Aureobasidum pullulans is a black yeast, particularly known for its biotechnological role in the production of the polysaccharide pullulan and of many extracellular enzymes. The use of these enzymes is currently limited, but recent sequencing of the genome of A. pullulans represents a good opportunity for identification and examination of its enzymes. A. pullulans has high tolerance for stress conditions like low water activity, high salt concentrations and low pH.
The yeast Saccharomyces cerevisiae is a well-established laboratory model system. Because S. cerevisiae is not pathogenic, we can handle with it in a microbiological laboratory without special precautions. Baker’s yeast is important in the food industry for rising the dough and in the alcohol drink industry. It is also important in energetics, where it is particularly used for production of bioethanol. For all these reasons S. cerevisiae is extensively studied and modifications, which would improve its biotechnologically important traits, are constantly being attempted.
The purpose of this master thesis was to select genes for extracellular enzymes with potential industrial use. Furthermore, the purpose was to express these genes in the laboratory strain of S. cerevisiae and their basic characterization.
In our research, we first identified genes for enzymes and amplified genes. Amplified genes were separated with agarose gel electrophoresis. The next step was purification of the amplifiers and insertion into the plasmid pBEVY-U. The transformation of S. cerevisiae followed. The transformants were verified by polymerase chain reaction and by determining the nucleotide sequence of the inserted genes. In the next step, the function of the inserted genes on the media was checked to detect the corresponding enzyme activities. Finally, we tested the activity of selected enzymes during the growth of genetically modified yeast under stress conditions. A. pullulans is known as an extremotolerant organism, it also grows at elevated salt concentrations and at different temperatures. Therefore, the activity of extracellular enzymes was checked at elevated salinity and at different temperatures.
We were able to express the genes for the extracellular enzymes of A. pullulans in S. cerevisiae. Last but not least, we were able to demonstrate that extracellular enzymes from A. pullulans also function under stress conditions with the addition of sodium chloride in culture medium.
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