Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Cellulose and hemicellulose represents the two most abundant groups of renewable
polysaccharides known to man. Apart from their presence in plant material, they also
contribute to a significant portion of inexpensive readily available material, such as
wastes and bypro ducts from forestry / agricultural origin. The chemical composition of
plant material varies, but the biomass content consists of approximately 75%
carbohydrate polymers (cellulose and hemicellulose) and 25% lignin.
The enzymes required for the degradation of cellulose and hemicellulose are
collectively called cellulases and hemicellulases. These enzymes have a broad spectrum
of industrial applications including the production of fuel ethanol through fermentations,
reducing the amount of chlorine required for bleaching in the pulp and paper industry,
increasing dough volume in the baking industry, improving digestion and nutritional
value of animal feed, increasing clarification and enhancing the filterability of wine, beer
and fruit juice, etc. Therefore, a large potential market exists for cellulases and
hemicellulases provided their production is economical and the product, authentic.
Aspergilli occur in a wide variety of habitats including soil, stored food and feed
products and decaying vegetation. The advantages for using A. niger as host for
heterologous enzyme production include good protein secretion, industrial fermentation
technology dating as far back as 1919, being a non-pathogenic fungus with GRAS status,
no special substrate or cultivation requirements, FDA approval of numerous enzymes
(homologous and heterologous) produced, etc.
In this study an Aspergillus expression vector was constructed using the
constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdp) of A. niger and
the glucoamylase terminator (glaAT) of Aspergillus awamori. The cDNA copies of the
eg! and xyn2 genes of Trichoderma reesei, cbhl-4 of Phanerochaete chrysosporium,
man! of Aspergillus aculeatus and xyn3 of Aspergillus kawachii were introduced into the
expression vector, respectively. All the plasmids were co-transformed with plasmid
p3SR2 to A. niger and transformants selected for stable plasmid integration into the
genome of the host. The recombinant enzymes EgI, Xyn2, Cbhl-4, Man! and XynC
were successfully expressed and secreted at activity levels of 2300, 8000, 500, 6000 and 900 nkatlml, respectively. The enzymes were produced as functional entities and were
subsequently characterized. The EgI, Xyn2 and ManI were evaluated as feed additives
for the possible use in the animal feed industry. Improved biomass gain was observed
with in vivo studies on poultry.
With the possible mass production of heterologous enzymes in mind, a simple
medium had to be devised for their inexpensive production. Molasses medium (available
from the South African sugar industry) was therefore evaluated and the cultivation
conditions optimized for it's possible use as cultivation substrate for A. niger. The
evaluation was done on the grounds of EgI and Xyn2 activity produced which was
monitored over time.
This study highlighted the possible use of A. niger for the heterologous
production of enzymes, the use of industrial substrate for cultivation and paved the way
for the high level expression of industrially important genes at low cost and a positive
environmental impact. / AFRIKAANSE OPSOMMING: Sellulose en hemisellulose verteenwoordig die twee vollopste herwinbare polisakkariede
bekend. Behalwe vir hul teenwoordigheid in plantmateriaal, dra hulle ook by tot 'n
beduidende fraksie van goedkoop, maklik bekombare materiaal soos afval- en
byprodukte van bosbou I landbou oorsprong. Soos te verwagte, varieër die chemiese
samestelling van die plantmateriaal, maar die biomassa-inhoud bestaan uit naastenby
25% lignien en 75% koolhidraatpolimere (sellulose and hemicellulose).
Die ensieme benodig vir die afbraak van sellulose en hemisellulose staan
gesamentlik as sellulases en hemisellulases bekend. Hierdie ensieme het 'n breë
spektrum van industriële toepassings insluitende die produksie van brandstofalkohol
d.m.v. fermentasies, vermindering in die hoeveelheid chloor benodig vir die bleikproses
in die pulp-en-papier industrie, toename in deegvolume in die bakkersindustrie,
verbetering van verteerbaarheid en verhoging van voedingswaarde van dierevoer,
toename in verheldering en verbeterde filtreerbaarheid van wyn, bier en vrugtesap, ens.
Dus bestaan daar 'n groot potensiële mark vir sellulases en hemisellulases, mits hul
produksie ekonomies en die produk outentiek is.
Aspergilli kom in 'n wye verskeidenheid van omgewings voor, insluitende grond,
gestoorde voedsel- en voerprodukte asook ontbindende plante materiaal. Die voordele
vir die gebruik van A. niger as gasheer vir heteroloë ensiemproduksie sluit in 'n goeie
proteïen produseerder, industriële fermentasietegnologie dateer sover terug as 1919, 'n
nie-patogeniese fungus met GRAS-status, benodig geen spesiale substrate of
kwekingskondisies nie, FDA goedkeuring vir 'n groot aantal ensieme (homoloog sowel
as heteroloog) wat reeds geproduseer word, ens.
In hierdie studie is 'n Aspergillus uitdrukkingsvektor gekonstrueer deur van die
konstitutiewe gliseraldehied-3-fosfaat dehidrogenase promoter (gpdp) van A. niger en die
glukoamilase termineerder (glaAT) van Aspergillus awamori gebruik te maak. Die cDNA
kopiee van die die eg! en xyn2 van Trichoderma reesei, cbhl-4 van Phanerochaete
chrysosporium, man! van Aspergillus aculeatus en die xynC van Aspergillus kawachii
was onderskeidelik na die uitdrukkingsplasmied oorgedra. Alle plasmiede is gesamentlik
met die p3 SR2 plasmied na A. niger getransformeer en vir stabiele integrasie in die gasheergenoom geselekteer. Die rekombinante ensieme Egl, Xyn2, Cbhl-4, Manl en
Xyn3 is suksesvol uitgedruk en teen aktiviteitsvlakke van 2300, 8000, 500, 6000 en 900
nkat/ml, onderskeidelik uitgeskei. Die ensieme is as funksionele entiteite geproduseer en
vervolgens gekaraktiriseer. Die Egl, Xyn2 en Manl is as voertoevoegings vir die
moontlike gebruik in die dierevoerindustrie geëvalueer. Verbeterde biomassa toename is
in die in vivo studie op pluimvee waargeneem.
Met die moontlikheid van grootskaalse heteroloë ensiemproduksie in gedagte,
moes 'n eenvoudige substraat vir hul goedkoop produksie gevind word. Molasse
medium (verkrygbaar vanaf die Suid Afrikaanse suiker industrie) was derhalwe
geëvalueer en die kwekingskondisies geoptimiseer vir die moontlike gebruik as
kwekingssubstraat vir A. niger. Vir die evaluasie is die Egl en Xyn2 aktiwiteite onder
verskillende toestande geproduseer en oor tyd gemonitor.
Hierdie studie beklemtoon die moontlike gebruik van A. niger vir heteroloë
produksie van ensieme, die gebruik van industriële substrate as kwekingsmedium en baan
die weg vir ekonomiese, hoëvlakuitdrukking van industrieelbelangrike ensieme met 'n
positiewe implikasie op die omgewing.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53437 |
| Date | 03 1900 |
| Creators | Rose, Shaunita Hellouise |
| Contributors | Van Zyl, W. 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 | Unknown |
| Type | Thesis |
| Format | 202 p. : ill. |
| Rights | Stellenbosch University |
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