Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The need for a cost-effective and environmentally friendly substitute for fossil fuels has
resulted in significant attention to the production of bioethanol. Lignocellulose being the
most abundant renewable resource on the planet consists of cellulose, hemicelluloses and
lignin. It can be exploited as a source of fermentable sugars for the conversion to ethanol
which may serve as the ultimate fossil fuel replacement. Hemicelluloses, contributing one
third of lignocellulose, consists of xylan and mannan. Mannan consists of glucomannan,
galactomannan and galactoglucomannan. A cocktail of enzymes are required for its complete
hydrolysis, including β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and
acetyl-mannan esterases. A need has arisen for the development of a recombinant
microorganism capable of converting lignocelluloses to bioethanol through an economically
feasible process.
The yeast Saccharomyces cerevisiae naturally ferments hexose sugars into ethanol and has
been used in various industrial applications due to its robustness in industrial processes, its
well-developed expression systems, its frequent use as a model organism for heterologous
gene expression and its current GRAS (Generally Regarded As Safe) status. This yeast is
unable to naturally utilise complex lignocelluloses. Recombinant biotechnology can be
implemented to overcome this limiting factor. Due to certain restraints by the yeast
S. cerevisiae such as hyperglycosylation and poor secretion capacity, alternative hosts such as
Aspergillus niger has also been considered for heterologous protein production.
The Aspergillus aculeatus β-mannanase (man1) and Talaromyces emersonii α-galactosidase
(Agal) genes were expressed in S. cerevisiae Y294. The cDNA of A. niger β-mannosidase
(cAnmndA) and synthetic Cellvibrio mixtus β-mannosidase (CmMan5A) were expressed in
A. niger. The sequence coding for the native secretion signal from CmMan5A was removed
and replaced with the XYNSEC sequence (yielding XYNSEC-CmMan5A) and expressed in
E. coli DH5α. The recombinant Man1, Agal, cAnmndA, CmMan5A and
XYNSEC-CmMan5A displayed optimal pH of 5.47, 2.37, 3.4, 3.4 and 5.47, respectively, and
optimal temperatures of 70°C for Man1, Agal, cAnmndA and CmMan5A and 50°C for
XYNSEC-CmMan5A. Activity levels of Man1, Agal, cAnmndA, CmMan5A and
XYNSEC-CmMan5A peaked at 36.08, 256.83, 11.61, 7.58 and 2.14 nkat/ml, respectively.
Co-expression of Agal and man1 led to a decrease in enzyme secretion and therefore
individual expression of these genes should be considered rather than co-expression. The enzymatic activity of Man1, Agal and CmMan5A resulted in a significant decrease in the
viscosity of galactomannan when used synergistically. This study confirmed successful
production of galactomannan hydrolysing enzymes by the yeast S. cerevisiae and the fungus
A. niger, as well as providing insight into the synergistic effect of these enzymes on the
viscosity of galactomannan. / AFRIKAANSE OPSOMMING: Die behoefte vir 'n koste-effektiewe en omgewingsvriendelike plaasvervanger vir
fossielbrandstowwe het tot 'n beduidende belangstelling in die produksie van bio-etanol gelei.
Lignosellulose synde die volopste hernubare hulpbron op die planeet bestaan uit sellulose,
hemiselluloses en lignien. Dit kan as 'n bron van fermenteerbare suikers vir die omskakeling
na etanol benut word, wat kan dien vir uiteindelike fossielbrandstofvervanging.
Hemiselluloses, wat bydra tot 'n derde van lignosellulose, bestaan uit xilaan en mannaan.
Mannaan bestaan uit glukomannaan, galaktomannaan en galaktoglukomannaan. 'n Mengsel
van ensieme word vir die volledige hidroliese van mannaan benodig, insluitende
β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en asetiel-mannaan esterases. 'n
Behoefte bestaan vir die ontwikkeling van 'n rekombinante mikroörganisme wat in staat is tot
die omskakeling van lignoselluloses na bio-etanol deur middel van 'n ekonomies
lewensvatbare proses.
Die gis Saccharomyces cerevisiae kan heksoe suikers na etanol omskakel en word gebruik in
verskeie industriële toepassings as gevolg van sy robuustheid in industriële prosesse, goed
ontwikkelde uitdrukking sisteme, gereelde gebruik as 'n model-organisme vir heteroloë
uitdrukking van gene en huidige GRAS (Generally Regarded As Safe) status. Die gis is nie
daartoe in staat om komplekse lignosellulose te benut nie. Rekombinante biotegnologie kan
egter geïmplementeer word om hierdie beperkende faktor te oorkom. As gevolg van sekere
beperkinge van die gis S. cerevisiae soos hiperglikosilering en lae sekresie kapasiteit, is
alternatiewe gashere soos Aspergillus niger ook oorweeg vir heteroloë proteïenproduksie.
Die Aspergillus aculeatus β-mannanase (man1) en Talaromyces emersonii α-galaktosidase
(Agal) gene is in S. cerevisiae Y294 uitgedruk. Die cDNA van A. niger β-mannosidase
(cAnmndA) en sintetiese Cellvibrio mixtus β-mannosidase (CmMan5A) is in A. niger
uitgedruk. Die DNA volgorde wat kodeer vir die natuurlike sekresiesein van CmMan5A is
verwyder en vervang met die XYNSEC volgorde (gegewe XYNSEC-CmMan5A) en uitgedruk
in E. coli DH5α. Die rekombinante Man1, Agal, cAnmndA, CmMan5A en
XYNSEC-CmMan5A vertoon optimale pH kondisies van 5.47, 2.37, 3.4, 3.4 en 5.47,
onderskeidelik, en die optimale temperatuur van 70°C vir Man1, Agal, cAnmndA en
CmMan5A en 50°C vir XYNSEC-CmMan5A. Aktiwiteitsvlakke van Man1, Agal,
cAnmndA, CmMan5A en XYNSEC-CmMan5A het 'n maksimum bereik op 36.08, 256.83,
11.61, 7.58 en 2.14 nkat/ml, onderskeidelik. Gesamentlike uitdrukking van Agal en man1 het tot 'n afname in ensiemsekresie gelei en dus moet individuele uitdrukking van hierdie gene
eerder as gesamentlike-uitdrukking oorweeg word. Die ensiematiese aktiwiteite van Man1,
Agal en CmMan5A het tot 'n beduidende afname in die viskositeit van galaktomannaan gelei
wanneer dit sinergisties gebruik word. Hierdie studie bevestig suksesvolle produksie van
galaktomannaan hidrolitiese ensieme in die gis S. cerevisiae en die fungus A. niger, en
verskaf insig in die sinergistiese effek van hierdie ensieme op die viskositeit van
galaktomannaan.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79817 |
| Date | 03 1900 |
| Creators | Malherbe, Alexander Robert |
| Contributors | Van Zyl, W. H., Rose, S. H., 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 | 119 pages : illustrations |
| Rights | Stellenbosch University |
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