Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aim of this study was to develop a ZnO nanowire-array biosensor that would detect immunoglobulins and record changes in the concentration of an antibody. Early detection of disease-causing agents is essential for an early response. In contrast to conventional methods, biosensors may detect disease-associated agents much faster and more accurate, which holds specific benefits to rural communities. The development of such a biosensor would be favourable for diagnostics in underprivileged communities without infrastructure. The hypothesis was that binding of antibodies to the surface of ZnO nanowires would result in the generation of a piezoelectric potential that, when channelled through a Schottky barrier, would produce a constant voltage reading. Piezoelectricty would be generated due to the bending of the nanowires, or tensile stress applied to the nanowires due to binding of the antibodies. The performance of such a device largely depends on the methods used to construct the ZnO nanowires and methods used to funtionalize the sensor surface. The biggest challenge was thus to chemically modify the self-assembled monolayers (SAMs) and create intermediate monolayers that would react to primary amino groups of lysozyme and form a covalent amide bond. Lysozyme was selected as model antigen, since its structure and reaction with antibodies has been well studied. Alkanethiol and dialkyl disulphides were used to form SAMs. Different SAMs were compared to select the absorbate that would bind the highest concentration of lysozyme. Lysozyme was best immobilized onto Au film layers in the presence of SAM 3-mercaptopropionic acid. Weakest immobilization was in the presence of combined SAM 11-mercaptoundecanoic acid/1-nonanethiol. The sensitivity of the constructed ZnO nanowire biosensor was tested in vitro, in the presence of different concentrations of lysozyme antibodies. An increase in the dimension of the ZnO seed layer led to an increase in the mean diameter of the ZnO seed grains, and subsequently an increase in the mean diameter of the synthesized ZnO nanowires. Deposition of the ZnO seed layer, using the RF cylindrical magnetron sputtering technique, improved the c-axis alignment of the nanowires and produced nanowires with similar dimensions. However, deposition of the ZnO seed layer using the sol-gel spin coating technique, produced nanowires with irregular c-axis alignments and irregular diameters. An increase in the Au film thickness led to a decrease in the mean diameter of the synthesized ZnO nanowires and worsening of the c-axis alignment. In contrast to single crystalline Au (111) film layers, polycrystalline Au layers increased the mean diameter of the synthesized nanowires. The crystal orientation of the Au film layer had no effect on the c-axis alignment. Increased voltage readings were recorded with an increase in antibody binding, indicating that the ZnO nanosensor may be used to record changes in immunoglobulin levels. Antibody concentrations ranging from 10 ng/ml to 20 μg/ml were sensed. This is the first study showing that ZnO nanowires, conformed into piezoelectric transducers, may be used in the detection of antibodies. The current size of the chip with ZnO nanowires is approximately 1 cm², which is too big to incorporate into a compact monitoring device. Apart from the challenge to produce smaller nanowire-arrays, highly sensitive sensors and miniature amplifiers will have to be developed to increase the strength of the signals generated by the nanowires. The biosensor will also have to be optimised to detect a variety of immunoglobulins. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om ‘n ZnO nanodraad biosensor te ontwikkel wat immunoglobuliene kan opspoor en veranderinge in konsentrasies van die teenliggaampies sal reflekteer. Vroë deteksie van siekte veroorsaakende agente is belangrik vir n vroeg tydige respons. In teenstelling tot konvensionele metodes, kan biosensors siekte veroorsaakende agente vining en akkuraat opspoor, wat veral voordele vir gemeenskappe in landelike gebiede inhou. Die hipotese was dat binding van teenliggaampies aan die ZnO nanodrade ‘n piëzo-elektriese potensiaal sal skep, wat dan ‘n konstante leesbare spanningspotensiaal sal lewer nadat dit deur ‘n Schottky versperring gestuur is. Piëzo-elektrisiteit word gegenereer deur die buiging van die nanodrade, of deur spanning wat op die nanodrade geplaas word deur binding van die teenliggaampies. Die sukses van die ontwerp hang grootliks af van die metode wat gebruik word om die ZnO nanodrade te konstrueer en metodes wat gebruik word om die sensor oppervlak te funksionaliseer. Die grootste uitdaging was dus om die monolae wat outomaties saam groepeer (SAMs) chemies so te verander dat intermediêre monolae vorm wat aan primêre aminogroepe van lisosiem bind ten einde kovalente amied-bindings te vorm. Lisosiem is as model antigeen geselekteer omdat die struktuur en reaksie daarvan met teenliggaampies reeds goed bestudeer is. Alkaantiol en di-alkiel disulfied is gebruik om SAMs te vorm. ‘n Verskeidenheid SAMs is vergelyk ten einde die anker te selekteer waaraan die hoogste konsentrasie lisosiem sal bind. Lisosiem is die effektiefste aan Au film lae ge-immobiliseer in die teenwoordigheid van SAM 3-merkapto-propanoësuur. Die swakste immobilisasie is in die teenwoordigheid van kombineerde SAM 11-merkapto-dekanoësuur/1-nanotiol waargeneem. Die sensitiwiteit van die ZnO nanodrade is in vitro getoets, in die teenwoordigheid van verskillende konsentrasies van lisosiem teenliggaampies. ‘n Toename in die dimensie van die ZnO grondlaag het die gemiddelde deursnit van die ZnO grein verhoog en so ook die gemiddelde deursnit van die gesintetiseerde ZnO nanodrade. Toediening van die ZnO grondlaag deur gebruik te maak van die RF silindriese mikrogolf-verstuiwings tegniek het die orientasie van die c-aslyn van die nanodrade verbeter. Toediening met die sol-gel draai-bedekkings tegniek het ‘n onreëlmatige orientasie van die c-aslyn teweeg gebring, asook ‘n variasie in die afmetings van die nanodrade. ‘n Toename in die Au laag het ‘n afname in die gemiddelde afmetings van die nanodrade en ook ‘n onreelmatige oriëntasie van die c-aslyn veroorsaak. In teenstelling met enkel-kristallyne Au (111) het poli-kristallyne Au lagies ‘n toename in die gemiddelde deursnit van die nanodrade veroorsaak. Die kristal-oriëntasie van die Au laag het geen effek op die belyning van die nanodrade gehad nie. Die spanningspotensiaal het verhoog met ‘n toename in teenliggaampie binding. Hiervolgens kan die ZnO nanosensor gebruik word om veranderinge in immunoglobulien vlakke te monitor. Teenliggaampie konsentrasies wat wissel van 10 ng/ml tot 20 μg/ml is opgespoor. Hierdie is die eerste studie wat toon dat ZnO nanodrade, omskep tot piëzo-elektriese transduseerders, gebruik kan word in die opsporing van teenliggaampies. Die grootte van die skyfie met die ZnO nanodrade is tans ongeveer 1 cm² en is te groot om in ‘n kompakte biosensor in te bou. Benewens die uitdaging om kleiner nanodraad skyfies te ontwikkel, sal hoogs sensitiewe sensors en seinversterkers ontwikkel moet word om die sein afkomstig van die nanodrade te versterk. Die biosensor sal ook ge-optimiseer moet word om ‘n verskeidenheid immunoglobuliene op te spoor.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main vectors for the spread of antibiotic and heavy metal resistance genes in the environment. Functional and taxonomic characterization of novel plasmids is, therefore, central to our general understanding of plasmid biology and their contribution to microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring resistance to tetracycline were isolated from the opportunistic fish pathogens Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed the presence of 18 putative open reading frames (ORFs), of which the tetAR tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs with significant similarity to known sequences within the NCBI database. Putative functions were assigned to 10 of the ORFs based on their distant homology with proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to comprise the minimal region required for replication (minimal replicon) by means of deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were found to be essential for plasmid replication. Inactivation of ORF3 resulted in an increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7 days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown that ORF3 autoregulated its own promoter. These results, therefore, suggested that although ORF3 was not essential for replication, it may be involved in plasmid copy number regulation and control. Host range analysis indicated that p31T1 was able to replicate in two other members of the γ-proteobacteria group (Escherichia coli and Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus suggesting a limited host range. Furthermore, p31T1 was mobilized only at low frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability, host range and mobilization characteristics determined for p31T1 and that no other plasmid replication and mobilization systems with significant sequence similarity to these plasmids have yet been identified, it is likely that these two plasmids are the first representative members of a new family of plasmids found within aquacultureassociated Aeromonas species and which are involved in the spread of tetracycline resistance. / AFRIKAANSE OPSOMMING: Plasmiede vorm ‘n integrale deel van die horisontale geen poel en vorm daarom die hoof vektore vir die verspreiding van antibiotika- en swaarmetaal-weerstandbiedende gene in die omgewing. Funksionele en taksonomiese karakterisering van nuwe plasmiede is belangrik in die begrip van plasmied biologie en hul bydrae tot mikrobiese evolusie. Twee 14-kb mobiliseerbare plasmiedes, p31T1 en p36T2, met tetrasiklien weerstandigheid was vanaf die opportunistiese vis patogene Aeromonas sobria en Aeromonas hydrophila geïsoleer en het identiese restriksie fragment lengte polimorfisme (RFLP) patrone. DNA volgorde analise van die twee isogeniese plasmiede (slegs die volgorde van p36T2 was bepaal) het die teenwoordigheid van 18 moontlike oop leesrame (OLR) getoon. Die tetAR tetrasiklien weerstandbiedende gene, wat met ‘n verkorte Tn1721 transposon geassosieerd is, was die enigste OLR wat beduidende volgorde ooreenkoms met bekende volgordes binne die NCBI databasis getoon het. Moontlike funksies was toegeken aan 10 van die OLRe en was gebasseer op vêrlangse homologie met proteïene met bekende funksies. Ses van die 18 OLRe strek oor ‘n 5.7- kb minimale replikon fragment wat benodig word vir replisering en is deur middel van delesie analises van p31T1 derivate gevind. Van hierdie ses OLRe, word OLR2 en OLR4 benodig vir plasmied replisering. Inaktivering van OLR3 het ‘n toename in plasmied kopiegetal (PKG) vanaf ~3 tot ~7 plasmiede per kromosoom en ‘n afname in stabiliteit vanaf ~80% tot 16% oor 127 generasies (7 dae) tot gevolg gehad. Verder kon daar deur middel van β-galaktosidase fusie analises getoon word dat OLR3 sy eie promotor outoreguleer. Hierdie resultate stel dus voor dat alhoewel OLR3 nie benodig was vir replikasie nie, mag dit dalk by plasmied kopiegetal regulering en beheer betrokke wees. Bakteriële gasheer analises het getoon dat p31T1 in 2 addisionele lede van die γ-proteobakterieë groep (Escherichia coli en Pseudomonas putida) kon repliseer, maar nie in ‘n α-proteobacterium nie. Verder kon p31T1 teen ‘n lae frekwensie (5.4 x 105) gemobiliseer word deur ‘n IncP-1 konjugasie sisteem, maar dit mag wees dat die mobilisering eerder optimaal kan plaasvind met ‘n alternatiewe konjugasie sisteem. Na aanleiding van die unieke PKG, stabiliteit, gasheer en mobilisering eienskappe wat vir p31T1 bepaal is en die feit dat geen ander replisering en mobilisering sisteme met noemenswaardige volgorde homologie tot hierdie plasmiede gevind kon word nie, blyk dit dat hierdie van die eerste lede van ‘n nuwe familie van plasmiede binne die akwakultuur-geassosieerde Aeromonas spesies is, wat betrokke is by die verspreiding van tetrasiklien weerstandbiedendheid.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2005. / Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Research on probiotics has increased over the past years, which led to commercialization of a number of probiotic supplements and functional foods. In vitro assays such as tolerance to acid and bile, adhesion to mucus and epithelial cells, antimicrobial activity and antibiotic resistance tests are performed to screen lactic acid bacteria for probiotic properties. Enterococcus mundtii ST4SA produces an antimicrobial peptide (peptide ST4SA) with activity against Gram-positive and Gram-negative bacteria. Lactobacillus plantarum 423 produces plantaricin 423, a typical class II bacteriocin, active against a number of Gram-positive bacteria. A gastro-intestinal model (GIM) simulating the gastro-intestinal tract (GIT) of infants, was developed to study the survival of E. mundtii ST4SA and L. plantarum 423 and evaluate them as possible probiotics. Growth of the two strains in the GIM was compared to the growth of commercially available probiotics. Infant milk formulations were used as growth medium. Changes in pH, the addition of bile salt and pancreatic juice, and intestinal flow rates were controlled by peristaltic pumps linked to a computer with specifically designed software. Strain ST4SA was sensitive to low pH and high concentrations of bile salts. Growth of strain ST4SA was repressed in the first part of the GIM, however, the cells recovered in the ileum. Strain 423 was also sensitive to acidic conditions. However, the cells withstood the presence of bile and pancreatin in the first part of the GIT. Neither of the two strains displayed bile salt hydrolase (BSH) activity. Both strains were resistant to amoxicillin, ampicillin, chloramphenicol, cefadroxil, roxithromycin, meloxicam, doxycycline, erythromycin, novobiocin, rifampicin, tetracyclin, bacitracin, oflaxacin and cephazolin, anti-inflammatory drugs Na+- diklofenak and ibuprofen, and painkillers codeine terprim hydrate aminobenzoic acid, metamizole aspirin and paracetamol. Strain 423 was resistant to ciprofloxacin. Genes encoding cytolysin, non-cytolysin β-hemolysin and cell aggregation substances were detected on the genome of strain ST4SA but they were not expressed. L. plantarum 423 does not contain genes encoding gelatinase, cell aggregation, enterococcus surface protein, hemolysin, non-cytolysin β- hemolysin and enterococcus endocarditis antigen. Both strains inhibited the growth of Listeria monocytogenes ScottA in the GIM. Survival of the strains improved when used in combination and compared well with the survival of commercially available probiotics. Adhesion to epithelial cells is an important prerequisite for bacterial colonization in the GIT. The adhesion of E. mundtii ST4SA and L. plantarum 423 was studied using Caco-2 (human colon carcinoma epithelial) cells. Both strains revealed good adhesion compared to other probiotic strains. No correlation was found between hydrophobicity, auto-aggregation and adhesion to Caco-2 cells. Antibiotics and anti-inflammatory medicaments had a negative effect on adhesion. Different combinations of proteins were involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. E. mundtii ST4SA, L. plantarum 423 and L. monocytogenes ScottA were stained with fluorescent dyes to visualize adhesion to Caco-2 cells. Adhesion of L. monocytogenes ScottA to Caco-2 cells was not reduced in the presence of strains ST4SA and 423. Cell-free culture supernatants of both strains inhibited the invasion of L. monocytogenes ScottA. The cell structure of Caco-2 cells changed in the presence of L. monocytogenes ScottA. Strains ST4SA and 423 protected Caco-2 cells from deforming. / AFRIKAANSE OPSOMMING: Navorsing op probiotika het die afgelope tyd drasties toegeneem en aanleiding gegee tot die kommersialisering van ‘n groot hoeveelheid probiotiese supplemente en funksionele voedselsoorte. In vitro studies, soos bv. weerstand teen suur en gal, vashegting aan mukus en epiteelselle, antimikrobiese aktiwiteit en weerstand teen antibiotika word uitgevoer om te bepaal of melksuurbakteriëe aan probiotiese standaarde voldoen. Enterococcus mundtii ST4SA produseer ’n peptied met antimikrobiese werking teen Grampositiewe en Gram-negatiewe bakteriëe. Lactobacillus plantarum 423 produseer ‘n tipiese klas II bakteriosien, plantarisien 423, met aktiwiteit teen sekere Gram-positiewe bakteriëe. ’n Gastro-intestinale model (GIM) wat die spysverteringskanaal (SVK) van babas simuleer, is ontwikkel om die oorlewing van E. mundtii ST4SA en L. plantarum 423 te bepaal en hul eienskappe met dié van kommersiële probiotiese stamme te vergelyk. Babamelk formules is as groeimedium gebruik. Verandering in pH, byvoeging van galsoute en pankreassappe, en intestinale vloei is met behulp van peristaltiese pompe gereguleer wat seine vanaf ‘n spesiaal ontwikkelde rekenaarprogram ontvang. E. mundtii ST4SA was sensitief vir lae pH en hoë galsoutkonsentrasies en groei is in die eerste deel van die GIM onderdruk. Selgetalle het wel in die ileum herstel. Stam 423 was ook sensitief vir lae pH, maar het die galsout- en pankreatienvlakke in die laer deel van die SVK weerstaan. Geen galsout-hidrolase aktiwiteit is by enige van die twee stamme gevind nie. Beide stamme het weerstand getoon teen amoksillien, ampisillien, chloramfenikol, cefadroksiel, roksitromisien, meloksikam, doksisiklien, eritromisien, novobiosien, rifampisien, tetrasiklien, basitrasien, oflaksasien, kefazolien, die anti-inflammatoriese medikamente Na+-diklofenak en ibuprofen, en die pynstillers kodeïenterprimhidraataminobensoësuur, metamisoolaspirien en parasetamol. L. plantarum 423 was bestand teen ciprofloksasien. Gene wat kodeer vir sitolisien, nie-sitolisien β-hemolisien III en sel-aggregasie is op die genoom van E. mundtii ST4SA gevind, maar word nie uitgedruk nie. L. plantarum 423 besit nie die gene wat vir gelatinase, selaggregasie substansies, enterokokkus selwandproteïen, hemolise, nie-sitolisien β-hemolisien en enterokokkus endokarditis antigeen kodeer nie. Albei stamme inhibeer die groei van Listeria monocytogenes ScottA in die GIM. Die twee stamme in kombinasie het tot beter oorlewing in die GIM gelei. Stamme ST4SA en 423 vergelyk goed met kommersieël beskikbare probiotika. Vashegting van probiotiese stamme aan epiteelselle is belangrik vir kolonisering in die SVK. Vashegting van E. mundtii ST4SA en L. plantarum 423 is bestudeer deur van Caco-2 (kolon epiteel) selle van die mens gebruik te maak. Die aanhegting van beide stamme aan Caco-2 selle het goed vergelyk met kommersieël beskikbare probiotiese stamme. Geen korrelasie is gevind tussen hidrofobisiteit, aggregasie en vashegting aan Caco-2 selle nie. Antibiotika en antiinflammatoriese medikamente het ‘n negatiewe effek op vashegting gehad. Verskillende kombinasies van proteïene is betrokke in die vashegting van E. mundtii ST4SA en L. plantarum 423 aan Caco-2 selle. E. mundtii ST4SA, L. plantarum 423 en L. monocytogenes ScottA is met fluoreserende kleurstowwe gemerk om vashegting aan Caco-2 selle te monitor. Vashegting van L. monocytogenes ScottA aan Caco-2 selle is nie deur die teenwoordigheid van stamme ST4SA en 423 beïnvloed nie. Sel-vrye kultuursupernatante van beide stamme het die binnedring van L. monocytogenes ScottA verhoed. Die selstruktuur van Caco-2 selle het in die teenwoordigheid van L. monocytogenes ScottA van vorm verander. E. mundtii ST4SA en L. plantarum 423 het die Caco-2 selle teen vervorming beskerm.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Heterologous gene expression is of considerable interest for the production of proteins of therapeutic and industrial importance. As the nature of recombinant proteins has become more complex and as transformation systems have been established in more species, so the variety of hosts available for expression has increased. Every system available has both advantages and disadvantages. The research presented here highlights the advantages of selecting the most appropriate expression system for different recombinant proteins. Expression of different biocatalytically-relevant enzymes, epoxide hydrolases, halohydrin dehalogenase, laccase and mannanase, in different host systems is undertaken, and expression levels and activity are compared. The development of Yarrowia lipolytica as a whole-cell biocatalyst is described. Y. lipolytica is used for the functional expression of epoxide hydrolases (EHs) and halohydrin dehalogenases. EHs are hydrolytic enzymes that convert epoxides to vicinal diols by ring-opening. Two new fungal EHs from Rhodosporidium toruloides NCYC 3181 and NCYC 3158 (a putative Cryptococcus curvatus strain) were identified and cloned. Additional EHs from different sources, including bacteria, yeasts, fungi and plants, were chosen for expression in Y. lipolytica, in order to determine its suitability as the expression system of choice for the production of EHs. Multi-copy integrants were developed, with the genes under control of the growthphase dependent hp4d promoter. A Saccharomyces cerevisiae strain was developed, expressing the EH from Rhodotorula araucariae1, to compare as a whole-cell biocatalyst with Y. lipolytica. This strain proved to be an exceptionally poor wholecell biocatalyst. All the Y. lipolytica strains developed showed varying levels of activity towards different classes of epoxides. Some strains displayed opposite enantioselectivities, allowing for potential complete conversions of racemic epoxides to the desired enantiomeric product. Halohydrins can be considered direct precursors of epoxides. Halohydrin dehalogenases catalyse the nucleophilic displacement of a halogen ion in halohydrins by a vicinal hydroxyl group, yielding an epoxide, a proton and a halide ion. The HheC gene from Agrobacterium radiobacter AD1, codon-optimised to match the codon usage of Y. lipolytica, was over-expressed in Y. lipolytica by generation of multi-copy integrants, further expanding the use of this organism as a host strain for heterologous production of enzymes. Expression levels were maximised by creating tandem repeats of the introduced HheC gene. The ring-closure activity with 2-chloro- 1-phenylethanol as substrate was demonstrated to be broadly dose-dependent. The b-mannanase gene (man1) from Aspergillus aculeatus MRC11624 was expressed in Y. lipolytica with effective secretion in the presence of its native secretion signal, using the hp4d promoter. The same gene was expressed in Aspergillus niger2 under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and the Aspergillus awamori glucoamylase terminator (glaT). Following optimisation with copy numbers and culture conditions, maximal activity levels of 26,140 nkat.ml-1 for Y. lipolytica, and 16,596 nkat.ml-1 for A. niger were obtained. Laccases are important enzymes for bioremediation, and the best characterised enzymes are from the fungus Trametes versicolor. The objective of this research was to optimise expression of T. versicolor laccases (lcc1 and lcc2) in A. niger D15 and Pichia pastoris3. The Lcc1 enzyme was less active than Lcc2 in both hosts. P. pastoris secreted 0.4 U.L-1 Lcc1 and 2.8 U.L-1 Lcc2, compared to 2,700 U.L-1 produced by A. niger. The Lcc2 enzyme from recombinant A. niger was subsequently purified and characterised in terms of molecular weight and glycosylation, and compared to the wild-type enzyme purified from T. versicolor. The work presented underscores the requirement for experimentation before finalising the choice of an expression system for the optimal production of the desired protein. Every system available has both advantages and disadvantages, and when considering which system to use for producing a recombinant protein, various factors must be taken into consideration. However, the choice is broad and each decision needs to be made empirically. 1 The construction of the S. cerevisiae epoxide hydrolase production strain was carried out by Dr Neeresh Rohitlall of CSIR Biosciences. The Y. lipolytica epoxide hydrolase strains were constructed by the author. 2 The construction of Man1-producing A. niger strain was done by Dr Shaunita Rose of the University of Stellenbosch. The construction of Y. lipolytica Man1 production strains was done by the author. 3 The expression of T. versicolor laccases in P. pastoris was done by Christina Bohlin of Karlstad University. A niger laccase production strains were created by the author. / AFRIKAANSE OPSOMMING: Heteroloë geen uitdrukking is van groot belang vir die produksie van proteïene wat van terapeutiese en industriele belang is. Soos die aard van rekombinante proteïene meer ingewikkeld raak en getransformasie-sisteme vir verskeie spesies gevestig raak, is daar ’n groter verskeidenheid van gashere beskikbaar vir geenuitdrukking. Elke sisteem het beide sy voor- en nadele. Hierdie navorsing beklemtoon die voordele wanneer die mees gepaste uitdrukkingssiteem gekies word. Die uitdrukking van verskeie ensieme van biokatalities belang, epoksiedhidrolases, halohidrien dehalogenase, lakkase en mannanase in verskillende gasheersisteme is onderneem en die uitdrukkingsvlakke en aktiwiteite vergelyk. Die ontwikkeling van Yarrowia lipolytica as ’n heelsel biokatalis word beskryf. Y. lipolytica word gebruik vir die funksionele uitdrukking van epoksiedhidrolases (EHs) en halohidrien dehalogenases. EHs is hidroliseringsensieme wat die epoksiede omskakel na aangrensende diole deur middel van ring-opening. Twee nuwe fungi EHs vanaf Rhodosporidiom toruloides NCYC 3181 en NCYC 3158 (’n moontlike Cryptococcus curvatus) is geïdentifiseer en gekloneer. Verdere EHs van verskillende bronne, insluitend bakterieë, giste, fungi en plante, is gekies vir uitdrukking in Y. lipolytica ten einde sy geskiktheid vir die produksie van EHs te bepaal. Multikopie integrante is ook ontwikkel met gene onder beheer van die groei-fase afhanklike hp4d promotor. ’n Saccharomyces cerevisiae ras is ook ontwikkel vir die uitdrukking van die EH van Rhodotorula araucariae4 sodat dit met Y. lipolytica as ’n heelsel biokatalis vergelyk kan word. Hierdie ras was ‘n buitengewone swak heelsel biokatalis. Al die Y. lipolytica rasse wat ontwikkel is het wisselende aktiwiteitsvlakke teenoor verskillende klasse van epoksiede getoon. Sommige rasse het teenoorgestelde enantio-selektiwiteit getoon en het die potensiaal om rasemiese epoksiede volledig na die gewensde enantiomeriese produk om te skakeling. Halohidriene kan as direkte voorgangers van epoksiede beskou word. Halohidrien dehalogenases kataliseer die nukleofiliese vervanging van ’n halogeen-ioon in halohidriene deur ’n aangrensende hidroksiel groep, wat ’n epoksied, ’n proton en ’n halied-ioon lewer. Die HheC geen van Agrobacterium radiobacter AD1 is kodon– geöptimiseer om te pas by die kodon gebruik van Y. lipolytica en was uitgedruk in Y. lipolytica deur die skep van mulitkopie integrante, ’n verdere verbreeding van die toepaslikheid van die organisme as gasheerras vir die heteroloë produksie van ensieme. Maksimum uitdrukkingsvlakke is bereik deur die skep van opeenvolgende herhalings van die ingevoegde HheC-geen. Daar is ook gewys dat die ring-sluitingsaktiwiteit met 2-chloro-1-pheniel-etanol as substraat meestal dosis-afhanklik is. Die -mannanase geen (man1) van Aspergillus aculeatus MRC11624 is uitgedruk en effektief in Y. lipolytica mbv sy eie uitskeidings sein uitgeskei, met die gebruik van die groei-fase afhanklike hp4d promotor. Dieselfde geen is uitgedruk in Aspergillus niger5 onder beheer van die A. niger gliseraldehied-3-fosfaat dehidrogenase promotor (gpdp) en die Aspergillus awamori glikoamilase termineerder (glaT). Verdere optimisering van kopiegetal en voedingskondisies het gelei tot maksimum aktiwiteitsvlakke van 26,140 nkat.ml-1 vir Y. lipolytica en 16,596 nkat.ml-1 vir A. niger. Lakkases is belangrike ensieme vir bio-remediëring, en die ensieme van die fungus Trametes versicolor is die beste gekarateriseer. Die doelwit van hierdie navorsing was die optimisering van die uitdrukking van T. versicolor lakkases (lcc1 en lcc2) in A. niger en Pichia pastoris6. Die Lcc1 ensiem was minder aktief as Lcc2 in altwee die gashere. P. pastoris het 0.4 U.L-1 Lcc1 en 2.8 U.L-1 Lcc2 onderskeidelik uitgeskei, in vergelyking met 2,700 U.L-1 Lcc2 wat deur A. niger geproduseer is. Die Lcc2 ensiem afkomstig van die rekombinante A. niger is vervolgens gesuiwer en gekarakteriseer met betrekking tot molekulêre massa en glikosilering, en daarna vergelyk met die wilde-tipe ensiem wat deur T. versicolor geproduseer word. Die werk wat hier aangebied word, beklemtoon die vereistes vir eksperimentering voor die finale keuse met betrekking tot ’n gepaste uitdrukkingsisteem gemaak kan word vir die optimale produksie van die gewensde proteïen. Elke sisteem het beide voordele en nadele, en wanneer ’n sisteem oorweeg word is daar verskeie faktore wat in ag geneem moet word. ’n wye verskeidenheid van keuses is beskikbaar en elke besluit moet empiries gemaak word. 4 Die konstruksie van die S. cerevisiae epoksiedhidrolase-produserende ras is deur Dr Neeresh Rohitlall van CSIR Biosciences gedoen. Die Y. lipolytica epoxied hydrolase rasse is deur die outeur gemaak. 5 Die konstruksie van die Man1-produserende A. niger ras is deur Dr Shaunita Rose van die Universiteit van Stellenbosch gemaak. Die Y. lipolytica Man1 ras is gemaak deur die outeur. 6 Die uitdrukking van T. versicolor lakkases in P. pastoris is gedoen deur Christina Bohlin van Karlstad University. Die A niger lakkase produksie ras is geskep deur die outeur.

Dissertation (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The acidophilic, chemolithoautotrophic bacterium, Acidothiobaci/lus ferrooxidans is one of a consortium of bacteria involved in biornining, including the recovery of gold from arsenopyrite ores. The genes conferring arsenic resistance to At. ferrooxidans were cloned and sequenced and shown to be chromosomally located. Homologues to the arsB (membrane located arsenite efflux pump), arsC (arsenate reductase) and arsH (unknown function) genes from known arsenic resistance (ars) operons were identified. A fourth gene was found to have weak homology to the ArsR-family of regulators. The arsenic resistance genes of At. ferrooxidans are arranged in an unusual manner, with the arsRC and arsBH genes divergently transcribed. This divergent arrangement was found to be conserved in all four of the At. ferrooxidans strains we tested. All of the At. ferrooxidans ars genes were expressed in Escherichia coli and the arsB and arsC genes conferred arsenite (and antimonite) and arsenate resistance, respectively, to an E. coli ars mutant (AW311 0). Analysis of the putative amino acid sequences of these ars genes revealed that the ArsB from At. ferrooxidans is closely related to the ArsB proteins from other Gram-negative bacteria. However, the ArsC protein is more closely related to the ArsC proteins from Gram-positive bacteria. Furthermore, a functional thioredoxin (trxA) gene was required for ArsC-mediated arsenate resistance in E. coli. This suggests that reduction of arsenate by At. ferrooxidans has a similar reaction mechanism as that by Gram-positive ArsC proteins. While arsH was expressed in an E. coli-derived in vitro transcription-translation system, the presence of this gene was not required for, nor enhanced, arsenite or arsenate resistance in E. coli. We predict that the function provided by this gene is not required in E. coli. While the putative ArsR from At. ferrooxidans does contain a potential DNA-binding helix-turn-helix (HTH) domain, it does not contain the arsenite binding motif (ELCVCDL), required for response to the presence of inducer. Instead, the ArsR-like protein from At. ferrooxidans is related to a group of unstudied ArsR-like proteins that have been associated with other ars-like genes identified during genome sequencing projects. Using arsB-lacZ, arsC-lacZ, and arsR-lacZ fusions, it has been shown that this atypical ArsR protein from At. ferrooxidans did repress expression from the arsBH and arsRC promoters and that this repression was relieved by the presence of either arsenite or arsenate. Deletion of 19 amino acids from the C-terminus of the ArsR protein did not affect regulation, while deletion of a further 28 amino acids inactivated ArsR. Northern blot hybridization confirmed that expression of the arsRC and arsBH transcripts is increased in the presence of either arsenite or arsenate. This study is the first to show that the ars genes from the acidophilic biorning bacterium At. ferrooxidans are able to be studied in the neutrophilic bacterium, E. coli. We have also shown that the atypical ArsR found in this ars operon is able to regulate expression of these genes in response to arsenic, despite not containing the arsenite binding domain, suggesting that this protein senses arsenic by a different mechanism to that used by the ArsR family members already studied. / AFRIKAANSE OPSOMMING: Acidothiobacillus ferrooxidans, 'n asidofiliese, chemolitotrofiese bakterium, is een van 'n konsortium bakterieë betrokke by biologiese ontgunnig ("biomining") asook by die herwinning van goud uit arsenopiriet erts. Die gene wat aan At. ferrooxidans weerstandbiedendheid teen arseen verleen, is gekloneer. Die DNA-volgorde van hierdie gene is bepaal en daar is bewys dat die gene op die chromosoom geleë is. Homoloë van die arsB (membraan geleë pomp wat arseniet uitpomp), arsC (arsenaat reduktase) en die arsH (funksie onbekend) gene is in bekende arseenweerstanbiedheidsoperons (arsoperons) geïdentifiseer. Verder is daar 'n vierde geen geïdentifiseer wat lae homologie met die ArsR-familie van reguleerders toon. At. ferrooxidans se ars gene is op 'n ongewone manier gerangskik met twee van die gene, arsRC en arsBH wat lil teenoorgestelde rigtings getranskribeer word. Hierdie rangskikking van gene IS waargeneem in al vier die At. ferrooxidans rasse wat getoets is. Al die At. ferrooxidans ars gene is in Escherichia coli uitgedruk. Die arsB en arsC gene het aan 'n E. coli ars mutant (AW311 0) weerstandbiedendheid teen aseniet, antimoniet en arseen verleen. Analiese van die afgeleide aminosuurvolgorde van die ars proteïene het getoon dat die At. ferrooxidans ArsB naby verwant aan die ArsB-proteïene van ander Gram negatiewe bakterieë is. In teenstelling hiermee, is gevind dat die ArsC-proteïene nader verwant aan die ArsC-proteïene van Gram positiewe bakterieë is. Daar is ook gevind dat 'n funksionele tioredoksien (trxA) geen vir ArsC-bemiddelde arsenaat weerstandbiedendheid in E.coli benodig word. Dit dui daarop dat die meganisme van arsenaatreduksie deur At. ferrooxidans soortgelyk is aan die ArsC-proteïen-meganisme van Gram positiewe bakteriee. In vitro studies met behulp van 'n E. coli gebaseerde transkripsie-translasie sisteem het getoon dat arsH nie nodig is vir arsenaat of aseniet weerstanbiedendheid in sensitiewe E.coli rasse nie en ook nie help om weerstand in hierdie rasse te verhoog nie. Daarom kan daar aangeneem word dat die funskie van die arsH geen nie deur E. coli benodig word nie. Die vermeende ArsR van At. ferrooxidans bevat 'n potensiële DNA-binding heliks-draaiheliks motief, maar nie die arsiniet binding motief (ELCVCDL) wat nodig is vir reaksie in die teenwoordigheid van 'n induseerder nie. Die ArsA-proteïen van At. ferrooxidans is soortgelyk aan 'n groep ArsA-proteïene wat tydens genoom DNA- volgordebepalingsprojekte geïdentifiseer is. Hierdie groep gene is egter nog nie verder bestudeer nie. Deur gebruik te maak van 'n stel fusie gene, arsB-IacZ, arsC-IacZ en arsRlacZ kon daar bewys word dat die ongewone ArsH-proteïen van At. ferrooxidans uitdrukking van arsBH en arsRC onderdruk en dat die onderdrukking deur arseniet of arsenaat opgehef kan word. Delesie van die eerste 19 aminosure vanaf die C-terminus van die ArsA-proteïen het geen uitwerking op die regulering van die proteïen nie, maar delesie van 'n vedere 28 aminosure het ArsR geïnaktiveer. Verhoogde vlakke van transkripsie van arsRC en arsBH in die teenwoordigheid van arseniet en arsenaat is met behulp van Noordelike kladanalise bewys. Hierdie is die eerste studie waarin daar bewys word dat die ars gene van die asidofiliese bakterium Atferrooxidans in die neutrofiliese bacterium E. coli bestudeer kan word. Daar is ook bewys dat ten spyte daarvan dat die ArsR in die ars operon nie 'n arseniet bindingsdomein het nie, dit die uitdrukking van die gene in hierdie operon reguleer in reaksie op arseen. Dit dui dus daarop dat hierdie proteïen op arseen in die omgewing reageer met behulp van 'n meganisme wat verskil van die ArsR-proteïene wat tot dusver bestudeer is.

Thesis (PhD)--Stellenbosch University, 2012. / Please refer to full text for abstract.

Thesis (PhD)--University of Stellenbosch, 1999. / ENGLISH ABSTRACT: Hemicellulose, consisting mainly of xylan, ranks after cellulose, as the most abundant group of renewable polysaccharides in agricultural biomass. Xylan is a complex polymer consisting of a β D 1,4 linked xylopyranoside backbone, which may contain substituents. Enzymatic hydrolysis of xylan involves the action of a number of different hydrolytic enzymes. The yeast Saccharomyces cerevisiae has been used extensively in traditional food and beverage processes (baking, brewing and winemaking), as well as for the production of ethanol (potable alcohol and fuel extenders) and single-cell protein (protein supplements in food and animal feed). S. cerevisiae therefore has complete GRAS (Generally Regarded as Safe) status. However, the yeast S. cerevisiae can neither degrade nor utilize complex polysaccharides, including xylan. Through recombinant DNA technology, S. cerevisiae can be complemented by heterologous polysaccharase-encoding genes, thereby broadening its substrate range and facilitating a direct bioconversion of polysaccharides to valuable commodities, such as potable ethanol, protein supplements and industrial enzymes. In this study, the successful expression and co-expression of a β xylanase gene (Trichoderma reesei xyn2) and two β xylosidase genes (Bacillus pumilus xynB and A. niger xlnD) in S. cerevisiae, is described. Expression of these genes was obtained with the aid of multi-copy episomal yeast plasmids pJC1, pDLG1, pDLG4 and pRLR1. These plasmids contain either the derepressible alcohol dehydrogenase 2 (ADH2) or the constitutive phosphoglycerate kinase 1 (PGK1) promoter and terminator sequences. The enhanced production of recombinant enzymes by S. cerevisiae in a rich medium, without the risk of losing the episomal vector, was obtained by disrupting the uracil phosphoribosyltransferase (FUR1) gene in the plasmid-containing S. cerevisiae strains. This step ensured auto-selection of the URA3-bearing expression plasmids in rich growth medium. High level expression of the T. reesei β xylanase gene in S. cerevisiae enabled the yeast to degrade xylan to short xylo-oligosaccharides, but very little monomeric D xylose was formed. Both β xylosidase genes enabled S. cerevisiae to degrade short xylo-oligosaccharides like xylobiose and xylotriose. Co-expression of the β xylanase and the B. pumilus β xylosidase led to a small increase in the β xylanase activity, but a substantial decrease in the amount of β xylosidase activity. This recombinant yeast strain was unable to degrade xylan to D xylose. Expression of the T. reesei β xylanase with the A. niger β xylosidase gene enabled this strain to completely degrade xylan to its monomeric constituents, D xylose. / AFRIKAANSE OPSOMMING: Hemisellulose, wat hoofsaaklik uit xilaan bestaan, is ná sellulose, die volopste hernubare polisakkaried in landbouafval. Xilaan is 'n komplekse polimeer wat bestaan uit 'n β-D-1,4-gekoppelde xilopiranoseruggraat wat in sommige gevalle ook sykettings bevat. Ensimatiese afbraak van xilaan benodig die werking van hele aantal hidrolitiese ensieme. Die gis Saccharomyces cerevisiae word al vir baie jare in die voedsel- en drankbedryf (bak van brood en die maak van bier en wyn), asook vir die produksie van etanol (vir menslik gebruik en as brandstof aanvuller) en enkelselproteïene (proteïenaanvulling vir mens en dier) gebruik en het daarom volledige GRAS (Generally Regarded As Safe) status. Ongelukkig kan S. cerevisiae nie komplekse polisakkariede, xilaan ingesluit, afbreek of as koolstofbron benut nie. Met behulp van rekombinante-DNA-tegnologie kan S. cerevisiae gekomplementeer word met die nodige gene wat kodeer vir polisakkariedafbrekende ensieme om sodoende die gis in staat te stel om 'n wyer verskeidenheid van substrate af te breek en te benut. Dit sal lei tot die direkte bio-omskakeling van polisakkariede na bruikbare produkte soos etanol, proteïenaanvullers en ensieme vir industriële gebruik. In hierdie proefskrif word die suksesvolle uitdrukking asook die gesamentlike uitdrukking van 'n xilanasegeen (Trichoderma reesei xyn2) en twee β-xilosidasegene (Bacillus pumilus xynB en A. niger xlnD) in S. cerevisiae beskryf. Multikopie episomale plasmiede pJC1, pDLG1, pDLG4 en pRLR1 met die glukose onderdrukbare alkoholdehidrogenase 2 (ADH2) of die konstitutiewe fosfogliseraatkinase 1 (PGK1)- promoter en -termineerder is vir hierdie doel gebruik. Verhoogde produksie van die rekombinante ensieme deur S. cerevisiae in 'n ryk medium, sonder dat die gis die episomale plasmiedvektore verloor is moontlik gemaak deur die urasielfosforibosieltransferasegeen (FUR1) van hierdie giste te onderbreek met behulp van die LEU2-geen. Op hierdie manier word daar outomaties vir giste wat die URA3-uitdrukkingsplasmiede bevat geselekteer, selfs in ryk medium. Hoë vlak uitdrukking van T. reesei se xilanasegeen het S. cerevisiae in staat gestel om xilaan tot kort xilo-oligosakkariede af te breek, maar byna geen monomeriese D-xilose is gevorm nie. Albei die β-xilosidasegene het die gis in staat gestel om kort xilo-oligosakkariede soos xilobiose en xilotriose na D-xilose af te breek. Die gesamentlike uitdrukking van die xilanasegeen en B. pumilus se β-xilosidase geen het 'n klein toename in die xilanase-aktiwiteit tot gevolg gehad, maar 'n drastiese afname in die β-xilosidase-aktiwiteit. Hierdie rekombinante ras kon dus nie xilaan tot xilose afbreek nie. Uitdrukking van T. reesei se β-xilanasegeen saam met die β-xilosidasegeen van A. niger, het S. cerevisiae in staat gestel om xilaan tot sy monomeriese boustene, D-xilose, af te breek.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The need to mitigate the twin crises of peak oil and climate change has driven a headlong rush to biofuels. This study was aimed at the development of a process to efficiently convert steam explosion pretreated (STEX) sugarcane bagasse into ethanol by using combinations of commercial enzyme cocktails and recombinant Saccharomyces cerevisiae strains. Though enzymatic saccharification is promising in obtaining sugars from lignocellulosics, the low enzymatic accessibility of the cellulose and hemicellulose is a key impediment thus necessitating development of an effective pretreatment scheme and optimized enzyme mixtures with essential accessory activities. In this context, the effect of uncatalysed and SO2 catalysed STEX pretreatment of sugarcane bagasse on the composition of pretreated material, digestibility of the water insoluble solids (WIS) fraction and overall sugar recovery was investigated. STEX pretreatment with water impregnation was found to result in a higher glucose recovery (28.1 g/ 100 bagasse) and produced WIS with a higher enzymatic digestibility, thus was used in the optimization of saccharification and fermentation. Response surface methodology (RSM) based on the 33 factorial design was used to optimize the composition of the saccharolytic enzyme mixture so as to maximize glucose and xylose production from steam exploded bagasse. It was established that a combination of 20 FPU cellulase/ g WIS and 30 IU -glucosidases/ g WIS produced the highest desirability for glucose yield. Subsequently the optimal enzyme mixture was used to supplement enzyme activities of recombinant yeast strains co-expressing several cellulases and xylanases in simultaneous saccharification and fermentations SSFs. In the SSFs, ethanol yield was found to be inversely proportional to substrate concentration with the lowest ethanol yield of 70% being achieved in the SSF at a WIS concentration of 10% (w/v). The ultimate process would however be a one-step “consolidated” bio-processing (CBP) of lignocellulose to ethanol, where hydrolysis and fermentation of polysaccharides would be mediated by a single microorganism or microbial consortium without added saccharolytic enzymes. The cellulolytic yeast strains were able to autonomously multiply on sugarcane bagasse and concomitantly produce ethanol, though at very low titres (0.4 g/L). This study therefore confirms that saccharolytic enzymes exhibit synergism and that bagasse is a potential substrate for bioethanol production. Furthermore the concept of CBP was proven to be feasible. / AFRIKAANSE OPSOMMING: Die behoefte om die twee krisisse van piek-olie en klimaatsverandering te versag, het veroorsaak dat mense na biobrandstof as alternatiewe energiebron begin kyk het. Hierdie studie is gemik op die ontwikkeling van 'n proses om stoomontplofde voorafbehandelde (STEX) suikerriet bagasse doeltreffend te omskep in etanol deur die gebruik van kombinasies van kommersiële ensiem mengsels en rekombinante Saccharomyces cerevisiae stamme. Alhoewel ensiematiese versuikering belowend is vir die verkryging van suikers vanaf lignosellulose, skep die lae ensiematiese toeganklikheid van die sellulose en hemisellulose 'n hindernis en dus is die ontwikkeling van' n effektiewe behandelingskema en optimiseerde ensiemmengsels met essensiële bykomstige aktiwiteite noodsaaklik. In hierdie konteks, was die effek van ongekataliseerde en SO2 gekataliseerde stoomontploffing voorafbehandeling van suikerriet bagasse op die samestelling van voorafbehandelde materiaal, die verteerbaarheid van die (WIS) breuk van onoplosbare vastestowwe in water (WIS), en die algehele suikerherstel ondersoek. Daar was bevind dat stoomontploffing behandeling (STEX) met water versadiging lei tot 'n hoër suikerherstel (21.8 g/ 100g bagasse) en dit het WIS met ‘n hoër ensimatiese verteerbaarheid vervaardig en was dus gebruik in die optimalisering van versuikering en fermentasie. Reaksie oppervlak metodologie (RSM), gebasseer op die 33 faktoriële ontwerp, was gebruik om die samestelling van die ‘saccharolytic’ ensiemmengsel te optimaliseer om sodoende die maksimering van glukose en ‘xylose’ produksie van stoomontplofde bagasse te optimaliseer. Daar was bevestig dat ‘n kombinasie van 20 FPU sellulase/ g WIS en 30 IU ‘ -glucosidases/ g’ WIS die hoogste wenslikheid vir glukose-opbrengs produseer het. Daarna was die optimale ensiemmengsel gebruik om ensiemaktiwiteit van rekombinante gisstamme aan te vul, wat gelei het tot die medeuitdrukking van verskillende ‘cellulases’ en ‘xylanases’ in gelyktydige versuikering en fermentasie SSFs. In die SSFs was daar bevind dat die etanol-produksie omgekeerd proporsioneel is tot substraat konsentrasie, met die laagste etanolopbrengs van 70% wat bereik was in die SSF by ‘n WIS konsentrasie van 10% (w/v). Die uiteindelike proses sal egter 'n eenmalige "gekonsolideerde" bioprosessering (CBP) van lignosellulose na etanol behels, waar die hidrolise en fermentasie van polisakkariede deur' n enkele mikroorganisme of mikrobiese konsortium sonder bygevoegde ‘saccharolytic’ ensieme bemiddel sal word. Die ‘cellulolytic’ gisstamme was in staat om vanself te vermeerder op suikerriet bagasse en gelyktydig alkohol te produseer, al was dit by baie lae titres (0.4 g/L). Hierdie studie bevestig dus dat ‘saccharolytic’ ensieme sinergisme vertoon en dat bagasse 'n potensiële substraat is vir bio-etanol produksie. Daar was ook onder meer bewys dat die konsep van CBP uitvoerbaar is. / The National Research Foundation (NRF) for financial support

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Complete degradation of cellulose produces mainly glucose, which can be fermented to ethanol. Therefore cellulose presents an abundant renewable energy resource for the production of an alternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanases and -glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol. Therefore, a need has arisen to develop a recombinant microorganism with the ability to produce cellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single process step, referred to as “Consolidated Bioprocessing” (CBP). This would provide a cost-effective, economically feasible strategy for the production of bioethanol. The naturally fermentative yeast, Saccharomyces cerevisiae, is often used as host for the expression of recombinant proteins due to several characteristics, including its robustness in industrial processes, the well developed genetic tools available for manipulation and its proven safety status. A number of cellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. In this study, all three components of the cellulase system were co-expressed in S. cerevisiae to test the ability of the yeast to effectively produce the heterologous proteins, and consequently produce enough glucose for growth on an amorphous cellulosic substrate. The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by a S. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1 towards CMC and barley -glucan, respectively. The major endoglucanase gene, egI (Cel7B) from T. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. coli mismatch repair deficient strain. Screening of S. cerevisiae transformants revealed a strain, S. cerevisiae Y294[pLEM1], with improved levels of endoglucanase activity (21.8 nkat.ml-1), compared to S. cerevisiae Y294[pAZ40], expressing the wild type gene (10.3 nkat.ml-1). Through subcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it was established that the mutations located in both the promoter- and gene sequences were responsible for the improved levels of activity displayed by S. cerevisiae Y294[pLEM1]. The egII gene and the altered egI gene were co-expressed with a codon optimised T. reesei cellobiohydrolase (sCBHI) and a -glucosidase from Saccharomycopsis fibuligera. This resulted in a reduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast by co-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressing strains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enable growth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likely that a bottleneck exists at its conversion to glucose, suggesting inadequate -glucosidase activity. This study has provided insight into co-expression of cellulase enzymes by the yeast S. cerevisiae. The knowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulose degradation and eventual production of ethanol by recombinant yeast. It has also demonstrated the applicability of random mutagenesis for improving the activity of cellulases.