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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.
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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.
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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...
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Study of β-N-acetylhexosaminidase in tobacco plants
Valenta, Robert ; Tichá, Marie (referee) ; Ryšlavá, Helena (advisor)
β-N-acetylhexosaminidases are ubiquitous in all living organisms, but there is little information about these enzymes in plants, especially in leaves. Suggested functions of plant β-N-acetylhexosaminidases are participation in defence responses against fungal and other pathogens and also in degradation of storage glycoproteins. β-N-acetylhexosaminidase from tobacco leaves (Nicotiana tabacum L.) was purified to final specific activity 190 nmol min-1 mg-1 with p-NP-GlcNAc as substrate. Precipitation by ammonium sulphate, gel filtration chromatography on Sephacryl S-300 column and affinity chromatography on Con A- Sepharose column were used. Michaelis constant and maximal reaction rate of β-N- acetylhexosaminidases determined for p-NP-GlcNAc was 0.33 mM and 414 nmol min-1 mg-1 , respectively. The cleavage of diacetylchitobiose catalyzed by β-N-acetylhexosaminidase was studied using capillary electrophoresis. Determined activity of β-N-acetylhexosaminidase for diacetylchitobiose was more than 10-times lower compared to β-N-acetylhexosaminidase activity for p-NP-GlcNAc. These results indicate that chitobiose and probably other chitooligomers are not natural substrates of β-N-acetylhexosaminidase and thus this enzyme is most likely not involved in protection against fungal pathogens. Natural substrate...
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