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The study of extracellular enzymes produced by different species of yeast
Vršanská, Martina ; Ing.Jana Molnárová, Ph.D. (referee) ; Voběrková, Stanislava (advisor)
The thesis deals with the study of the different yeast strains from the point of view of extracellular lipolytic enzyme production. First part of this work consisting of appropriate yeasts was developed within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. From ten given strains three yeasts such as Pseudozyma fusiformata, Meyerozyma guilliermondii, Yarrowia lipolytica were chosen, these strains showed the highest lipolytic activity and cell growth on basal medium with Tween 80. These yeasts were used for optimization of cultivation conditions and characterization of lipolytic enzymes. The yeasts were cultivated on media with different carbon sources, which appeared to be a most suitable medium the basal medium with Tween. Tween acted as and inducer of lipase production. The substrate specificity was determined using three p-nitrophenylester substrates with varying sizes of the fatty acid site chains. The results showed that tested lipases are probably triacylglycerol-acyl-hydrolases which has the highest activity towards in the water insoluble substrates with medium long chains. The pH optimum and temperature optimum were measured. The results showed that the tested lipases had the highest activity in neutral and mild acid region around 30°C. By measuring of thermal stability has been demonstrated that extracellular lipases are relatively thermostable enzymes. Afterwards the storage stability was measured for 5 weeks when supernatant was kept in fridge at 4°C and in freezing box at -20°C. The results showed that in both cases tested lipases exhibited high storage stability which allows to store the samples without loss of activity for a longer time. Finally, the results of lipolytic and proteolytic activity, cell growth and pH of the medium of yeast Y. lipolytica were compared between the batch cultivation in L-tubes with the continual cultivation in the bioreactor. The highest lipases production was achieved in bioreactor due to the setting conditions of the continual proces to regulate the production and enzymatic stability.
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