Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine
properties. While the extracellular proteins of Saccharomyces cerevisiae have been
characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these
enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound
molecules. Another category of enzymes of oenological interest is represented by acid
proteases that are able to prevent or reduce protein haze, as reported in literature, while
simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino
acids from peptides and proteins that serve as aroma precursors may also have an indirect
effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology
(IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces
yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were
isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and
Candida apicola IWBT Y1384, respectively. However, no further characterization was
undertaken. This study aimed to clone these two genes into a recombinant bacterial host for
expression and purify the corresponding enzymes as a first step toward characterizing their
kinetic properties. Considering that some non-Saccharomyces species have been shown to
produce more than one acid protease, an additional aim was to identify novel acid proteases
within M. pulcherrima IWBT Y1123.
Cloning of the genes and transformation of the expression vectors into E. coli were achieved.
Optimal conditions for induced expression were established following extensive optimization.
Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing
conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated
into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column
system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity.
Purification was followed by refolding experiments, but yielded poor recovery of active enzymes.
Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be
elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1.
Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola
IWBT Y1384 revealed that both possess at least one additional protease other than those
previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT
Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH.
Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small
groups of proteins thereof did not allow the identification of these enzymes. / AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike
impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces
cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend.
Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van
glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie
word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos
voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud
kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma
voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie
wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee
aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit
Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie-
Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT
Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van
hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir
uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om
in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was
‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123.
Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was
suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende
optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol
was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat
die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was
gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied
fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was
gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe
ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir
redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille
tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises
uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat
voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen
geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima
IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë
het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie
van hierdie ensieme bevestig nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85704 |
| Date | 12 1900 |
| Creators | Theron, Louwrens Wiid |
| Contributors | Divol, Benoit, Zietsman, Anscha, Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Format | 103 p. : ill. |
| Rights | Stellenbosch University |
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