Cloning and expression of the Lactobacillus fermentum acid urease gene in Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: Arginine is one of the main amino acids present in grape must and is degraded by thewine yeast Saccharomyces cerevisiae to ornithine, ammonia and carbon dioxide. Urea isformed as an intermediate product and is secreted into the grape must, resulting in highconcentrations 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 tohuman 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 formationof urethane by degrading the urea produced during wine fermentation. The ureaseenzyme (not produced by S. cerevisiae) can degrade urea to ammonia and carbondioxide. When using an acid urease, this reaction can take place at the low pH conditionsassociated with wine fermentation. The lactic acid bacterium Lactobacillus Jermentumwas chosen as a source of the genes that encode the three structural subunits of the acidurease. 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 asthe linker sequences present in the jack bean urease gene were therefore constructed forexpression in S. cerevisiae.The production and activity of the recombinant protein were tested by expressing it firstin a urease-positive strain of Schizosaccharomyces pombe that should provide theessential 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 ureaseand are therefore essential for an active urease enzyme. The transcription of therecombinant gene was confirmed by northern blot analysis and the activity of therecombinant protein was tested under different pH conditions. However, the proteinproved to be unstable, making it extremely difficult to quantify the activity.

[发布机构] Stellenbosch University