Browsing by Author "Visser, Johanna Jacoba"
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- ItemCloning and expression of the Lactobacillus fermentum acid urease gene in Saccharomyces cerevisiae(Stellenbosch : Stellenbosch University, 1999-09) Visser, Johanna Jacoba; Van Vuuren, H. J. J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Arginine is one of the main amino acids present in grape must and is degraded by the wine yeast Saccharomyces cerevisiae to ornithine, ammonia and carbon dioxide. Urea is formed as an intermediate product and is secreted "into the grape must, resulting in high concentrations of urea in fermenting grape must. Ethanol, produced during fermentation, reacts with the urea during long term storage and forms ethyl carbamate (urethane). Urethane is a carcinogenic and mutagenic substance representing a potential hazard to human health and therefore has to be addressed by the wine and related industries. The aim of this study was to develop a wine yeast strain that could prevent the formation of urethane by degrading the urea produced during wine fermentation. The urease enzyme (not produced by S. cerevisiae) can degrade urea to ammonia and carbon dioxide. When using an acid urease, this reaction can take place at the low pH conditions associated with wine fermentation. The lactic acid bacterium Lactobacillus Jermentum was chosen as a source of the genes that encode the three structural subunits of the acid urease. The bacterial genes are found in an operon, whereas three open reading frames (ORF) separated by linker sequences encode the eukaryotic enzyme from jack bean. Expression cassettes containing an ORF comprised of the three bacterial genes, as well as the linker sequences present in the jack bean urease gene were therefore constructed for expression in S. cerevisiae. The production and activity of the recombinant protein were tested by expressing it first in a urease-positive strain of Schizosaccharomyces pombe that should provide the essential accessory proteins for its own urease, as well as for the recombinant protein. These accessory proteins are responsible for incorporating the nickel ions into the urease and are therefore essential for an active urease enzyme. The transcription of the recombinant gene was confirmed by northern blot analysis and the activity of the recombinant protein was tested under different pH conditions. However, the protein proved to be unstable, making it extremely difficult to quantify the activity.