|
|
Occurrence of β-rutinosidase in eukaryotic microorganisms
Adamcová, Kateřina ; Weignerová, Lenka (advisor) ; Králová, Blanka (referee)
Rutinosides are very common glycosidic aroma precursors. The glycosidic moiety influences wine aroma, flavour and taste of juices, so its cleavage has many consequences. These interesting insights led us to a diglycosidase - the extracellular β-rutinosidase from Aspergillus niger. The purified β- rutinosidase was partly analyzed by MALDI-TOF/TOF. The insert encoding for β-rutinosidase was ligated into the expression vector pPICZα A. Pichia pastoris KM71H was used as an expression system. It was find out, that β rutinosidase gene consists of a 1137 bp, encoding protein with 379 amino acids. The enzyme was determined to have relative molecule mass 60 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were found to be 3,0 and 50 řC, respectively. p-Nitrophenyl-β-rutinoside was used as a substrate Powered by TCPDF (www.tcpdf.org)
|
|
| |
|
|
Enzymatic modification of Glc-NAc-thiazoline inhibitors of hexosaminidase
Šmeringaiová, Ingrida ; Weignerová, Lenka (advisor) ; Šulc, Miroslav (referee)
This work deals with the problem of searching for effective derivatives of 1,2-dideoxy-2'- methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NGT), potential inhibitors of human β-N- acetylhexosaminidases. The work is targeted to production of inhibitors derived from NGT by the modification of its structure by free and immobilised lipases. Potential inhibitors of β-N- acetylhexosaminidases may be potent tools for studying of enzymes in cell processes and also open possibility to the discovery of new possible drugs for the treatment of some neurodegenerative diseases, such as Alzheimer disease. The thesis is focused on brief characterization of β-N-acetylhexosaminidases, e.g., glycosidases from enzymatic groups GH 20 and GH 84. β-N-Acetylhexosaminidases from microorganisms Bacteroides thetaiotaomicron, Aspergillus oryzae, Streptomyces plicatus, Talaromyces flavus and humans1 were used in the experiments. The enzymes produced were purified and then tested for their activity. We also tested inhibitory activity of potential inhibitor 6-O-acetyl-1,2-dideoxy-2'-methyl-α- D-glucopyranoso-[2,1-d]-Δ2'-thiazoline. Starting compound, NGT, was synthesized by the modified process (originally created by prof. Knapp2 ) using 2-acetamido-1,3,4,6-tetra-O- acetyl-2-deoxy-α-D-glucopyranose in the reaction with...
|
|
|
Sulphated metabolites of silybin and their interactions with sulphatases
Ježková, Zuzana ; Weignerová, Lenka (advisor) ; Kavan, Daniel (referee)
This bachelor thesis solves the question of sulphated metabolites of silybin by their interactions with sulfatases. Silybin is flavonolignan prepared from the seeds of milk thistle (Silybum marianum). Sulfate of silybin (metabolite) has not been isolated from organism so far thus its exact structure has not been determined. In this work synthesis of silybin-7,23-disulfate from natural silybin is presented. Silybin disulfate was isolated and spectrally characterized (NMR, MS). In the next step the kinetics of the reaction pNPS with sulfatase from Helix pomatia was measured: Km = 0.0494 mmol/l a Vmax = 0.0325 mmol/dm3 /min. Conditions for sulfatase reaction from Helix pomatia were selected according to literature. One of the main goal was to determine, whether silybin-7,23-disulfate is a substrate and/or an inhibitor of sulfatase from Helix pomatia. Activity measurements of the sulfatase from Helix pomatia in the presence of various concentrations of silybin-7,23-disulfate enabled us to determine that silybin disulfate at concentration higher than 0.2 mM is a strong inhibitor of the sulfatase tested. Series of reactions with sulfatase from Helix pomatia were performed to determine whether silybin-7,23-disulfate is a substrate. Reactions were monitored by HPLC and it was demonstrated that the...
|
|
|
Isolation and purification of the environmental DNA from the horizon of the capping: stratification study
Zahradník, Jiří ; Šulc, Miroslav (advisor) ; Weignerová, Lenka (referee)
According to our contemporary knowledge soil is the most abundant source of microbial biomass. Unfortunately, only one percent of the microorganism species is available by classical cultivation techniques. Soil metagenomic DNA is a collection of the whole DNA including also uncultivated microorganism in the soil sample and provides information to study molecular aspects of microorganism and their DNA sequences inaccessible by other techniques. This work is focused on characterization and isolation of soil metagenomic DNA from deep horizon of capping. Evaluation in term of the isolation and techniques of recombination of DNA and stratification study are included in this work. Obtained collection of samples was preliminary characterized with the view of quality - content of clay, humic compounds and crude number of microorganism. The purified soil metagenomic DNA was quantitatively and qualitatively characterized for each sample. The quantification method and DNA quality determined the next applications and procedures of DNA techniques. Also the soil quality was discussed from this point of view. According to the results of DNA analysis, the three selected DNA samples were processed to DNA library with 16S rRNA DNA loci and after DNA sequencing analysis the phylogenetic study was performed. This...
|
|
|
New possibilities of nitrilases in biocatalysis and bioremediation
Veselá, Alicja Barbara ; Bezouška, Karel (advisor) ; Weignerová, Lenka (referee)
Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...
|
|
| |
|
| |
|
| |
guest ::
