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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20322 |
| Date | 03 1900 |
| Creators | Reid, Vernita Jennilee |
| Contributors | Divol, B. T., Du Toit, M., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 84 p. : ill. |
| Rights | Stellenbosch University |
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