracted rapeseed meal could serve as a good source in livestock nutrition.
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