|
|
Investigation of the β-N-Acetylhexosaminidase Stucture from Penicillium oxalicum.
Krunclová, Tereza ; Novák, Petr (advisor) ; Hýsková, Veronika (referee)
in English β-N-Acetylhexosaminidase (EC 3.2.1.52) is exoglycosidase, which exhibits the unique properties in the filamentous fungi. Enzyme from these organisms are dimeric, inducible and secreted extracelluary. It is expresed as preproprotein, consists of a signal sequence, a large propeptid and a catalytic subunit. Although the enzyme is widely distributed, its structure differs in varies organisms. Bacteria have only monomeric hexosaminidase. Human enzymes are dimeric as well as fungal, but only hexosaminidase from filamentous fungi have the catalytic subunit noncovalently associated with the propeptide. Propeptide is a essential for the enzyme activity. It exists a homologues model of the catalytic subunit of β-N-acetylhexosaminidase from Penicillium oxalicum, but the structure of the propeptide has not yet been solved. The first part of this diploma thesis deals with the optimization of production and purification conditions. The second part deals with structural studies of β-N-acetylhexosaminidases from the filamentous fungi Penicillium oxalicum CCF 3438. These studies were carried out using chemical cross-linking and high resolution mass spectrometry. The combination of these methods revealed region of the noncovalent interaction of the catalytic subunit with the propeptide.
|
|
|
Applications of capillary electrophoresis in life sciences
Křížek, Tomáš ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Tůma, Petr (referee)
Tomáš Křížek: Applications of Capillary Electrophoresis in Life Sciences (Dissertation thesis) ABSTRACT This thesis is focused on the applications of capillary electrophoresis in two important areas of life sciences, proteomics and enzyme assays. In the first part, Pluronic F-127 copolymer was studied as a sieving matrix for proteomic applications of capillary gel electrophoresis. The effect of thermoassociation of Pluronic F-127 on the separation selectivity was investigated and no difference in selectivity of the separation below, inside and above the thermoassociation temperature region was observed. The performance of Pluronic F-127 in capillary gel electrophoresis was compared with dextran as a commonly used sieving matrix. The results showed, that Pluronic F-127 offers superior performance for low-molecular-mass proteins because it provides higher separation power than dextran with significantly lower viscosity of the background electrolyte. The lower viscosity makes the polymer easier to replace after each analysis, which leads to remarkably higher repeatability of the experiments. On the other hand, dextran, due to its higher viscosity, was shown to be more convenient for separations of protein digests, where extremely high separation efficiency is required. The second part focuses on...
|
|
|
Investigation of the β-N-Acetylhexosaminidase Stucture from Aspergillus oryzae
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Ryšlavá, Helena (referee)
in English β-N-acetylhexosaminidase (EC 3.2.1.52) belongs to exoglycosidase, and is one of the most abundant enzymes found in organisms from bacteria to human. The fungal β-N-acetylhexosaminidase from Aspergillus oryzae is composed of propeptide and catalytic domain. The propeptide is noncovalently associated with the catalytic domain of the enzyme. Propeptide is essential for the enzyme activity. While the structure of the catalytic domain was desidned by homology modeling, the structure of the propeptide has not been resolved yet. In this study, the position where the propeptide is associated with the catalytic domain, was uncovered. Presented work consists of two parts. First part deals with optimization of production conditions, purification and crystallization of β-N-acetylhexosaminidase from the filamentous fungi Aspergillus oryzae. Second part is devoted to the study of interaction between propeptide and catalytic domain, which was characterized by chemical cross-linking and high-resolution mass spectrometry. It was found that the structural changes of the catalytic domain depend on the presence of the propeptide molecule. Moreover the region of propeptide-catalytic domain interaction was revealed.
|
guest ::
