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

Visser, Johanna Jacoba (1999-09)

Thesis (M.Sc. Agric.) -- University of Stellenbosch, 1999.

Thesis

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.

AFRIKAANSE OPSOMMING: Arginien is die hoof aminosuur teenwoordig in druiwemos en word deur die wyngis Saccharomyces cerevisiae na ornitien, ammoniak en koolstofdioksied afgebreek. . Ureum word as 'n byproduk tydens hierdie reaksie gevorm en in die druiwemos uitgeskei, wat lei tot hoe konsentrasies ureum in die gistende druiwemos. Etanol wat gedurende die fermentasie geproduseer word, reageer met die ureum gedurende lang opbergingsperiodes en vorm etielkarbamaat. Etielkarbamaat is 'n karsinogeniese en mutageniese verbinding wat 'n potensiele gevaar vir menslike gesondheid inhou en dus deur die wyn- en verwante industriee aangespreek moet word. Die doel van hierdie studie was om 'n wyngis te ontwikkel wat die vormmg van etielkarbamaat kan voorkom deur die ureum wat tydens wyn fermentasie geproduseer word, af te breek. Die urease ensiem (wat nie deur S. cerevisiae geproduseer word nie) breek ureum af na ammoniak en koolstofdioksied. Indien 'n suur urease gebruik word, kan hierdie reaksie by die lae pH kondisies wat met wynfermentasie geassosieer word, plaasvind. Die melksuurbakterium Lactobacillus fermentum is gebruik as bron van die gene wat die drie strukturele subeenhede van die suur urease ensiem kodeer. Die bakteriese gene word as 'n operon uitgedruk, terwyl die drie homoloe gene in die "jack bean" deur verbindingsfragmente geskei word. Uitdrukkingskasette wat 'n oopleesraam bestaande uit die drie bakteriese gene, sowel as die verbindingsfragmente van die "jack bean" urease gene bevat, is vir uitdrukking in S. cerevisiae gekonstrueer. Die produksie en aktiwiteit van die rekombinante proteYen is eers in 'n urease-positiewe ras van Schizosaccharomyces pombe getoets wat die noodsaaklike hulpproteYene vir sy eie urease, sowel as vir die rekombinante proteien, kan verskaf. Hierdie hulpproteYene is verantwoordelik vir die inbouing van die nikkel-ione in die urease en is dus noodsaaklik vir ensiemaktiwiteit. Transkripsie van die rekombinante proteien is deur northernklad analise bevestig en die aktiwiteit van die rekombinante proteYen is onder verskillende pH toestande getoets. Die proteYen was egter onstabiel en gevolglik was dit uiters moeilik om die aktiwiteit te kwantifiseer.

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