Thesis (MScAgric) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass.
These biopolymers are therefore an important renewable source of food, fuels and
chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and
is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The
individual molecules are held together by hydrogen bonds, forming largely crystalline
fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide,
coats and binds the cellulose microfibrils, preventing the cellulose from becoming too
crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3-
and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named
according to the sugar type that forms the polymer backbone. Pectic substances
contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains
are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side
chains. Two groups of enzymes, cellulases and pectinases, are required for the
microbial utilisation of crystalline cellulose and pectin. Cellulases are
multicomponent complexes that are often composed of endoglucanases,
exoglucanases and cellobiases. Cellobiose is the major end product of concerted
endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose
by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying
action of the saponifying enzymes, pectinesterase, releasing the methyl
groups of the pectin molecule, and by hydrolase or lyase action of the
depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a-
1.4-glycosidic linkages in the polygalacturonate chain.
The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic
beverage industry for fermentations of wine, beer and other alcoholic beverages for
many years. However, it is unable to produce extracellular depolymerising enzymes
that can efficiently degrade polysaccharides, which are the main cause of
clarification and filtration problems. Enzyme preparations have been used in the
alcoholic beverage industries to degrade haze-forming polysaccharides, thereby
improving the filterability and quality of products such as beer and wine. An
alternative would be to develop S. cerevisiae strains that produce extracellular
polysaccharidases, enabling the yeast to degrade polysaccharides without the
addition of commercial enzyme preparations. These strains can also be very useful
in improving the quality of wine, as well as cutting the costs of the winemaking
process. The objective of this study was to investigate the effects of two transformed
S. cerevisiae strains on different wine grape varieties.
The following genes have been cloned and characterised previously: the
Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3-
1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and
the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene
terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast
mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to
compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated
END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t
(designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE).
Two yeast integrating plasmids were constructed, one containing the END1 and
XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two
plasmids were then integrated into the URA3 locus of two separate industrial wine
yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast
transformants in the absence of auxotrophic markers, the integrating plasmid
containing the END1 and XYN4 gene cassettes was issued with the dominant
selectable Geneticin G418-resistance {G f) marker. The integrating plasmid
harbouring the PEH1-PELE gene cassette was issued with the dominant selectable
sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids
into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and
PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4-
glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase.
These recombinant yeasts were capable of extracting more colour from grape
skins of certain varieties, as well as leading to more freeflow wine as a result of the
more effective degradation of glucans, xylans and pectins in the skins. They also led
to decreased turbidity in the wine, making it more filterable.
Future work will entail further investigation of the effects of these recombinant
yeasts on different white and red wine grape varieties.
Another objective of this study was to screen non-Saccharomyces wine yeasts for
the production of extracellular hydrolytic enzymes. The reason for this part of the
thesis was to determine the types of extracellular hydrolytic enzymes that are
produced and to determine which genera produce which kinds of extracellular
enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida,
Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and
Kluyveromyces, were screened for the production of extracellular pectinases,
proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases,
amylases and sulphite reductase activity. These yeasts were all isolated from
grapes and clarified grape juice to ensure that they were yeasts found in must during
the initial stages of fermentation. This information can be used to pave the way to
pinpoint the specific effects in wine of these enzymes produced by the so-called wild
yeasts associated with grape must. This information can also be used to transform
Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces
yeasts for the production of extracellular hydrolytic enzymes.
However, future research will have to be done to determine the extent of the
activity of these enzymes in wine fermentations and to obtain better knowledge of the
physiological and metabolical features of non-Saccharomyces yeasts. / AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere
is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee.
Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit
herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg
is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar
geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose,
wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose
vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie
predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane,
1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die
suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n
rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings
periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings,
onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur
mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is
multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en
sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde
aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot
glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind
deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas.
Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die
hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en
poligalakturonase), split die a-1,4-glukosidiese verbindings in die
poligalakturonaatketting.
Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf
vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit
egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die
effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat
die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn
en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om
waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate
in die alkoholiese bedryf vir die degradasie van die probleem
polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en
bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor
die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus
polisakkariede self sonder die byvoeging van eksterne kommersiele
ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die
verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde
aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases
produseer, op verskillende wyndruifvarieteite na te vors.
Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase-
geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl)
van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi
en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die
alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder
(ADH2T), die gistriptofaansintase geen se termineerder (TRP5t)
en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die
volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as
END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T
fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE).
Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4-
geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee
plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die
URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in
die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin
G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende
plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende
(SMR1) merker is in die integreringsplasmied, wat die PEH1-
PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie
plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-,
XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van
die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase,
pektaatliase en poligalaturonase.
Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding
gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook
tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die
glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde
turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak.
Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing
gedoen moet word om die presiese effekte van hierdie rekombinante giste op
verskillende rooi en wit druifvarieteite te bepaal.
‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die
produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is
om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word,
asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate
wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia,
Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie
van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase,
sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is
almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie
inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme,
wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van
fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms
gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste
te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van
die problematiese polisakkariede in wyn te produseer.
Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang
van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis
te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces
wyngiste.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53688 |
| Date | 03 1900 |
| Creators | Strauss, Marlene |
| Contributors | Pretorius, I. S., Van Rensburg, P., Lambrechts, M. G., 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 | 91 p. : ill. |
| Rights | Stellenbosch University |
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