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.

Thesis (MSc)--Stellenbosch University, 2002. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has established its importance as a wine yeast and also proven itself as a reliable starter culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly, its metabolic activities result in the production of higher alcohols, fatty acids and esters, which are important flavour and aroma compounds that are essential for consistent and predictable wine quality. There is a growing consumer demand for wine containing lower levels of alcohol and chemical preservatives. Glucose oxidase (GOX) has received considerable research interest regarding its potential application in the wine industry to reduce alcohol levels and as a biocontrol agent. Several physical processes are used for the removal or reduction of alcohol in wine and some of them are sometimes used in combination. These processes tend to involve expensive equipment and can be intensive from a processing point of view. An alternative approach was introduced with the concept of treating grape must with GOX to reduce the glucose content of the must, and therefore produce a wine with a reduced alcohol content after fermentation. Due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialised wine yeast strains possessing a wide range of optimised, improved or novel oenological properties. The first and main objective of this study was to genetically engineer wine yeasts to produce wine with a reduced alcohol content. In order to do this, the structural glucose oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5) containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox- PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of S. cerevisiae (~1278). Results obtained indicated the production of biologically active glucose oxidase and showed that it is secreted into the culture medium. This would mean that the enzyme will convert the glucose to gluconic acid in the medium before the yeast cells are able to metabolise the glucose to ethanol. Microvinifications performed with Chardonnay grapes showed that the laboratory yeast starter cultures transformed with GOX1 were indeed able to reduce the total amount of alcohol in the finished product. The second objective of this study involved the potential application of GOX as a biocontrol agent. Screening was performed for wine spoilage microorganisms, such as acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage microorganisms tested formed different sized inhibition zones, indicating varying degrees of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed under a scanning electron microscope. The total collapse of the bacterial cell wáll could be seen and might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a result of the peroxidation of the membrane lipid, and a strong oxidising effect on the bacterial cell, which is the cause of the destruction of basic molecular structures, such as nucleic acids and cell proteins. In this exciting age of molecular yeast genetics and modern biotechnology, this study could pave the way for the development of wine yeast starter culture strains for the production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs) within the wine industry is a limiting factor at present and credible means must be found to effectively address the concerns of traditionalists within the wine industry and the negative overreaction by some consumer groups. There is a vast potential benefit to the wine consumer and industry alike and the first recombinant wine products therefore should unmistakably demonstrate safe products free of potentially harmful compounds, and have organoleptic, hygienic and economic advantages for both the wine producer and consumer. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na wyne met 'n laer alkoholinhoud en minder preserveermiddels. Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en selfs unieke, wynkundige einskappe bevat. Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278) getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei. Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied (H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese molekulêre strukture, soos die nukleïensure en selproteïene. Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste, asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan. Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese voordele toon te opsigte van beide die wynprodusent en gebruiker.