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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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Enzymatic hydrolysis of wastes associated with coffee production
Kovářová, Markéta ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of potential production of extracellular hydrolytic enzymes by microorganisms – bacterium and moulds, which have been cultivated on spent coffee grounds. The theoretical part deals with characterization of coffee and utilization of coffee by-products. There are also subscribed microorganisms and enzymes which have been noticed. In experimental part coffee ground was used as the sole substrate for production of extracellular hydrolytic enzymes. Productions of protease, cellulase, mannase and lipase enzymes were observed. None-identified isolate of mould spontaneously contaminating spent coffee grounds was identified as the best producer of these enzymes. Subsequently the conditions of cultivation such as water content and shaking vs. static cultivation of this moulds were optimized. Further, we performed partial purification and pre-concentration of the enzyme cocktail by ultrafiltration, ultradialysis and PAGE-SDS characterization of extracellular enzymes was performed as well.
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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 and characteritzation of extracellular hydrolases from selected moulds
Skoumalová, Petra ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on study of potential production of extracellular hydrolytic enzymes. The theoretical part deals with characterization of selected hydrolytic enzymes, their catalytic properties, the possibility of extracellular hydrolase production by fungi and their applications. In experimental part production strains Aureobasidium pullulans, Fusarium solani and Phanerochaete chrysosporium were used. Productions of cellulase, amylase, xylanase, lipase, protease and lignin-degraded enzymes (laccase, manganese- dependent peroxidase, lignin peroxidase) were observed. Cultivations were carried out in submersed mode in mineral medium supplemented by waste co-substrates such as wheat bran, corn bran, rice bran and oat bran, sawdust, rice, apple fiber, egg pasta and egg-free pasta. Production of enzymes depended on the substrate type and time of cultivation. The highest cellulase, xylanase and amylase activities were measured in the first period of cultivation (3 to 7 day). Lignin-degraded enzymes and proteases were produced at the end of cultivation (7 to 10 days). Lipolytic activity was detected only in A. pullulans, where the activity increased with time of cultivation. The highest value was determined during cultivation on wheat bran (3.6 nmol/ml.min). The highest xylanase and celulase activity (170.3 nmol/ml.min, 248.0 nmol/ml.min) were determined during cultivation of F. solani on corn bran. The highest amylase activity (111.8 nmol/ml.min) was reported in P. chrysosporium during the cultivation on rice. The highest protease activity (68.0 nmol/ml.min) was determined in F. solani grown on wheat bran. The best producer of laccase was A. pullulans, the highest production was recorded for egg-free pasta (27.0 nmol/ml.min). The maximum lignin peroxidase activity (12.5 nmol/ml.min) was measured during the cultivation of F. solani on egg pasta, while the highest yield of Mn-dependent peroxidase (7.7 nmol/ml.min) was achieved during the cultivation of A. pullulans on wheat bran. Lignin-degraded enzymes behaved as inductive, while the other enzymes were produced in mineral medium too. Activity of cellulase in the mineral medium was in A. pullulans strain higher than in media with waste substrates. Enzymes produced into A. pullulans medium were purified by ultrafiltration, ion exchange chromatography and gel filtration.
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Degradace syntetických barviv pomocí Phanerochaete chrysosporium
Blahutová, Andrea
Azo dyes are the most important and largest group of commercially produced synthetic dyes. Their degradation by white-rot fungi Phanerochaete chrysosporium is induced by a lignin-degrading enzyme system, which is capable of degrading xenobiotic compounds. This bachelor thesis focuses on the degradation of the five most commonly used azo dyes by Phanerochaete chrysosporium. The process has been monitored not only on individual dyes (E102 Tartrazine, E110 Yellow, E122 Azorubin, E124 Ponceau 4R and E123 Amaranth), but also on two alimentary products (Vitacit – lemon and Vitacit – strawberry) containing E102 Tartrazine or E122 Azorubin azo dye. The absorbance of the solutions was measured spectrophotometrically at four-day intervals across a twenty-day cultivation period. The color loss was calculated based on the recorded values and the results were expressed as a percentage of decolorization. At the end of the measurement, the Azorubin dye showed the highest decolorization (90 %), whether the Ponceau 4R dye was decolorized the least (20 %). The level of decolorization of the powdered beverage solutions was compared with the decolorization of the individual dyes. The degradation was mainly effected by the various azo dyes structure. The composition of powdered beverages also played a role in their degradation.
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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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Production and characteritzation of extracellular hydrolases from selected moulds
Skoumalová, Petra ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on study of potential production of extracellular hydrolytic enzymes. The theoretical part deals with characterization of selected hydrolytic enzymes, their catalytic properties, the possibility of extracellular hydrolase production by fungi and their applications. In experimental part production strains Aureobasidium pullulans, Fusarium solani and Phanerochaete chrysosporium were used. Productions of cellulase, amylase, xylanase, lipase, protease and lignin-degraded enzymes (laccase, manganese- dependent peroxidase, lignin peroxidase) were observed. Cultivations were carried out in submersed mode in mineral medium supplemented by waste co-substrates such as wheat bran, corn bran, rice bran and oat bran, sawdust, rice, apple fiber, egg pasta and egg-free pasta. Production of enzymes depended on the substrate type and time of cultivation. The highest cellulase, xylanase and amylase activities were measured in the first period of cultivation (3 to 7 day). Lignin-degraded enzymes and proteases were produced at the end of cultivation (7 to 10 days). Lipolytic activity was detected only in A. pullulans, where the activity increased with time of cultivation. The highest value was determined during cultivation on wheat bran (3.6 nmol/ml.min). The highest xylanase and celulase activity (170.3 nmol/ml.min, 248.0 nmol/ml.min) were determined during cultivation of F. solani on corn bran. The highest amylase activity (111.8 nmol/ml.min) was reported in P. chrysosporium during the cultivation on rice. The highest protease activity (68.0 nmol/ml.min) was determined in F. solani grown on wheat bran. The best producer of laccase was A. pullulans, the highest production was recorded for egg-free pasta (27.0 nmol/ml.min). The maximum lignin peroxidase activity (12.5 nmol/ml.min) was measured during the cultivation of F. solani on egg pasta, while the highest yield of Mn-dependent peroxidase (7.7 nmol/ml.min) was achieved during the cultivation of A. pullulans on wheat bran. Lignin-degraded enzymes behaved as inductive, while the other enzymes were produced in mineral medium too. Activity of cellulase in the mineral medium was in A. pullulans strain higher than in media with waste substrates. Enzymes produced into A. pullulans medium were purified by ultrafiltration, ion exchange chromatography and gel filtration.
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Enzymatic hydrolysis of wastes associated with coffee production
Kovářová, Markéta ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of potential production of extracellular hydrolytic enzymes by microorganisms – bacterium and moulds, which have been cultivated on spent coffee grounds. The theoretical part deals with characterization of coffee and utilization of coffee by-products. There are also subscribed microorganisms and enzymes which have been noticed. In experimental part coffee ground was used as the sole substrate for production of extracellular hydrolytic enzymes. Productions of protease, cellulase, mannase and lipase enzymes were observed. None-identified isolate of mould spontaneously contaminating spent coffee grounds was identified as the best producer of these enzymes. Subsequently the conditions of cultivation such as water content and shaking vs. static cultivation of this moulds were optimized. Further, we performed partial purification and pre-concentration of the enzyme cocktail by ultrafiltration, ultradialysis and PAGE-SDS characterization of extracellular enzymes was performed as well.
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