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Imobilizace galaktosidasy a její využití v potravinářské technologii
Dobešová, Natálie
This master‘s thesis deals with the issue of galactosidase immobilization and its use in food technology. β-galactosidase from Kluyveromyces lactis was immo-bilized using two methods, specifically encapsulation and cross-linking. The aim of the encapsulation was to obtain hydrogel capsules formed by a biopolymer matrix based on sodium alginate, crosslinked with calcium chloride for repeated catalytic use. The second chosen method was the enzyme cross-linking on a carrier formed by mag-netic particles modified with chitosan. Enzyme activity was determined spectropho-tometrically using the substrate o-nitrophenyl-β-D-galactopyranoside and p-nitrophenol as a standard. Enzyme activity was evaluated in milk samples and the amount of lactose formed was determined polarimetrically. The experimental results showed that the immobilized enzyme on the chitosan magnetic carrier showed higher activity compared to the sys-tem based on alginate capsules, which was also confirmed during the lactose hydroly-sis process. Lactose hydrolysis was the most efficient using a commercial enzyme and the enzyme immobilized on magnetic chitosan. There was no statically significant difference in the lactose content in milk samples without enzyme and with the use of alginate calcium capsules.
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Production of human milk oligosaccharides in the cell factory of E. coli
Havrdová, Jana ; Bojarová, Pavla (advisor) ; Smrček, Stanislav (referee)
Human milk oligosaccharides (HMOs) are among the most abundant components of human breast milk and are essential for the development of neonatal health. It is very challenging to isolate these oligosaccharides from animal milk, especially the less abundant ones. Therefore, different approaches are to be sought after. Chemical and enzymatic syntheses of these compounds are labor-intensive, and expensive affording low yields. A newly adopted approach to HMO synthesis is through bac- terial cell factories, in which genetically engineered bacterial strains can use cheap carbohydrate substrates and convert them into specific oligosaccharides. The aim of this thesis is to examine the feasibility of using selected bacterial strain (E. coli) for the production of HMOs. With lactose as a glycan substrate, the bacterial host has to be β-galactosidase deficient, otherwise, the substrate would be degraded. In order to generate higher lactose intake in the cell, a crp gene that encodes for the positive transcriptional regulator (Catabolite Activator Protein - CAP) can be incorporated into the host organism. The cloned plasmid - pRSFDuet- 1-crp was used for the transformation of into the selected bacterial strain. Lactose from the cells was purified by gel chromatography and the influence of CAP over expression...
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Engineering of microbial glycosidases for modifying synthetic potential
Hovorková, Michaela ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
Glycosidases (EC 3.2.1.) alias glycoside hydrolases are enzymes that catalyze the cleavage of a glycosidic bond between two carbohydrates or between a carbohydrate and an aglycone. Under suitable conditions (especially reduction of water activity in the reaction mixture), these enzymes are also able to synthesize a glycosidic bond. By targeted mutagenesis of the catalytic centre of the enzymes, it is possible to suppress or completely abolish their hydrolytic activity. Enzyme synthesis using glycosidases makes it possible to prepare bioactive galactosides, for example galectin ligands. The present work deals mainly with β-galactosidase from Bacillus circulans, its recombinant expression and mutagenesis. In the first part of the work, the commercially prepared plasmid of -galactosidase from B. circulans isoform A that I designed was used for recombinant expression in E. coli. It was necessary to optimize the conditions of the enzyme production. As it is a large protein (189 kDa), the expression vector pCOLD II and cold production at 15 ř C were used. The enzyme is specific for the formation of the β-1,4 glycosidic bond and has been used to synthesize complex tri- and tetrasaccharide ligands that cannot be prepared with a crude commercial preparation containing undesirable enzyme activities....
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