Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage or regarded as undesired yeasts in wine. However, these yeasts have recently come under investigation for their positive contribution towards wine aroma especially when used in sequential or co-inoculated fermentations with Saccharomyces cerevisiae. These yeasts are also known to secrete a number of enzymes that could be applicable in wine biotechnology. Amongst these enzymes are aspartic proteases. The secreted proteases from some non-Saccharomyces yeast may play a role in protein haze reduction, as demonstrated by some authors, while simultaneously increasing the assimilable nitrogen content of the wine for the utilization and growth of fermentative microorganisms. Moreover, the proteases may have an indirect effect on wine aroma by liberating amino acids that serve as aroma precursors. Although many screenings have been performed detecting protease activity in non-Saccharomyces yeasts, no attempts have been made to characterize these enzymes. This study set out to isolate and characterize genes encoding extracellular aspartic proteases from non-Saccharomyces yeasts. An enzymatic activity screening of a collection of 308 Saccharomyces and non-Saccharomyces yeasts, isolated from grape must, was performed. The aspartic protease-encoding genes of two non- Saccharomyces yeasts, which showed strong extracellular proteolytic activity on plate assays, were isolated and characterized by in silico analysis. The genes were isolated by employing degenerate and inverse PCR. One gene was isolated from Metschnikowia pulcherrima IWBT Y1123 and named MpAPr1. The other putative gene was isolated from Candida apicola IWBT Y1384 and named CaAPr1. The MpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecular weight of 40.1 kDa. The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putative protein with a predicted molecular weight of 39 kDa. These features are typical of extracellular aspartic proteases. The deduced protein sequences showed less than 40% homology to other yeast extracellular aspartic proteases. By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that the gene encodes an extracellular acid protease. The expression of MpAPr1 was shown to be induced in media containing proteins as sole nitrogen source and repressed when a preferred nitrogen source was available. The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grape juice proteins and repressed in the presence of ammonium sulphate. Expression was most induced in the presence of grape juice proteins, which was expected since these proteins are present in the natural habitat of the yeast. A genetic screening confirmed the presence of the MpAPr1 gene in 12 other M. pulcherrima strains isolated from grape juice. The extracellular protease activity of the strains was also visualized on plates. As far as we know, this is the first report on the genetic characterization of secreted aspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides the groundwork for further investigations. / AFRIKAANSE OPSOMMING: Nie-Saccharomyces giste is voorheen met wynbederf geassosieer en hul teenwoordigheid in wyn is ongewens. Hierdie giste is onlangs ondersoek vir hulle positiewe bydrae tot wyn aroma, in veral sekwensiële en ko-inokulerings met Saccharomyces cerevisiae. Sommige van die nie-Saccahromyces giste skei ‘n verskeidenhied ensieme af wat moontlik vir die wynmaker van nut kan wees. Een groep van hierdie ensieme is die aspartiese suurproteases. Soos deur sommige navorsers aangetoon word, kan die proteases die vorming van proteïenwaasverlaging, terwyl dit terselfdertyd die assimilerende stikstofinhoud van die wyn vir die gebruik en groei van fermentasie-mikroörganismes verhoog. Die proteases kan moontlik ook ‘n indirekte uitwerking op die aromaprofiel van die wyn hê deur die vrystelling van aminosure wat as aromavoorlopers dien. Alhoewel baie studies gedoen is wat die ekstrasellulêre teenwoordigheid van proteases bevestig in nie-Saccharomyces giste wat van druiwesap/wyn afkoms is, is daar geen dokumentasie oor die genetiese karakterisering van hierdie ensieme beskikbaar nie. Die doel van hierdie studie was om gene wat aspartiese proteases in nie-Saccharomyces giste enkodeer, te isoleer en gedeeltelik te karakteriseer. ‘n Versameling van 308 Saccharomyces en nie-Saccharomyces giste wat uit druiwe sap geïsoleer is, is gesif vir ensiematiese aktiwiteit deur plaattoetse uit te voer. Twee gene wat aspartiese protease enkodeer, is geïsoleer van twee nie-Saccharomyces giste. Dit hetpositief gedurende die aktiwiteitstoetse getoets en is deur in silico–analise gekarakteriseer. Die gene is deur die uitvoering van gedegenereerde en inverse PKR geïdentifiseer. Een geen is vanaf Metschnikowia pulcherrima IWBT Y1123 geïsoleer en is MpAPr1 genoem, terwyl die ander van Candida apicola IWBT Y1384 geïsoleer en CaAPr1 genoem is. Die MpAPr1-geen is 1137 bp lank en enkodeer ‘n proteïen wat uit 378 aminosure bestaan met ‘n voorspelde molekulêre massa van 40.1 kDa. Daar teenoor is die CaAPr1-geen 1101 bp lank en enkodeer vir ‘n proteïen wat uit 367 aminosure met ‘n molekulêre massa van 39 kDa bestaan. Hierdie eienskappe is kenmerkend van aspartiese protease. Die afgeleide proteïenvolgorde het minder as 40% homologie met ander ekstrasellulêre aspartiese proteases vertoon, wat dui op die nuwigheid van hierdie ensieme. Die MpAPr1-geen is heterologies in S. cerevisiae YHUM272 uitgedruk en dit het bevestig dat die geen inderdaad ‘n ekstrasellulêre aspartiese protease enkodeer. Die MpAPr1-geen is uitgedruk in media wat alleenlik proteïen as stikstofbron bevat het, terwyl dit onderdruk is in gevalle waar ‘n verkose stikstofbron beskikbaar was. Die geen is uitgedruk in die teenwoordigheid van kaseïen, BSA en proteïene afkomstig vanaf druiwesap en in die teenwoordigheid van ammoniumsulfaat onderdruk. Die hoogste uitdrukking was in die teenwoordigheid van druifproteïene. Hierdie proteïene is teenwoordig in die natuurlike habitat van die gis en is dus dalk ‘n bekende stikstofbron vir die gis. ‘n Genetiese sifting het die teenwoordigheid van die MpAPr1-geen in 12 ander M. pulcherrima–rasse, wat ook van wynkundige oorsprong is, bevestig. Die aspartiese protease-aktiwiteit van die 12 rasse is ook op agarplate waargeneem. Na ons wete, is dit die eerste verslag oor die genetiese karakterisering van afgeskeide aspartiese proteases van nie- Saccharomyces giste van wynkundige oorsprong en verskaf die grondslag vir verdere ondersoek.