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Partial purification and characterization of polygalacturonases of Geotrichum candidum.
Jäger, Jakub ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
This work discusses the possibilities of using microbial degradation of grape pomace, main waste material from wine production, to preparate industrially important enzymes. The issue is focused on the production of pectolytic enzymes, particularly polygalacturonase, by Geotrichum candidum CCY 16-1-29 via solid state fermentation on grape pomace. The theoretical part of the bachelor thesis focuses on studying plant cells and saccharides from which the plant cell wall is made of, mainly pectin. Cell wall sacharides were used as a carbon source for solid state fermentation (SSF) and pectin as an inductor of pectolytic enzymes. This bachelor thesis also deals with the enzymatic degradation of cell wall polysacharides. The greatest attention is paid to degrade pectin and pectolytic enzyme function. Production of pectolytic enzymes is mentioned subsequently. The last chapter from the theoretical part is dedicated to technical use of pectolytic enzymes. In the experimental part of this work I deal with the partial purification and characterization of majority polygalacturonase produced on the seventh day of cultivation, when another increase of extracellular polygalacturonase activity occurred. The yield of cultivation was 43,5 mg of protein extract /100 g of grape pomace. The extract contained protein, and its activity was lyophilisate. Its specific activity was protein. The enzyme was produced in at least four forms differing in pH optimum (4,0; 4,4; 4,8; 5,2). The pH optimum for majority polygalacturonase was 4,8. Action pattern of this enzyme determined as the dependence of polymeric substrate viscosity decrease on its degradation showed that the enzyme is a typical polygalacturonase with random action pattern (EC 3.2.1.15).Value of Km reached indicating a high affinity for this substrate. The amino acid sequence "SNNVVSNVNILSSQVVNSDNGVR" obtained by mass spectrometry after SDS-PAGE and tryptic digestion, was identified as a stretch of primary structure of polygalacturonase of Ap2PG1 G. candidum based on the comparison with proteins from the Uniprot database. It shows the highest similarity with other polygalacturonases of G. candidum S31PG1, S31PG2 and G. klebahnii PSE3. On the basis of this similarity to enzymes produced by phytopathogenic strains of G. candidum and the fact that this enzyme was not produced only in the early stages of cultivation, it can be assumed, that the strain of G. candidum CCY 16-1-29 acted also as a phytopathogenic strain.
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Optimization of ethanol bioproduction from waste materials using SSF method
Filová, Dagmar ; Vránová, Dana (referee) ; Babák, Libor (advisor)
Presented diploma thesis is dealing with the problematics of fuel ethanol production. Relevant basic terminology is explained in the theoretical part, methods of lignocellulose pre-treatments and their conversion to bioethanol are introduced. Attetion is also given to microorganisms used for bioethanol production on industrial scale, as well as analytical instrumental techniques for glucose and ethanol detection. In experimental part, we are focusing on substrate composition analysis – contents of dry matter, cellulose and ash was investigated. Waste paper was chosen as substrate, as it does not find any other use beside recycling these days. Chosen production microorganism, that conversts sugars into etanol was the unknown strain of Saccharomyces cerevisiae. Primal substrate pre-treatment – removal of rigid parts was performed in several physical and physical – chemical ways. Substrate with such pre-treatment was ready for enzymatic hydrolysis, during which monomers from polymer matrix were formed. Ethanol was produced using method of simultaneous saccharification and fermentation, when enzymatic hydrolysis and fermentation take place at the same time and the same container.
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Enzymatic hydrolysis of waste cardboard using the SSF method - a source of raw materials for the production of liquid biofuels.
Hlaváček, Viliam ; Stloukal, Radek (referee) ; Gabriel, Petr (advisor)
This master’s thesis discusses the useof enzymatic hydrolysis process of waste cardboard using simultaneous saccharification and fermentation (SSF) as a source of raw materials for production of liquid biofuels. This thesis is based on theses written by Ing. Brummer and Ing.Lepař.Thus, results gained in these works have been used and also further developed. The theoretical part summarizes the reasons for further development of SSF method and discusses, as well, the achievements reached in the processing of lignocellulosic waste materials by the SSF method so far.This section also discusses the general characteristics of lignocellulosic materials and also of the cellulolytic enzymes. It focusses also on individual pretreatment methods of lignocellulosic material and options of increasing the yield of the whole process. The experimental part verifies the particular results reached in previous theses and at the same time a further optimization of the method has been carried out because of the transfer of the whole process into a fermenter. Cardboard was set as the substrate for the experiments as it was evaluated by Ing. Brummer as the best one for enzymatic hydrolysis which was carried out by enzymes from Novozymes®. Parameters such as temperature, pH and kind of used buffer, the loading concentration of substrate and enzymes, were set according to the thesis of Ing. Lepař, which was aimed to their optimization. The SSF process done in fermenter of 2.0 l volume confirmed the previous results and furthermore it has been more effective through optimization of the added inoculum volume. It has been confirmed that the best substrate is cardboard finely grinded by vibrating mill. Also experiments with added nutrients had been done as an effort to increase the ethanol concentration, but these haven’t resulted insatisfying results. The maximal concentration of ethanol was 23,49 g/l, which was achieved after further optimization of various conditions. This result equals to experimental yield of 84,79 %.
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Study of growth and optimization of selected metabolites production by Zymomonas mobilis
Lukačková, Adéla ; Vránová, Dana (referee) ; Babák, Libor (advisor)
In the diploma thesis are discussed the process of enzymatic hydrolysis of waste paper as a source for the production of bioethanol by bacteria Zymomonas mobilis. In the theoretical part summarize basic information about particular methods of hydrolysis, about paper used as a raw material for enzymatic hydrolysis, about possibilities of the fermentative production of bioethanol focusing on the method of simultaneous saccharification and fermentation comparison with enzymatic hydrolysis and fermentation. Suitable microorganisms for ethanolic fermentation and simultaneous saccharification and fermentation and their advantages and disadvantages, are further discussed in this part as well. The theoretical part ends with the suggestion of the technological process for production of bioetanol. It covers all necessary steps from the input of raw material to the separation of produced ethanol. In the experimental part various parameters of hydrolysis, fermentation and simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the Zymomonas mobilis CCM2770 and Zymomonas mobilis LMG457 bacterium. The conversion rate of paper cellulose to gluckose and production of ethanol were observed by HPLC/RI method. Type of buffer, quantity of cells, enzyme and substrate were optimized in order to maximize the efficiency of the process. All experiments were performed on paper containing high amount of cellulose and for comparison on standard medium which contains gluckose. The highest yields was achieved with the use of Novozymes® Cellulosic ethanol enzyme Kit. The strain Zymomonas mobilis LMG457 has demonstrated as a better producer.
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Modern way of calculation of CAPM coefficient: Beta hedging application
Šopov, Daniel ; Andrlíková, Petra (advisor) ; Gapko, Petr (referee)
Model CAPM je považován za základní model při oceňování systematického risku aktiv a jeho provázanosti s výnosností trhu. Tato práce využívá této struktury a použitím různých metod, mezi které patří OLS, DCC MGARCH a SSF modelovaní, se snaží najít nejvhodnější metodu z výše zmíněných, která dokáže nejlépe odhadnout koeficienty systematického risku. Tyto koeficienty jsou dále použity pro zajištění rizika portfolií, které jsou vytvořeny z akcií obchodovaných na různých burzách- NYSE Composite a NASDAQ Composite. Na základě obdržených výsledků o výkonu zajištění rizika v každém portfoliu budeme schopni vyhodnotit, která z metod je nejvhodnější pro odhad systematické risku v modelu CAPM. Klíčová slova: CAPM, Systematický risk, Portfolio risk hedge, OLS, DCC MGARCH, SSF model JEL Classification: C22, C58, G11, G12, G15 Author's e-mail: danielsopov@email.cz Supervisor's e-mail: andrlikova@gmail.com
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Modern way of calculation of CAPM coefficient: Beta hedging application
Šopov, Daniel ; Andrlíková, Petra (advisor) ; Gapko, Petr (referee)
Model CAPM je považován za základní model při oceňování systematického risku aktiv a jeho provázanosti s výnosností trhu. Tato práce využívá této struktury a použitím různých metod, mezi které patří OLS, DCC MGARCH a SSF modelovaní, se snaží najít nejvhodnější metodu z výše zmíněných, která dokáže nejlépe odhadnout koeficienty systematického risku. Tyto koeficienty jsou dále použity pro zajištění rizika portfolií, které jsou vytvořeny z akcií obchodovaných na různých burzách- NYSE Composite a NASDAQ Composite. Na základě obdržených výsledků o výkonu zajištění rizika v každém portfoliu budeme schopni vyhodnotit, která z metod je nejvhodnější pro odhad systematické risku v modelu CAPM. Klíčová slova: CAPM, Systematický risk, Portfolio risk hedge, OLS, DCC MGARCH, SSF model JEL Classification: C22, C58, G11, G12, G15 Author's e-mail: danielsopov@email.cz Supervisor's e-mail: andrlikova@gmail.com
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Optimization of ethanol bioproduction from waste materials using SSF method
Filová, Dagmar ; Vránová, Dana (referee) ; Babák, Libor (advisor)
Presented diploma thesis is dealing with the problematics of fuel ethanol production. Relevant basic terminology is explained in the theoretical part, methods of lignocellulose pre-treatments and their conversion to bioethanol are introduced. Attetion is also given to microorganisms used for bioethanol production on industrial scale, as well as analytical instrumental techniques for glucose and ethanol detection. In experimental part, we are focusing on substrate composition analysis – contents of dry matter, cellulose and ash was investigated. Waste paper was chosen as substrate, as it does not find any other use beside recycling these days. Chosen production microorganism, that conversts sugars into etanol was the unknown strain of Saccharomyces cerevisiae. Primal substrate pre-treatment – removal of rigid parts was performed in several physical and physical – chemical ways. Substrate with such pre-treatment was ready for enzymatic hydrolysis, during which monomers from polymer matrix were formed. Ethanol was produced using method of simultaneous saccharification and fermentation, when enzymatic hydrolysis and fermentation take place at the same time and the same container.
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Enzymatic hydrolysis of waste cardboard using the SSF method - a source of raw materials for the production of liquid biofuels.
Hlaváček, Viliam ; Stloukal, Radek (referee) ; Gabriel, Petr (advisor)
This master’s thesis discusses the useof enzymatic hydrolysis process of waste cardboard using simultaneous saccharification and fermentation (SSF) as a source of raw materials for production of liquid biofuels. This thesis is based on theses written by Ing. Brummer and Ing.Lepař.Thus, results gained in these works have been used and also further developed. The theoretical part summarizes the reasons for further development of SSF method and discusses, as well, the achievements reached in the processing of lignocellulosic waste materials by the SSF method so far.This section also discusses the general characteristics of lignocellulosic materials and also of the cellulolytic enzymes. It focusses also on individual pretreatment methods of lignocellulosic material and options of increasing the yield of the whole process. The experimental part verifies the particular results reached in previous theses and at the same time a further optimization of the method has been carried out because of the transfer of the whole process into a fermenter. Cardboard was set as the substrate for the experiments as it was evaluated by Ing. Brummer as the best one for enzymatic hydrolysis which was carried out by enzymes from Novozymes®. Parameters such as temperature, pH and kind of used buffer, the loading concentration of substrate and enzymes, were set according to the thesis of Ing. Lepař, which was aimed to their optimization. The SSF process done in fermenter of 2.0 l volume confirmed the previous results and furthermore it has been more effective through optimization of the added inoculum volume. It has been confirmed that the best substrate is cardboard finely grinded by vibrating mill. Also experiments with added nutrients had been done as an effort to increase the ethanol concentration, but these haven’t resulted insatisfying results. The maximal concentration of ethanol was 23,49 g/l, which was achieved after further optimization of various conditions. This result equals to experimental yield of 84,79 %.
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Study of growth and optimization of selected metabolites production by Zymomonas mobilis
Lukačková, Adéla ; Vránová, Dana (referee) ; Babák, Libor (advisor)
In the diploma thesis are discussed the process of enzymatic hydrolysis of waste paper as a source for the production of bioethanol by bacteria Zymomonas mobilis. In the theoretical part summarize basic information about particular methods of hydrolysis, about paper used as a raw material for enzymatic hydrolysis, about possibilities of the fermentative production of bioethanol focusing on the method of simultaneous saccharification and fermentation comparison with enzymatic hydrolysis and fermentation. Suitable microorganisms for ethanolic fermentation and simultaneous saccharification and fermentation and their advantages and disadvantages, are further discussed in this part as well. The theoretical part ends with the suggestion of the technological process for production of bioetanol. It covers all necessary steps from the input of raw material to the separation of produced ethanol. In the experimental part various parameters of hydrolysis, fermentation and simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the Zymomonas mobilis CCM2770 and Zymomonas mobilis LMG457 bacterium. The conversion rate of paper cellulose to gluckose and production of ethanol were observed by HPLC/RI method. Type of buffer, quantity of cells, enzyme and substrate were optimized in order to maximize the efficiency of the process. All experiments were performed on paper containing high amount of cellulose and for comparison on standard medium which contains gluckose. The highest yields was achieved with the use of Novozymes® Cellulosic ethanol enzyme Kit. The strain Zymomonas mobilis LMG457 has demonstrated as a better producer.
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Partial purification and characterization of polygalacturonases of Geotrichum candidum.
Jäger, Jakub ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
This work discusses the possibilities of using microbial degradation of grape pomace, main waste material from wine production, to preparate industrially important enzymes. The issue is focused on the production of pectolytic enzymes, particularly polygalacturonase, by Geotrichum candidum CCY 16-1-29 via solid state fermentation on grape pomace. The theoretical part of the bachelor thesis focuses on studying plant cells and saccharides from which the plant cell wall is made of, mainly pectin. Cell wall sacharides were used as a carbon source for solid state fermentation (SSF) and pectin as an inductor of pectolytic enzymes. This bachelor thesis also deals with the enzymatic degradation of cell wall polysacharides. The greatest attention is paid to degrade pectin and pectolytic enzyme function. Production of pectolytic enzymes is mentioned subsequently. The last chapter from the theoretical part is dedicated to technical use of pectolytic enzymes. In the experimental part of this work I deal with the partial purification and characterization of majority polygalacturonase produced on the seventh day of cultivation, when another increase of extracellular polygalacturonase activity occurred. The yield of cultivation was 43,5 mg of protein extract /100 g of grape pomace. The extract contained protein, and its activity was lyophilisate. Its specific activity was protein. The enzyme was produced in at least four forms differing in pH optimum (4,0; 4,4; 4,8; 5,2). The pH optimum for majority polygalacturonase was 4,8. Action pattern of this enzyme determined as the dependence of polymeric substrate viscosity decrease on its degradation showed that the enzyme is a typical polygalacturonase with random action pattern (EC 3.2.1.15).Value of Km reached indicating a high affinity for this substrate. The amino acid sequence "SNNVVSNVNILSSQVVNSDNGVR" obtained by mass spectrometry after SDS-PAGE and tryptic digestion, was identified as a stretch of primary structure of polygalacturonase of Ap2PG1 G. candidum based on the comparison with proteins from the Uniprot database. It shows the highest similarity with other polygalacturonases of G. candidum S31PG1, S31PG2 and G. klebahnii PSE3. On the basis of this similarity to enzymes produced by phytopathogenic strains of G. candidum and the fact that this enzyme was not produced only in the early stages of cultivation, it can be assumed, that the strain of G. candidum CCY 16-1-29 acted also as a phytopathogenic strain.
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