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.

The master‘s thesis is focused on the study of biodegradation of polyhydroxyalkanoates, namely polymer polyhydroxybutyrate. The first part of the thesis is focused on the study of biodegradation of polyhydroxybutyrate in the form of crystalline granules of PHB and PHB films using selected species of microorganisms from bacteria, yeasts and fungi. As a representative of bacteria was chosen microorganism Delftia acidorovans, as yeast was selected Aureobasidium pullulans and Aspergillus fumigatus as fungi. PHB depolymerase activity was measured employing turbidemtiric method with suspension of PHB granules as substrate. The results showed that D. acidorovans can partially degrade PHB. On the contrary A. pullulans cannot effectively degrade PHB. The most significant degradation ability revealed A. fumigatus, which was able to degrade PHB completely. Extracellular enzymes excreted by these microorganisms when cultivated on PHB materials as sole carbon sources were analyzed by SDS-PAGE. The second part of the thesis deals with the biodegradation of PHB in the form of PHB film, PHB hardened foil and PHB Nanoul fabric using standard composting test. Semi-solid cultivation showed positive results. In the interval from 14 days to two months were all forms of the PHB completely biodegraded. With semi-solid cultivation was also studied biodegradation rate of the polyurethane elastomeric films which were modified by partial replacement of polyester polyol by PHB. The test samples were prepared using PHB from Sigma and the PHB samples prepared at the Faculty of chemistry VUT. Samples with different concentrations of the dispersed PHB (1 %, 5 % and 10 %) in the polyurethane were also object of the study. At the end of the cultivation (after 2 months) were measured mechanical properties in tension of the material, then efficiency of biodegradation by gravimetric analysis and modification of the material surface by microscopic analysis.

Production of cellulase and polygalacturonase by Aspergillus niger and Aureobasidium pullulans was studied in submerged (SmF) and solid state fermentation (SSF) systems. Substrates used in fermentation systems were mandarin peels and grape pomace. With Aspergillus niger used on grape pomace as a sole carbon source, cellulase production was detected after 72 hours in SSF and after 24 hours in SmF systems. The activity of cellulase per gram of substrate was higher in a submerged than in a solid state fermentation system. The longer time for higher polygalacturonase production was necessary in submerged fermentation systems and polygalacturonase activity was higher in SmF. The SSF fermentation with mandarin peels as a sole carbon source was similar, the highest detected activity of cellulase was determined after 72 hours. Different production of polygalacturonase was observed on mandarin peels in SmF systems. A comparison of enzyme productivities on grape pomace and mandarin peels showed that polygalacturonase activity per gram of substrate is highest in SmF system with mandarin peels as a sole carbon source. With Aureobasidium pullulans used on grape pomace as a sole carbon source, cellulase production was detected after 48 hours in SmF and SSF fermentation systems. The activity of cellulase per gram of substrate was higher in solid state system than in a submerged fermentation system. Longer time for higher polygalacturonase production was necessary in both fermentation systems. Polygalacturonase activity was higher in SmF. The SSF fermentation with mandarin peels as a sole carbon source was similar, the highest detected activity of cellulase was determined after 48 hours. Different production of polygalacturonase was observed on mandarin peels in SmF systems. A comparison of enzyme productivities on grape pomace and mandarin peels showed that polygalacturonase activity per gram of substrate is highest in SmF system with mandarin peels as a sole carbon source. For both systems and both substrates manganese-dependent peroxidase was detected for the first time. Differences in the enzyme synthesis by Aspergillus niger and Aureobasidium pullulans depend on both the substrates used as well as on the fermentation system.

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.

Lagent de lutte biologique Aureobasidium pullulans souche Ach1-1 a présenté une grande potentialité dans le contrôle de Penicillium expansum, lagent causal de la pourriture bleue des pommes en conservation. Les mécanismes daction qui sont les plus impliqués dans son activité antagoniste ont été analysés, au cours de ce travail. Daprès des essais de protection réalisés sur pommes blessées, il apparaît que lefficacité de cette souche nest pas liée essentiellement à la sécrétion des métabolites toxiques dans le milieu ou à linduction de la résistance de fruit. Par contre, le mécanisme de la compétition pour la nutrition semble jouer un rôle important. Dans les essais in vitro, la souche antagoniste Ach1-1 a eu un important effet inhibiteur de la germination des conidies de P. expansum dans des milieux de jus de pomme à des faibles concentrations. Mais cet effet était réversible et les conidies inhibées étaient capables de germer une fois remises dans des conditions favorables en éléments nutritifs. Sur pommes blessées, leffet protecteur de la souche Ach1-1 vis-à-vis de P. expansum a été significativement affaibli par lajout dans les blessures de concentrations élevées des principaux composants des pommes en sucres, en vitamines et particulièrement en acides aminés. Il apparaît que lantagoniste exerce une activité fongistatique plus que fongicide vis-à-vis de P. expansum et agit par une compétition efficace pour les éléments nutritifs des blessures des pommes sans affecter la viabilité des conidies du pathogène. Une application exogène des acides aminés des pommes avec des concentrations croissantes dans les blessures a progressivement réduit lactivité antagoniste de la souche Ach1-1 sans altérer son développement dans les blessures, montrant que la compétition pour les acides aminés joue un rôle important dans la suppression de P. expansum. Ce résultat a été appuyé par lanalyse biochimique de la cinétique de lépuisement des acides aminés dans les blessures des pommes qui a montré que ces composés et particulièrement la sérine, la glycine et lacide glutamique sont mieux métabolisés par la souche antagoniste que par le pathogène. Lajout en excès de ces trois acides aminés en groupe ou individuellement dans les blessures des pommes a fortement réduit lefficacité de la souche Ach1-1 vis-à-vis de P. expansum. De plus, la présence de la sérine et la glycine avec des concentrations élevées dans des milieux synthétiques ne présentant aucune source azotée, a réduit leffet inhibiteur de la germination des conidies de P. expansum par la souche Ach1-1. Ainsi, ces acides aminés semble être parmi les éléments les plus limitants dans le mécanisme de la compétition./The biocontrol agent Aureobasidium pullulans strain Ach1-1 was very effective against Penicillium expansum, the causal agent of blue mold on stored apple. Modes of action that could be involved in its biocontrol activity were analysed in this work. According to some biocontrol trials on wounded apples, it appears that neither the production of metabolites nor the induction of fruit resistance were the principal modes of action of this strain. However the mechanism of nutrient competition appears to play an important role. In in vitro assays, the strain Ach1-1 had an important inhibitory effect of conidial germination of P. expansum in apple juice at low concentrations. However this inhibitory effect was suppressed when inhibited conidia were placed in favourable nutrients conditions. On wounded apples the protective activity of strain Ach1-1 against P. expansum was significantly reduced by adding, in the wounds, high concentrations of major apple compounds of sugar, vitamins and most particularly amino acids. It appears that the antagonist exerts a fungistatic rather than fungicidal activi ty on P. expansum as it can deplete limiting nutrient available at the infection site and inhibit conidia germination without affecting their viability. Moreover, an exogenous application of increasing apple amino acids concentrations in wounds had progressively reduced the antagonist activity of strain Ach1-1 without altering its development in wounds, suggesting that competition for apple amino acids by strain Ach1-1 plays an important role in suppressing P. expansum. This finding was strengthened by a time-course analysis of wounds amino acids during apple incubation in witch the strain Ach1-1 was able to assimilate apple amino acids better than P. expansum, most particularly Serine, Glycin and Glutamic acid. Exogenous additions of these three amino acids at high concentrations on apple wounds as a mixture or individually, strongly lowered the Biocontrol activity of strain Ach1-1. Moreover, the existence of amino acids serine and glycin at high concentration in synthetic media, without any nitrogen source, was able to reduce the inhibitory effect of conidial germination of P. expansum by the strain Ach1-1.Therefore these amino acids could be among the most limited nutrients in the mechanism of competition.

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.