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Production of microbial enzymes and their stabilization by encapsulation
Hazuchová, Eva ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The present thesis deals with the production of microbial enzymes and their subsequent stabilization through encapsulation. The theoretical part focuses on microbial enzymes, especially extracellular hydrolases, their producers and characteristics. Within the theory is also discussed the possibility of the application of enzymes in the field of pharmacy and medicine. Experimental work was focused on the actual production of microbial enzymes and methods for their to stabilization. The production of proteolytic and lipolytic enzymes in dependence on time and the used culture substrate were followed. The highest enzyme production was observed in Aspergillus oryzae when cultured on wheat bran at the third day of cultivation. In the experimental part was further carried out the identification, isolation and purification of enzymes. A substantial part of the experiment was to stabilize produced microbial enzymes by encapsulation. Enzymes were entrapped into alginate particles with encapsulation efficiency in the range of 55-70 %. The highest efficiency exhibited encapsulated enzymes from Aspergillus oryzae. Subsequently, long-term stability of the encapsulated enzyme in two environments (in water and gel) was followed during six weeks incomparison with free enzyme. During storage of free enzyme a significant decrease in enzyme activities occured, especially between the fourth and sixth week of storage. On the contrary, in encapsulated increased enzyme activities were observed. Empty particles exhibited higher stability during storage in the gel than in water. In this thesis potential use of enzymes in the pharmaceutical industry as agents promoting digestion was tested too. According to the results, particles with encapsulated microbial enzymes could be considered as suitable for some pharmaceutical applications.
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Characetrization of selected microbial enzymes
Bradáčová, Kristína ; Hlaváček, Viliam (referee) ; Márová, Ivana (advisor)
This bachelor´s thesis is focused on controlled production and identification of extracellular microbial hydrolytic enzymes by fungi. Theoretical part deals with characterization of selected hydrolytic enzymes, their properties, possibility of production and application. In experimental part the production of enzymes by fungal strains Phanerochaete chrysosporium, Aureobasidium pullulans and Aspergillus oryzae was performed. Cultivation was conducted in submersed mode in mineral medium and in media with waste co-substrates such as wheat bran, sawdust, rapeseed cake (lipids content 2,55 %) and rapeseed cake rich in lipids (9 %). The activity of cellulases, xylanases, amylases, ligninperoxidase, manganese-dependent peroxidase and laccase was monitored during cultivation process and regularly on 3rd, 7th, 10th and 15th day of cultivation. Production of enzymes depended on time and the subsrate type. Cellulases and xylanases were produced mainly on 3rd and 7th day of cultivation, amylases on 3rd and 15th day and lignolytic enzymes on 7th and 15th day. Samples were further separated and analyzed by ultrafiltration, gel filtration and PAGE-SDS electroforesis.
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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.
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Characetrization of selected microbial enzymes
Bradáčová, Kristína ; Hlaváček, Viliam (referee) ; Márová, Ivana (advisor)
This bachelor´s thesis is focused on controlled production and identification of extracellular microbial hydrolytic enzymes by fungi. Theoretical part deals with characterization of selected hydrolytic enzymes, their properties, possibility of production and application. In experimental part the production of enzymes by fungal strains Phanerochaete chrysosporium, Aureobasidium pullulans and Aspergillus oryzae was performed. Cultivation was conducted in submersed mode in mineral medium and in media with waste co-substrates such as wheat bran, sawdust, rapeseed cake (lipids content 2,55 %) and rapeseed cake rich in lipids (9 %). The activity of cellulases, xylanases, amylases, ligninperoxidase, manganese-dependent peroxidase and laccase was monitored during cultivation process and regularly on 3rd, 7th, 10th and 15th day of cultivation. Production of enzymes depended on time and the subsrate type. Cellulases and xylanases were produced mainly on 3rd and 7th day of cultivation, amylases on 3rd and 15th day and lignolytic enzymes on 7th and 15th day. Samples were further separated and analyzed by ultrafiltration, gel filtration and PAGE-SDS electroforesis.
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Production of microbial enzymes and their stabilization by encapsulation
Hazuchová, Eva ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The present thesis deals with the production of microbial enzymes and their subsequent stabilization through encapsulation. The theoretical part focuses on microbial enzymes, especially extracellular hydrolases, their producers and characteristics. Within the theory is also discussed the possibility of the application of enzymes in the field of pharmacy and medicine. Experimental work was focused on the actual production of microbial enzymes and methods for their to stabilization. The production of proteolytic and lipolytic enzymes in dependence on time and the used culture substrate were followed. The highest enzyme production was observed in Aspergillus oryzae when cultured on wheat bran at the third day of cultivation. In the experimental part was further carried out the identification, isolation and purification of enzymes. A substantial part of the experiment was to stabilize produced microbial enzymes by encapsulation. Enzymes were entrapped into alginate particles with encapsulation efficiency in the range of 55-70 %. The highest efficiency exhibited encapsulated enzymes from Aspergillus oryzae. Subsequently, long-term stability of the encapsulated enzyme in two environments (in water and gel) was followed during six weeks incomparison with free enzyme. During storage of free enzyme a significant decrease in enzyme activities occured, especially between the fourth and sixth week of storage. On the contrary, in encapsulated increased enzyme activities were observed. Empty particles exhibited higher stability during storage in the gel than in water. In this thesis potential use of enzymes in the pharmaceutical industry as agents promoting digestion was tested too. According to the results, particles with encapsulated microbial enzymes could be considered as suitable for some pharmaceutical applications.
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