|
|
Selected microbial processes in a bioreactor
Klinková, Lucie ; doc.RNDr.Petr Zbořil, CSc. (referee) ; doc.Ing.Martin Mandl, CSc. (advisor)
This thesis focuses on the study of the influence of selected parameters on the course of microbial cultivation and evaluation of bioprocess. It is divided into two parts. The first part deals with the production of mutant forms of the protein cryptogein yeast Pichia pastoris. The theoretical part summarizes the findings of the yeast P. pastoris and its expression. It also deals with cryptogein that induces defense reactions in plants. In the experimental part was produced mutant cryptogein X24, in which the concentration of each fraction and the ability to transfer sterols. The second part of this thesis is focused on aerobic and anaerobic oxidation of elemental sulfur by Acidithiobacillus ferrooxidans. In the theoretical section, our knowledge on A. ferrooxidans, its metabolism and the importance of ATP in cell metabolism was summarized. In the experimental part, the above bioprocess was monitored using pH, biomass concentration, the rate of oxidation of elemental sulfur the cellular ATP content.
|
|
|
Rutinosidase from Aspergillus niger - analysis of active site and its mutagenesis
Šidáková, Anna ; Bojarová, Pavla (advisor) ; Palyzová, Andrea (referee)
Rutinosidases (α-L-rhamnosyl-β-D-glucosidases) from Aspergillus niger (AnRut) are glycosidases (EC 3.2.1) that catalyze the hydrolysis of the glycosidic bond between the aglycone and the disaccharide residue rutinose. The dual substrate specificity of this enzyme group describes the parallel activity towards the substrates rutin (carrying a rutinosyl disaccharide residue) and isoquercitrin (carrying a glucosyl residue). The active site of AnRut is more complex than that of other glycosidases and is composed of the catalytic amino acids Glu210 and Glu319 in the active-site cleft and a side tunnel. This untraditional structure with distinct interactions in the tunnel and active-site cleft is the probable reason for the enzyme exceptional substrate specificity. Through point or multiple mutations of the enzyme, we can modify its primary and secondary structure, thus causing a significant shift in substrate specificity. The main goal of this thesis is the analysis of three distinct mutant variants of AnRut rutinosidase; their production, purification, and the study of the influence of the mutations on the substrate specificity of the enzymes. All variants were designed based on molecular modeling. The substrate specificity was determined by reactions of the mutant variants with previously unstudied...
|
|
|
Mutant glycosidases with a high substrate specificity and their analysis
Nekvasilová, Pavlína ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
β-N-acetylhexosaminidases (EC 3.2.1.52, GH 20) are retaining exo-glycosidases that in vivo cleavage both β-N-acetylglucosamine (GlcNAc) or β-N-acetylgalactosamine (GalNAc) residues fom glycostructures. Under suitable reaction conditions, these enzymes are able to synthesize the glycosidic bond in good yields. Substitution of selected amino acid(s) in the emzyme active site by site-directed mutagenesis may change the enzyme's substrate specificity or suppress the hydrolytic activity of the enzyme in favor of synthesis. The present thesis deals with three mutant β-N-acetylhexosaminidases from Talaromyces flavus, in which the amino acid residues responsible for binding to C-4 hydroxyl of the substrate (Arg218, Glu546) were exchanged for amino acids proposed on the basis of molecular modeling. The effect of introduced single point mutations on substrate specificity of prepared enzymes was studied. Mutant β-N-acetylhexosaminidases were heterologously expressed in Pichia pastoris and characterized. Furthermore, transglycosylation reactions with these enzymes were performed. The prepared carbohydrate products were characterized by NMR.
|
|
| |
|
|
Studies on NKR-P1A and NKR-P1B receptors expressed in eukaryotic organisms
Ivanova, Lyubina ; Bezouška, Karel (advisor) ; Rychlovský, Petr (referee)
NK (natural killer) cells, with their ability to identify antigens and extraneous substances, available in the organism through various moleculary receptors, are an important component of the immune system. The NKR-P1A and NKR-P1B proteins belong to the lectin receptors of natural killer cells. Primary ligands of lectin receptors comprise terminal oligosaccharides of glycoproteins on the surface of target (e.g. tumor) cells. The interaction between carbohydrate structures on the surface of antigens and their binding partners on NK receptors is followed by triggering the effector function of NK cells against the targets. The NK cells and NK receptors findings and their interactions with ligands are greatly utilized in the treatment of cancer, viral and autoimmune diseases. Heterologous protein production in the eukaryotic organism brings a lot of advantages. Unlike the prokaryotic organism, the methylotrophic yeast Pichia pastoris has the capability of performing many posttranslational modifications resulting in production of biological active protein molecule. Usually, the P. pastoris expression system disposes of high level protein expression and is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from other eukaryotes. In this thesis, I...
|
|
|
Integrated development of a bioprocess: From the soil enzyme to the yeast production platform
Borčinová, Martina ; Kyslík, Pavel (advisor) ; Uhlík, Ondřej (referee) ; Hasal, Pavel (referee)
For a sustainable future, there is a call to increase the market share of bio-based technologies and materials. Microbial-based technologies have the potential and the ability to contribute substantively on many levels to global efforts to achieve sustainability. Development and utilization of microbial technologies is, however, an extensive process involving numerous steps, including the discovery of novel technologies and the development of industrially viable production systems. In the presented thesis, individual steps of microbial biotechnology development were addressed. In the first part of the study, a variety of methodological approaches were employed in order to study the effect of the anthropogenic activity (i.e., decades lasting production of penicillin G) on the structure of soil microbial communities. Moreover, both cultivable and non-cultivable fractions of populations were subjected to functional screening in order to unravel the biotechnological potential of the microorganisms in terms of production of enzymes involved in biotransformation of beta-lactam antibiotics: penicillin G acylase (PGA) and alpha amino acid ester hydrolase (AEH). Our results indicated that the impacted communities harbour a microbial community with increased diversity and richness. However, on the...
|
|
| |
|
|
Mutant glycosidases with a high substrate specificity and their analysis
Nekvasilová, Pavlína ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
β-N-acetylhexosaminidases (EC 3.2.1.52, GH 20) are retaining exo-glycosidases that in vivo cleavage both β-N-acetylglucosamine (GlcNAc) or β-N-acetylgalactosamine (GalNAc) residues fom glycostructures. Under suitable reaction conditions, these enzymes are able to synthesize the glycosidic bond in good yields. Substitution of selected amino acid(s) in the emzyme active site by site-directed mutagenesis may change the enzyme's substrate specificity or suppress the hydrolytic activity of the enzyme in favor of synthesis. The present thesis deals with three mutant β-N-acetylhexosaminidases from Talaromyces flavus, in which the amino acid residues responsible for binding to C-4 hydroxyl of the substrate (Arg218, Glu546) were exchanged for amino acids proposed on the basis of molecular modeling. The effect of introduced single point mutations on substrate specificity of prepared enzymes was studied. Mutant β-N-acetylhexosaminidases were heterologously expressed in Pichia pastoris and characterized. Furthermore, transglycosylation reactions with these enzymes were performed. The prepared carbohydrate products were characterized by NMR.
|
|
|
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)
|
|
|
Studies on NKR-P1A and NKR-P1B receptors expressed in eukaryotic organisms
Ivanova, Lyubina ; Rychlovský, Petr (referee) ; Bezouška, Karel (advisor)
NK (natural killer) cells, with their ability to identify antigens and extraneous substances, available in the organism through various moleculary receptors, are an important component of the immune system. The NKR-P1A and NKR-P1B proteins belong to the lectin receptors of natural killer cells. Primary ligands of lectin receptors comprise terminal oligosaccharides of glycoproteins on the surface of target (e.g. tumor) cells. The interaction between carbohydrate structures on the surface of antigens and their binding partners on NK receptors is followed by triggering the effector function of NK cells against the targets. The NK cells and NK receptors findings and their interactions with ligands are greatly utilized in the treatment of cancer, viral and autoimmune diseases. Heterologous protein production in the eukaryotic organism brings a lot of advantages. Unlike the prokaryotic organism, the methylotrophic yeast Pichia pastoris has the capability of performing many posttranslational modifications resulting in production of biological active protein molecule. Usually, the P. pastoris expression system disposes of high level protein expression and is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from other eukaryotes. In this thesis, I...
|
guest ::
