The enzymes phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were studied in the yeast Sporobolomyces roseus (Kluyver and van Neil). Cells grown on a glucose-salts medium were ground with alumina, and the cell-free buffer extract was fractionated with ammonium sulfate. Enzyme activity was assayed by measuring spectrophotometrically the cinnamic acid and p-coumaric acid produced from phenylalanine and tyrosine respectively. Further attempts at purification resulted in the inactivation of the TAL. Although the two enzymes were not separated by the purification procedures used, there is some evidence that the deamination of phenylalanine and tyrosine are catalyzed by different proteins, and not by a single enzyme with wide specificity. TAL appears to be precipitated by lower concentrations of ammonium sulfate than is PAL. The pH curves of the two enzymes are different. The specific activities of the two enzymes can be changed relative to one another in the cell by changing the medium upon which the cells were grown. The rates of production of the two enzymes vary independently during the growth of the cells. While the proteins are probably distinct, the production and activity of each enzyme seem to be under common control. Peak production of both enzymes occurs during late logarithmic-early stationary phase in the growth of a batch culture. Replacement media containing either phenylalanine or tyrosine stimulate the production of both PAL and TAL. Similarly, media containing cinnamic acid or p-coumaric acid repress the formation of the enzymes. Studies using labelled substrate show that both products inhibit the action of both enzymes. / Science, Faculty of / Botany, Department of / Graduate

Carotenoids are naturally occurring pigments produced by bacteria, yeasts, filamentous fungi and plants. They exhibit significant biological effects and are widely used in the food industry, pharmacy and cosmetics. The aim of this diploma thesis proposed as a comparative study was regulation of carotenoid and ergosterol production in red yeasts using several waste substrates as whey, corn germs, wheat, apple fiber and pasta. To selected production media extracellular hydrolytic enzymes degrading polysaccharide were added. These enzymes were obtained from the cultivation media of four fungal strains. In this study three carotenogenic yeast strains were used: Rhodotorula glutinis, Sporobolomyces roseus and Cystofilobasidium capitatum. All strains were cultivated simultaneously and changes in biomass and carotenoid production in different production media were monitored and compared. As the best waste substrate apple fiber was utilized, particularly in Rhodotorula glutinis, which exhibited mainly biomass production increase. In Sporobolomyces roseus increased production of biomass and carotenoids have been reported in media with hydrolyzed fiber and pasta as well. Beta-carotene production in this strain reached 4776,38 mg/g of dry weight. The strain Cystofilobasidium capitatum exhibited in waste media a decerase of biomass production accompanied with increased production of carotenoids, especially in wheat mush and pasta medium hydrolyzed by enzyme preparative from Aureobasidium pullulan. It can be concluded that hydrolyzed waste substrates are very hopeful as cheap nutrient sources for yeast strains producing carotenoids and ergosterol. Nevertheless, further study of substrate processing for individual strains is needed.

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.