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Production of extracellular polymeric substances by Aureobasidium pullulans
Horáček, Pavel ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
The diploma thesis is focused on the study of the influence of cultivation conditions and arrangement for the production of extracellular polymeric substances by using yeast-like microorganism Aureobasidium pullulans. In the theoretical part a brief description of A. pullulans, its use in biotechnology and produced exobiopolymers, especially pullulan and poly-L-malic acid are presented. The first aim of the experimental part was to set the most appropriate cultivation conditions for A. pullulans CCM 8182. Growth and production properties in optimum conditions were compared with cultivation on waste substrates - oat bran, buckwheat husks, apple fiber and others. Waste substrates can be used as cheap nutrient sources which enable reducing cost of potential biotechnological production. As a further part of this work, optimization of HPLC/RI method for analysis of exobiopolymers has been done. Optimal mobile phase composition and chromatography conditions were proposed. Column Roa organic acid H+ was the most suitable for simultaneous separartion of glucose and malic acid. Before HPLC analysis hydrolysis of polymers was done. Sulphuric acid (5 mmol/L) was used as a mobile phase at flow rate 0.5 mL/min and temperature 60 °C. The highest production of pullulan occurred using oat bran as a substarate (13.03 g/L) at an initial pH 7.5. Maximum production of poly-L-malic acid was observed during the cultivation on apple peels (2.89 g/L) at pH 6. It was found that the higher production of poly-L-malic acid occurred at pH 6, while higher production of pullulan was at pH 7.5.
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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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Extracellular enzyme activites of soil yeasts
Pavlatovská, Barbora ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Yeasts form significant and important part of pedosphere microbiota. They keep nature balance, participate in cycles of elements and nutrients, are antagonists of various pathogens and as important decomposers, they produce the whole spectrum of different extracellular enzymes. The aim of this study was to determine the ability of yeasts, isolated from the soil adjacent to the fruit trees in Southwest Slovakia as well as from the contaminated soil (Pernek area, Slovakia), to produce extracellular enzymes. In total, 68 strains belonging to 45 different species were tested for the production of starch-like polysaccharide and for extracellular enzyme activities: polygalacturonases, lipases, proteases, cellulases, chitinases, -glucosidases and -amylases. This work was also focused on optimization of method for the yeast chitinase assay. Four methods were proved; two of them utilized liquid medium with chitin (colloidal and insoluble) as the sole carbon source and two others used solid plate methods with agar medium containing chitin. Based on results, cultivation in colloidal liquid chitin medium, terminated by the chitinase assay according to Ehrlich, was evaluated as the best method for detection of predominant exochitinase activity of yeasts. More than 75 % of tested yeasts exhibited some extracellular activity. Generally, the yeasts isolated from the soil under the fruit trees showed broader spectrum of enzyme activities than those originated from contaminated soils. Lipases, proteases and -glucosidases were found to be the most common activities. Only small proportion of yeasts was able to produce chitinases and/or cellulases. Aureobasidium pullulans, CCY 27-1-134, from the soil adjacent to the apple tree, showed the widest range of activities from all tested strains and it possessed all examined activities. On the other side, it did not produce starch-like polysaccharide. Tausonia (Trichosporon) pullulans and Cystofilobasidium macerans were the second most active producers of extracellular enzymes with variations in production of cellulases and -amylases. Representatives of the former polyphyletic genus Cryptococcus exhibited lipases, -glucosidases, -amylases and they were producers of the starch, but the interspecies differences were also noted. All strains of the genus Galactomyces were positive for polygalacturonases and the genera Candida and Cyberlindnera were positive for -glucosidases. All strains of Galactomyces candidum were tested for the production of polygalacturonases during 168 hours long cultivation on pectin media. Strain CCY 16-3-4 showed very stable growth on this medium and simultaneously exhibited significant amount of extracellular polygalactouronases. It has a potential to be very suitable producer of these enzymes but particular characterization of properties is necessary for its future use. Results of the screening showed that the production of extracellular enzymes is mostly strain-dependent and not species-dependent.
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Random mutagenesis and selection of red yeast mutants capable to utilize particular waste substrates
Čačková, Katarína ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.
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Study of carotenogenic yeasts doring growth by using advanced instrumental techniques
Vaněk, Martin ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
This work is dealing with application of advanced fluorescence techniques for gaining knowledge about culture development during fermentation of red yeasts. Flow cytometry was used for auto-fluorescence measurement a carotenoids quantitation. It was resolved that while carotenoids are stored mainly in membranes the technique was feasible. If red yeast starts to accumulate carotenoids into lipid bodies mainly throughout the course of stationary phase, then the method starts to fail. Flow cytometric method using cell size measurement and light scatter for lipid quantitation was proved as applicable, too. However, it works only if cells are not starved. Individual calibration for each species is needed for elimination inter-species variations of intracellular structures. Fluorescence lifetime imaging microscopy was also used for studying of red yeast. Inherent ability to resolve different fluorescent species of the same molecule, which arise due to different molecular environment, helps with quantitation of cellular lipidic structures changes through the course of fermentation. Increase in the levels of carotenoids and/or rigidity of membranes was found as mechanism of protection during metabolic shifts, when intracellular content is vulnerable to damage.
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Regulation of production of lipids and lipid compounds in yeasts
Rapta, Marek ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
Oleogenic yeasts under appropriate conditions produce and accumulate lipids and lipid-soluble metabolites in high amounts. This attribute is characteristic also for red yeasts that except lipids accumulate also carotenoids – natural pigments used in food industry and dietary supplements. The aim of this diploma thesis was designed as a comparative screening study of production properties of strains Cystofilobasidium capitatum, Rhodotorula glutinis, Sporobolomyces roseus and Sporobolomyces shibatanus. Choosen carbon sources were glucose and glycerol as waste by-product in biofuel industry. The best production properties were found in Cystofilobasidium capitatum and Rhodotorula glutinis. These two strains produced increased amounts of lipids as well as higher amounts of carotenoids. Strains were tested by FTIR spectroscopy that enables high-throughput, uncomplicated and accurate analysis.
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Effect of medium composition on the mass spectra of the yeast species of Cryptococcus laurentii and Cryptococcus flavescens
Ledvina, Vojtěch ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.
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Biotyping of Cryptococcus laurentii group using mass spectrometry
Jäger, Jakub ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“
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Extracellular enzyme activites of soil yeasts
Pavlatovská, Barbora ; Breierová, Emília (referee) ; Stratilová, Eva (advisor)
Yeasts form significant and important part of pedosphere microbiota. They keep nature balance, participate in cycles of elements and nutrients, are antagonists of various pathogens and as important decomposers, they produce the whole spectrum of different extracellular enzymes. The aim of this study was to determine the ability of yeasts, isolated from the soil adjacent to the fruit trees in Southwest Slovakia as well as from the contaminated soil (Pernek area, Slovakia), to produce extracellular enzymes. In total, 68 strains belonging to 45 different species were tested for the production of starch-like polysaccharide and for extracellular enzyme activities: polygalacturonases, lipases, proteases, cellulases, chitinases, -glucosidases and -amylases. This work was also focused on optimization of method for the yeast chitinase assay. Four methods were proved; two of them utilized liquid medium with chitin (colloidal and insoluble) as the sole carbon source and two others used solid plate methods with agar medium containing chitin. Based on results, cultivation in colloidal liquid chitin medium, terminated by the chitinase assay according to Ehrlich, was evaluated as the best method for detection of predominant exochitinase activity of yeasts. More than 75 % of tested yeasts exhibited some extracellular activity. Generally, the yeasts isolated from the soil under the fruit trees showed broader spectrum of enzyme activities than those originated from contaminated soils. Lipases, proteases and -glucosidases were found to be the most common activities. Only small proportion of yeasts was able to produce chitinases and/or cellulases. Aureobasidium pullulans, CCY 27-1-134, from the soil adjacent to the apple tree, showed the widest range of activities from all tested strains and it possessed all examined activities. On the other side, it did not produce starch-like polysaccharide. Tausonia (Trichosporon) pullulans and Cystofilobasidium macerans were the second most active producers of extracellular enzymes with variations in production of cellulases and -amylases. Representatives of the former polyphyletic genus Cryptococcus exhibited lipases, -glucosidases, -amylases and they were producers of the starch, but the interspecies differences were also noted. All strains of the genus Galactomyces were positive for polygalacturonases and the genera Candida and Cyberlindnera were positive for -glucosidases. All strains of Galactomyces candidum were tested for the production of polygalacturonases during 168 hours long cultivation on pectin media. Strain CCY 16-3-4 showed very stable growth on this medium and simultaneously exhibited significant amount of extracellular polygalactouronases. It has a potential to be very suitable producer of these enzymes but particular characterization of properties is necessary for its future use. Results of the screening showed that the production of extracellular enzymes is mostly strain-dependent and not species-dependent.
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Study of carotenogenic yeasts doring growth by using advanced instrumental techniques
Vaněk, Martin ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
This work is dealing with application of advanced fluorescence techniques for gaining knowledge about culture development during fermentation of red yeasts. Flow cytometry was used for auto-fluorescence measurement a carotenoids quantitation. It was resolved that while carotenoids are stored mainly in membranes the technique was feasible. If red yeast starts to accumulate carotenoids into lipid bodies mainly throughout the course of stationary phase, then the method starts to fail. Flow cytometric method using cell size measurement and light scatter for lipid quantitation was proved as applicable, too. However, it works only if cells are not starved. Individual calibration for each species is needed for elimination inter-species variations of intracellular structures. Fluorescence lifetime imaging microscopy was also used for studying of red yeast. Inherent ability to resolve different fluorescent species of the same molecule, which arise due to different molecular environment, helps with quantitation of cellular lipidic structures changes through the course of fermentation. Increase in the levels of carotenoids and/or rigidity of membranes was found as mechanism of protection during metabolic shifts, when intracellular content is vulnerable to damage.
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