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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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Prospects of Microbial Degradation and Waste Utilization from Food Processing Industry
Illková, Kateřina ; Karovičová, Jolana (referee) ; Jarošová, Alžběta (referee) ; Omelková, Jiřina (advisor)
This work deals with the problem of microbial degradation of the waste materials from food industry. This work is focused on the production of technological significant enzymes producing by microorganisms, which were able to use the waste as a sole carbon source. In the first part of this work, the attention was focused on the production of pectolytic enzymes. This part was made within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. The grape pomace as the waste form winegrowing was used as a sole carbon source for microbial growth and enzymes production. The production of pectolytic enzymes was tested on this waste. After screening the most suitable microorganisms was chosen with the highest production of polygalacturonase activity. Produced enzymes were isolated by extraction techniques, purified and then identified proteomically. The aim of the second part of this work was the waste water treatment containing lipids and lipolytic enzymes. The reason was the cooperation with the company constructing grease traps. The characterization of supplied commercial preparations was the subject of this work and the other reason was the characterization of conditions for lipases secreting by microorganisms, identification of microorganisms present in the commercial preparation and testing of new microbial cultures for the development of new preparation for the grease traps.
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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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Prospects of Microbial Degradation and Waste Utilization from Food Processing Industry
Illková, Kateřina ; Karovičová, Jolana (referee) ; Jarošová, Alžběta (referee) ; Omelková, Jiřina (advisor)
This work deals with the problem of microbial degradation of the waste materials from food industry. This work is focused on the production of technological significant enzymes producing by microorganisms, which were able to use the waste as a sole carbon source. In the first part of this work, the attention was focused on the production of pectolytic enzymes. This part was made within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. The grape pomace as the waste form winegrowing was used as a sole carbon source for microbial growth and enzymes production. The production of pectolytic enzymes was tested on this waste. After screening the most suitable microorganisms was chosen with the highest production of polygalacturonase activity. Produced enzymes were isolated by extraction techniques, purified and then identified proteomically. The aim of the second part of this work was the waste water treatment containing lipids and lipolytic enzymes. The reason was the cooperation with the company constructing grease traps. The characterization of supplied commercial preparations was the subject of this work and the other reason was the characterization of conditions for lipases secreting by microorganisms, identification of microorganisms present in the commercial preparation and testing of new microbial cultures for the development of new preparation for the grease traps.
|
|
|
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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|
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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