Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The argument of whether to use food for biofuel (bioethanol) production prompted the search for an
alternative non-food biomass, such as lignocellulose, as feedstock for bioethanol production. However, a
hindrance in producing bioethanol from lignocellulose on an industrial scale is the cost associated with
hydrolysing the lignocellulose to its respective sugar monomers. Improving enzyme production and
enhancement of enzyme cocktails for efficient lignocellulose hydrolysis is, therefore, a necessary
prerequisite.
In this study, a yeast culture collection from the Wine and Fermentation Technology Division (ARC Infruitec-
Nietvoorbij, Stellenbosch, South Africa), isolated from fruit from various regions in South Africa, was
screened for β-glucosidase and β-xylosidase enzyme activities. β-glucosidases catalyse the hydrolysis of
cellobiose and by doing so prevents end-product inhibition of cellobiohydrolases and endoglucanases
during cellulose degradation. Similarly, β-xylosidases hydrolyse xylobiose and prevents end-product
inhibition of endoxylanases during hemicellulose degradation. After initially screening 2180 non-
Saccharomyces yeasts, two yeast isolates were selected that could potentially serve as enzyme source for
lignocellulose hydrolysis; one as a producer of a β-glucosidase and another as a β-xylosidase producer. The
yeasts were identified as a β-glucosidase producing Rhodotorula slooffiae-like yeast isolate 131B2 and a
β-xylosidase producing Aureobasidium pullulans isolate 23B25, respectively.
The production of β-glucosidase by Rhodotorula slooffiae-like yeast isolate 131B2 and of β-xylosidase by
Aureobasidium pullulans isolate 23B25 was optimised using response surface methodology according to a
central composite design. Subsequently, the crude and partially purified enzymes were characterised
based on molecular mass, pH optima and stability, temperature optima and stability and inhibition by
lignocellulose hydrolysis end-products, such as glucose, xylose and ethanol. The crude β-glucosidase from
Rhodotorula slooffiae-like yeast isolate 131B2 was also compared to the commercial Aspergillus niger βglucosidase
preparation (Novozyme 188) based on the characteristics mentioned above and as βglucosidase
supplement during Avicel (microcrystalline cellulose) hydrolysis by the commercial cellulase
preparation (Celluclast). The crude β-xylosidase by Aureobasidium pullulans isolate 23B25 could not be
compared to a commercial β-xylosidase as none was available at the time of the study. During the study,
the crude β-glucosidase 131B2 and β-xylosidase 23B25 showed potential as lignocellulose hydrolytic
enzymes. Attempts were made to obtain the β-glucosidase and β-xylosidase genes from the respective
yeast isolates using PCR-based approaches and by constructing cDNA libraries. However, cloning the
β-glucosidase and β-xylosidase genes using these methods proved after several attempts to be
unsuccessful, although, during this section of the study valuable information was obtained about the
obstacles involved with using these approaches when the desired gene sequence is unknown and novel. / AFRIKAANSE OPSOMMING: Die debat oor die toepaslikheid van voedsel vir bio-brandstofproduksie (bio-etanol), het daartoe gelei dat
alternatiewe nie-voedsel grondstowwe, soos lignosellulose, as voermateriaal vir bio-ethanol ondersoek
word. Die koste geassosieer met die hidrolise van lignosellulose na die onderskeie suiker monomere
belemmer industriële-skaal toepassing van lignosellulose vir bio-etanolproduksie. Verbeterde
ensiemproduksie en verhoogde doeltreffendheid van ensiemmengsels vir lignosellulose hidrolise is dus ‘n
noodsaaklik voorvereiste.
In hierdie studie is 'n giskultuurversameling geisoleer vanaf vrugte van verskillende streke in Suid-Afrika
deur die Wyn en Fermentasie Tegnologie Afdeling (ARC Infruitec-Nietvoorbij, Stellenbosch, Suid-Afrika) vir
β-glukosidase en β-xilosidase ensiemaktiwiteite gesif. β-glukosidases wat die hidrolise van sellobiose
kataliseer voorkom eindprodukinhibisie van sellobiohidrolases en endoglukanases tydens sellulose afbraak.
β-xilosidases, op hul beurt, hydroliseer xilobiose en voorkom eindprodukinhibisie van endoxilanases tydens
hemisellulose afbraak. Na afloop van die aanvanklike sifting van 2180 nie-Saccharomyces giste, is twee giste
wat potensiëel as 'n ensiembron vir lignosellulose hidrolise kan dien geselekteer; een vir β-glukosidase en
‘n ander vir β-xilosidase produksie. Die giste is as ʼn β-glukosidase-produserende Rhodotorula slooffiaeagtige
gisras 131B2 en ʼn β-xilosidase-produserende Aureobasidium pullulans gisras 23B25 onderskeidelik
geïdentifiseer.
Die Rhodotorula slooffiae-agtige gisras 131B2 se produksie van β-glukosidase en die Aureobasidium
pullulans gisras 23B25 produksie van β-xylosidase was geoptimiseer met behulp van “response surface
methodology” volgens 'n “central composite design”. Daarna was die gedeeltelik-gesuiwerde kru-ensieme
volgens molekulêre massa, pH optima en stabiliteit, temperatuur optima en stabiliteit, en inhibisie deur
lignocelluloses hidrolise end-produkte soos glukose, xylose en etanol, gekarakteriseer. Die kru βglukosidase
van die Rhodotorula slooffiae-agtige gisras 131B2 is ook met die kommersiële Aspergillus niger
β-glukosidase (Novozyme 188) volgens die eienskappe vroeër genoem vergelyk en as β-glukosidase
aanvulling tydens die kommersiële sellulase (Celluclast) se hidrolise van Avicel (mikrokristalline sellulose).
Die kru β-xylosidase van die Aureobasidium pullulans gisras 23B25 kon nie vergelyk word met 'n
kommersiële β-xylosidase nie, aangesien daar nie een beskikbaar was tydens die studie nie. Gedurende die
studie het altwee, die kru β-glukosidase 131B2 en β-xylosidase 23B25, potensiaal getoon as lignosellulose
hidrolitiese ensieme. Pogings was aangewend om die β-glukosidase en β-xilosidase gene vanuit die
onderskeie gis isolate met behulp van PKR-gebaseerde tegnieke en die opstel van cDNA biblioteke te
kloneer. Hierdie klonering strategieë was egter na verskeie pogings onsuksesvol, maar waardevolle
inligting oor die struikelblokke betrokke by die gebruik van hierdie benaderings wanneer die gewenste geen
se DNS basispaarvolgorde onbekend en uniek is, was verkry.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80152 |
| Date | 03 1900 |
| Creators | Omardien, Soraya |
| Contributors | Volschenk, Heinrich, Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | v, 123 p. : ill. |
| Rights | Stellenbosch University |
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