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Immobilization of pectinase on selected carriers
Reichstädter, Marek ; Zichová, Miroslava (referee) ; Omelková, Jiřina (advisor)
The theoretical part of this bachelor thesis deals with the definition of pectin substances as substrates for pectolytic enzymes. It also describes the changes in properties of enzymes due to enzyme immobilization, various methods of immobilization and application of immobilized enzymes. The experimental part examines the preparation by immobilization of commercially used pectolytic complex Rohament P by sorption on the polymeric carrier made of polyethylene tereftalate (PETE) bottles, what is a new patent of BUT. The main observed property was the enzyme activity of all preparations, it was determinated by Somogyi method and spectrometry – for both free and immobilized enzymes in dependence on the quantity of immobilized enzyme adsorbed onto specific weight of carries. The difference in effects change between the free and immobilized enzyme by measuring the viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.
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Immobilization of selected glycanohydrolases
Reichstädter, Marek ; Trachtová, Štěpánka (referee) ; Omelková, Jiřina (advisor)
The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.
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Study enantioselectivity and synthesis of β-lactam antibiotics catalyzed by penicilin G acylase: Biocatalysis and in-silico experiments
Grulich, Michal ; Kyslík, Pavel (advisor) ; Kotík, Michal (referee) ; Ettrich, Rüdiger (referee)
11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...
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Study of enantioselectivity and synthesis of β-lactam antibiotics catalyzed by penicilin-G-acylase: Biocatalysis and in-silico experiments
Grulich, Michal
11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...
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Study enantioselectivity and synthesis of β-lactam antibiotics catalyzed by penicilin G acylase: Biocatalysis and in-silico experiments
Grulich, Michal ; Kyslík, Pavel (advisor) ; Kotík, Michal (referee) ; Ettrich, Rüdiger (referee)
11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...
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Study of enantioselectivity and synthesis of β-lactam antibiotics catalyzed by penicilin-G-acylase: Biocatalysis and in-silico experiments
Grulich, Michal
11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...
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Characterization of enzyme reactor with immobilized alkaline phosphatase
Plecitá, Denisa ; Kučerová, Zdenka (advisor) ; Miarková, Eva (referee)
Phosphorylation is one of the most common of all post-translational modifications of proteins and has been found in nearly all cellular processes. Abnormal phosphorylation is associated with many serious human diseases. One of the approaches used for the identification of protein phosphorylation sites is based on the application phosphatases and the comparison of MS analysis of samples before and after the sample treatment with the enzyme. The use of phosphatase immobilized to magnetic carriers is advantageous in comparison with the application of soluble enzyme: e.g. easy manipulation of samples, an increase of enzyme stability and a possibility of repeated use of immobilized enzyme. Investigation of properties of enzyme reactor - bovine alkaline phosphatase from intestinal mucosa immobilized to magnetic particles is a subject of this Bachelor Thesis. The enzyme was coupled to cellulose magnetic particles after activation with divinyl sulfone via the protein free amino groups. p-Nitrophenylphosphate was used as a substrate for the phosphatase activity determination. The effect of different conditions on the activity of soluble and immobilized forms of alkaline phosphatase was compared: the effect of pH and Mg2+ ions, storage stability and thermostability and possibility of repeated use of the...
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Epoxide hydrolases expressed from environmental DNA: characteristics of soluble and immobilized enzyme forms
Grulich, Michal ; Bezouška, Karel (advisor) ; Čabala, Radomír (referee)
8 Abstract Epoxide hydrolases (EHs) demonstrating high degree of enantioselectivity or enantioconvergence are useful biocatalysts for the production of optically active epoxides and vicinal diols, which can serve as chiral building blocks for syntheses of biologically active drugs. EHs can play an important role also in degradations of xenobiotics. Genes encoding EHs Kau2 and Kau8 were expressed in E. coli host strains TOP10 and RE3. Enantioselectivities and regioselectivities of Kau2 and Kau8 in supernatants of desintegrated cells were determined for four substrates: tert-butylglycidyl ether, para-chlorostyrene oxide, para-nitrostyrene oxide, α-methylstyrene oxide. The highest values of enantioselectivity and regioselectivity were achieved with Kau2 and para-nitrostyrene oxide as a substrate. The Kau2 was chosen for further experiments on the basis of these results. Kau2 was overexpressed in the recombinant strain RE3(pSEKau2). We performed two batch cultures and one fed-batch culture in stirred bioreactor. The highest volumetric activity of 4500 U/l was obtained in the case of fed-batch culture. Two phase system consisting of polyethylenglycole 6000 and sodium citrate (pH 7.7) was used for Kau2 purification from the supernatant of desintegrated cells. Purification factor 2.6 +/- 0.3 was achieved and...
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Immobilization of selected glycanohydrolases
Reichstädter, Marek ; Trachtová, Štěpánka (referee) ; Omelková, Jiřina (advisor)
The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.
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Immobilization of pectinase on selected carriers
Reichstädter, Marek ; Zichová, Miroslava (referee) ; Omelková, Jiřina (advisor)
The theoretical part of this bachelor thesis deals with the definition of pectin substances as substrates for pectolytic enzymes. It also describes the changes in properties of enzymes due to enzyme immobilization, various methods of immobilization and application of immobilized enzymes. The experimental part examines the preparation by immobilization of commercially used pectolytic complex Rohament P by sorption on the polymeric carrier made of polyethylene tereftalate (PETE) bottles, what is a new patent of BUT. The main observed property was the enzyme activity of all preparations, it was determinated by Somogyi method and spectrometry – for both free and immobilized enzymes in dependence on the quantity of immobilized enzyme adsorbed onto specific weight of carries. The difference in effects change between the free and immobilized enzyme by measuring the viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.
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