Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry. / National Research Foundation

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry. / National Research Foundation

Thesis submitted in compliance with the requirements for the Master's Degree in Technology: Industrial Engineering, Department of Industrial Engineering, Durban University of Technology, 2009. / This research focuses on the design and implementation of an intelligent machine vision and sorting system that can be used to sort objects in an industrial environment. Machine vision systems used for sorting are either geometry driven or are based on the textural components of an object’s image. The vision system proposed in this research is based on the textural analysis of pixel content and uses an artificial neural network to perform the recognition task. The neural network has been chosen over other methods such as fuzzy logic and support vector machines because of its relative simplicity. A Bluetooth communication link facilitates the communication between the main computer housing the intelligent recognition system and the remote robot control computer located in a plant environment. Digital images of the workpiece are first compressed before the feature vectors are extracted using principal component analysis. The compressed data containing the feature vectors is transmitted via the Bluetooth channel to the remote control computer for recognition by the neural network. The network performs the recognition function and transmits a control signal to the robot control computer which guides the robot arm to place the object in an allocated position. The performance of the proposed intelligent vision and sorting system is tested under different conditions and the most attractive aspect of the design is its simplicity. The ability of the system to remain relatively immune to noise, its capacity to generalize and its fault tolerance when faced with missing data made the neural network an attractive option over fuzzy logic and support vector machines.

Thesis (MSc) -- University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Rooibos tea plant (Aspalathus linearis) is indigenous to South Africa and occurs only in the Western Cape's Cedarberg region. Rooibos tea is produced from the leaves and fine stems of the plant. The tea is normally prepared by brewing the leaves and consuming the liquor. However, the Rooibos plant is not only used to prepare tea; the plant extracts are also used in various neutraceutical and pharmaceutical products, including health drinks, iced tea, soaps and moisturising creams. Although the tea plant contains native enzymes responsible for the colour and aroma development of Rooibos tea, the disruption and maceration of the plant material during processing is insufficient to allow these enzymes proper access to the substrates responsible for Rooibos tea's characteristics. The current processing of Rooibos tea is also time consuming and is done under uncontrolled conditions, leading to unnecessary loss in aroma and antioxidant content. The addition of enzymes could improve the maceration of the plant material, shorten the processing time and improve the extraction of aroma, colour and antioxidant components. During this study, 16 commercially available microbial enzymes were evaluated on three different Rooibos substrates for the improvement of aroma and colour development, as well as the extraction of soluble solids (SS) and total polyphenols (TP). Thirteen enzymes were evaluated on spent tea for the enhanced extraction of soluble solids and to determine the best candidates for further evaluation on fermented and green Rooibos tea. Seven of the enzymes improved the yield in SS from spent tea. Up to 232% improvement was obtained, depending on the type of enzyme and dosage applied. The best six enzyme preparations were further evaluated on fermented Rooibos tea. For Depol™ 670L at 20 ul/g tea, the laboratory treatment increased the yield in SS by 44%, while small-scale industrial simulations increased the SS by 26%. However, an increase in the yield in SS was usually accompanied by a decrease in the %TP/SS ratio, indicating that mainly inactive compounds were extracted. Based on the results with the commercial enzymes, twelve "synthetic" enzyme cocktails, consisting of different combinations of commercial enzymes were designed, of which three cocktails released increased amounts of SS without decreasing the %TP/SS ratio significantly. Thirteen enzymes were evaluated on dried and freshly cut green Rooibos tea, with three enzymes (Depol™ 670L, Pectinex Ultra SP-L and Depol™ 692L) increasing the yield in SS between 21% and 66%, and the TP content between 11% and 47%. Laccase was the best candidate in improving colour development from green tea, with the improvement being slightly better at 50°C than at 40°C. All the "synthetic" cocktails containing laccase improved the colour extract of all three substrates evaluated, but also significantly decreased the TP and antioxidant content. However, lower dosages of laccase resulted in colour development with little loss in the antioxidant content. Due to the promising results obtained with the treatments of Rooibos tea with laccases, it was decided to clone and express the laccase gene (lacA) of Pleurotus ostreatus into Aspergillus niger. The gene was successfully transformed into A. niger, but the expression of the recombinant gene was not effective. / AFRIKAANSE OPSOMMING: Die Rooibostee plant (Aspalathus linearisi is inheems tot Suid-Afrika en kom slegs in die Sederberg-omgewing in die Wes-Kaap voor. Rooibostee word van die blare en fyn stingels van die plant geproduseer. Die tee word normaalweg voorberei deur die blare in kookwater te laat trek en dan die aftreksel te drink. Die Rooibos plant word nie net gebruik om tee te maak nie; die tee ekstrak word ook gebruik vir verskeie neutraseutiese en farmaseutiese produkte, insluitende gesondheidsdrankies, ystee, seep en bevogtigingsrome. Ten spyte daarvan dat die teeplant sy eie ensieme vir die kleur en aroma ontwikkeling van Rooibostee bevat, is die verbreking en maserasie van die plantmateriaal tydens prosessering onvoldoende om die ensieme genoeg toe gang tot die substrate verantwoordelik vir die kenmerkende eienskappe van Rooibostee te gee. Die huidige prosessering van Rooibostee is ook tydrowend en geskied onder onbeheerde toestande, wat tot 'n onnodige verlies in aroma en antioksidante lei. Die toevoeging van ensieme kan die afbraak van die plantmateriaal verbeter, die behandelingsproses verkort en die aroma, kleur en antioksidant inhoud van ekstrakte verbeter. Tydens hierdie studie is 16 kommersieel-beskikbare mikrobiese ensieme op drie verskillende Rooibos substrate vir die verbetering van aroma, kleur en ekstraksie van oplosbare vastestowwe (SS) en totale polifenole (TP) getoets. Dertien ensieme is op oorskot tee vir die verbeterde ekstraksie van oplosbare vastestowwe geevalueer, waama die beste kandidate vir evaluering op gefermenteerde en ongefermenteeede Rooibostee gekies is. Sewe ensieme het die SS vanaf oorskot tee verhoog. Tot 232% verhoging is waargeneem, afhangende van die tipe ensiem en die dosis wat gebruik is. Die beste ensiern preparate IS verder op gefermenteerde Rooibostee geevalueer, Labarotoriurn behandelings met Depol™ 670L teen 20 ul/g tee het die SS inhoud met 44% verhoog, terwyl die kleinskaalse industriele simulasie die SS inhoud met 26% verhoog het. 'n Verhoging in SS het egter gewoonlik met 'n afname in die %TP/SS verhouding gepaard gegaan, wat aandui dat hoofsaaklik onaktiewe stowwe vrygestel IS. Na aanleiding van die resultate met die kommersiele ensieme, is twaalf "sintetiese" ensiemmengsels met verskillende ensiemkombinasies getoets, waarvan drie mengsels ook meer SS vrygestel het met byna geen verlaging in die %TP/SS verhouding nie. Dertien ensieme was op gedroogde en vars gekerfde groen Rooibostee getoets met drie ensieme (Depol™ 670L, Pectinex Ultra SP-L en Depol™ 692L) wat die SS met tussen 21% en 66%, en die TP inhoud met tussen 11% en 47% verhoog het. Lakkase was die beste kandidaat vir die verbetering van kleur ontwikkeling by groen Rooibostee met die verbetering effens beter by 50°C as by 40°C. Al die "sintetiese" ensiem mengsels wat lakkase bevat het, het die kleur by al die verskillende substrate verbeter, maar het ook die TP en antioksidant inhoud aansienlik verlaag. Laer lakkase dosisse het goeie kleurontwikkeling tot gevolg gehad met minimale verlies in die antioksidant inhoud. Vanwee die goeie resultate wat met die lakkase behandelings verkry is, is daar besluit om die lakkase geen (lacA) van Pleurotus ostreatus te kloneer en in Aspergillus niger uit te druk. Die geen is suksesvol in A. niger getransformeer, maar die uitdrukking daarvan was nie effektief nie.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: A severely polluted industrial effluent is generated by the local gelatinmanufacturing industry. Due to increasingly stringent restrictions on discharge qualities enforced by the National Water Act of 1998 and National Environmental Management Act of 1998, as well as increasing trade-effluent charges implemented via the Local Municipal Bylaws, the industry is compelled to consider a system to pre-treat the polluted effluent. A study was undertaken to examine the viability of anaerobic treatment of the gelatin-manufacturing effluent, since the anaerobic digestion technology is well recognised for the high success rate in the treatment of high-strength, complex wastewaters. Various laboratory and pilot-scale studies were done, using different hybrid Upflow Anaerobic Sludge Blanket (UASB) and contact designs. Two mesophilic laboratory-scale hybrid UASB digester designs, fitted with polyethylene (AD-1) and polyurethane (AD-2), performed well at a hydraulic retention time (HRT) of 1.0 d. Chemical oxygen demand (COD) removal efficiencies of up to 90% (avg. 53%) for AD-1 and 83% (avg. 60%) for AD-2 at organic loading rates (OLR) of 9.56 and 4.62 kg COD.m-3.d-1, respectively, were obtained. High sulphate (S04) removal efficiencies of up to 96% (avg. 86%) for AD-1 and 98% (avg. 82%) for AD-2 were also achieved, respectively. A maximum total solid (TS) removal of 65% (avg. 25%) for AD-1 and 62% (avg. 28%) for AD-2 was reported. An average methane content of 80% (AD-1) and 79% (AD-2) with average methane yields per COD removed of 2.19 and 1.86 m3. kg CODremoved.df-o1r AD-1 and AD-2 were found, respectively. When the same digesters (AD-1 and AD-2) were combined in a muItiphase series configuration, a total COD removal efficiency of up to 97% (avg. 80%) at an OLR of 8.32 kg COD.m-3.d-1,was achieved. Excellent total S04 removals of 96% (avg. 69%) were accomplished. Up to 82% TS (avg. 29%) was also removed during this study and the biogas consisted of 89% methane (avg. 79%). For this multi-phase combination up to 92% volatile fatty acids (VFA) (avg. 48%) were removed, indicating possible selective phase separation of the respective fatty acid producing/utilising bacterial populations. The use of a laboratory-scale UASB bioreactor with recirculation, resulted in COD removal efficiencies of up to 96% (avg. 51%) at an HRT of 3.0 d, and 95% (avg. 54%) at a HRT of 1.0 d. Low performances were generally found, with average S04 and TS removals of 59% (max. 97%) and 26% (max. 67%), respectively at an HRT of 1.0 d. The biogas production was very low throughout the study (0.05 - 0.63 I,d-1 ). A pilot-scale UASB reactor (300 I) was constructed and performed satisfactory with a 58% average COD removal and maximum of 96%. S04 and TS removals up to 96% (avg. 44%) and 93% (avg. 63%), respectively, were obtained. The methane content of the biogas was 85%. The pilot-scale studies were conducted under actual field conditions, where various shock and organic loads had to be absorbed by the system. The pilot-scale contact configuration (300 I) did not perform satisfactory as a result of continuous blockages experienced in the feed and recirculation lines. Maximum COD, S04, VFA and TS removal efficiencies of 41% (avg. 27%), 62% (avg. 41%), 64% (avg. 27%) and 39% (avg. 21%), respectively, were obtained. The results of all the studies indicated acceptable COD removals with increasing OLR's. Indications of the presence of active methanogenic and sulphate-reducing bacterial populations were apparent throughout the studies. One possibility for the successful start-up and commissioning of the anaerobic reactors was the use of a well-adjusted biomass, which consisted of highly selected and adapted microbial consortium for the specific gelatinmanufacturing effluent. It was clear from this study that gelatin-manufacturing effluent can be treated successfully, especially with the use of the UASB design. A welldefined data base was constructed which could be of great value for further upscaling to a full-scale digester. / AFRIKAANSE OPSOMMING: 'n Hoogs besoedelde industriele uitvloeisel word gegenereer deur die plaaslike gelatien-vervaardigings industrie. As gevolg van toenemende streng beperkings op die kwaliteit van uitvloeisels wat bepaal word deur die Nasionale Water Wet van 1998 en Nasionale Omgewings Bestuurs Wet van 1998, asook toenemende munisipale heffings wat geimplementeer word via Plaaslike Munisipale Wette, word die industrie verplig om die uitvloeisel vooraf te behandel. 'n Studie is onderneem om die lewensvatbaarheid van anaërobe behandeling van gelatien-vervaardigings uitvloeisel te ondersoek, aangesien anaërobe verterings tegnologie alombekend is vir die goeie sukses behaal in die behandeling van hoë-sterkte, komplekse uitvloeisels. Verskeie laboratorium- en loods-skaal studies is gedoen, met verskillende hibried Opvloei Anaërobe Slykkombers (OAS) en kontak ontwerpe. Goeie werksverrigting was verkry by 'n hidroliese retensie tyd (HRT) van 1.0 d met twee mesofiliese laboratorium-skaal hibried OAS verteerder ontwerpe wat uitgevoer was met poli-etileen (AD-1) en poli-uretaan (AD-2) materiaal. Chemiese suurstof behoefte (CSB) verwyderings van so hoog as 90% (gem. 53%) vir AD-1 en 83% (gem. 60%) vir AD-2 by organiese ladingstempo's (OLT) van 9.56 en 4.62 kg CSB.m-3.d-1,was onderskeidelik verkry. Hoë sulfaat (S04) verwyderings van tot 96% (gem. 86%) vir AD-1 en 98% (gem. 82%) vir AD-2 was ook onderskeidelik verkry. 'n Maksimum totale vaste stof (TVS) verwydering van 65% (gem. 25%) vir AD-1 en 62% (gem. 28%) vir AD-2 is gerapporteer. 'n Gemiddelde metaan inhoud van 80% (AD-1) en 79% (AD-2) met 'n gemiddelde metaan opbrengs per CSB verwyder van 2.19 en 1.86 m3.kg CSBverwyder.dv-i1r AD-1 en AD-2, was onderskeidelik gevind. Met die aanwending van dieselfde twee verteerders (AD-1 en AD-2) in 'n series gekoppelde multi-fase konfigurasie, is 'n totale CSB verwydering so hoog as 97% (gem. 80%) verkry by 'n OLT van 8.32 kg CSB.m-3.d-1. Uitstekende totale S04 verwydering van 96% (gem. 69%) is behaal. Tot 82% TVS (gem. 29%) was vewyder gedurende die studie en die biogas het uit 89% metaan (gem. 79%) bestaan. Vir die multi-fase kombinasie is 'n maksimum van 92% vlugtige vetsure (WS) (gem. 48%) verwyder, wat dui op die moontlike skeiding van selektiewe fases van die onderskeie vetsuur produserende/verbruiker bakteriële populasies. CSB verwydering van tot 96% (gem. 51%) by 'n HRT van 3.0 d en 95% (gem. 54%) met 'n HRT van 1.0 d was verkry, tydens die gebruik van In laboratorium-skaal OAS bioreaktor met hersirkulasie. Lae werksverrigting was oor die algemeen waargeneem, met gemiddelde S04 en TVS verwyderings van 59% (maks. 97%) en 26% (maks. 67%) by In HRT van 1.0 d. Die biogas produksie was baie laag gedurende die studie (0.05 - 0.63 I,d-\ In Loods-skaal OAS verteerder was opgerig en bevredigende resultate was verkry met In gemiddeld van 58% CSB verwydering en maksimum van 96%. S04 en TVS verwyderings so hoog as 96% (gem. 44%) en 93% (gem. 63%) is onderskeidelik verkry. Die metaan inhoud van die biogas was 85%. Die loods-skaal studie was uitgevoer gedurende ware veld kondisies, waartydens verskeie skok en organiese ladings deur die sisteem geabsorbeer is. Die loods-skaal kontak konfigurasie (300 I) het nie bevredigende resultate getoon nie, as gevolg van voortdurende blokkasies wat ondervind is in die toevoer en hersirkulasie pype. Maksimum CSB, S04, WS en TVS verwyderings van 41% (gem. 27%), 62% (gem. 41%), 64% (gem. 27%) en 39% (gem. 21%) was onderskeidelik verkry. Die resultate van al die studies het aanvaarbare CSB verwydering aangedui by toenemende OLT's. Indikasies van aktiewe metanogene en sulfaat-reduserende bakteriële populasies was ook teenwoordig gedurende die studies. Die suksesvolle aansit-prosedure en begin van die anaërobe verteerders kan toegeskryf word aan die gebruik van In goed aangepaste biomassa, wat uit hoogs selektiewe en aangepaste mikrobiese populasies vir die spesifieke uitvloeisel bestaan. Hierdie studie het getoon dat gelatien-vervaardigings uitvloeisel suksesvol met die OAS ontwerp behandel kan word. In Goed gedefinieerde data basis kan voorsien word, wat van groot waarde sal wees vir verdere opgradering na In volskaalse verteerder.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma of grapes and wine. This is especially applicable to wines of a muscat variety, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement other varietal flavours and aromas. These monoterpenols can be found in grapes and wine as free, volatile and odorous molecules, as well as in flavourless, nonvolatile glycosidic complexes. These complexes most often occur as 6-0-a-L-arabinofuranosyl-p-D-glucopyranosides (vicianosides), 6-0-P-D-xylopyranosyl- P-D-gluco-pyranosides (primverosides), 6-0-P-D-glucopyranosyl-p-D-glucopyranosides (gentio-biosides ), 6-0-a-L -rhamnopyra nosyl-p-D-g lucopyra nos ides (rutinos ides), or 6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol and hotrienol. These precursors are, however, hydrolyzed only to a limited extent by endogenous glycosidases during the fermentation process, as they exhibit very low activity in wine conditions. The monoterpenols can be released from their sugar moieties by one of two methods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanism is fully understood, and the process functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, an a-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomyces cerevisiae show very low activity towards these aromatic precursors during the handling of the juice and winemaking processes, the focus has increasingly fallen on introducing exogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases and a-L-arabinofuranosidases have been cloned from various organisms, including bacteria, fungi and yeasts. However, the activities and properties of these enzymes are not always suitable for exploitation under winemaking conditions, where a low pH, low temperatures, and high ethanol and glucose concentrations prevail. A genetically engineered wine yeast strain of S. cerevisiae that expresses glycosidases that are active in these conditions would be useful in improving the flavour and aroma of wines, thereby adding to the complexity and value of the wine. Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, were subcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable marker gene (SMR1) was also inserted onto these plasmids. These plasmids were designated pBGL 1 (containing the BGL 1 gene) and pBGL2 (containing the BGL2 gene) respectively. Introduction of the two plasmids into two strains of S. cerevisiae then followed. A laboratory strain, L1278, was transformed to confirm the effective secretion of the expressed protein. An industrial yeast strain, VIN13, was subsequently transformed by making use of the selectable marker (resistance against sulfometuron). Enzyme assays with the synthetic substrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine the activity of the j3-glucosidases over a period of days, as well as at certain temperatures and pH values. The stability of the enzymes was also investigated. These recombinant yeasts were able to degrade the pNPG efficiently. They showed promising results concerning pH optima, with a substantial amount of activity found at the pH levels as found in the wine environment. There was also a slight increase in specific activity at lower temperatures. The recombinant yeast strains were also tested in smallscale fermentations. Three wines were made, of which two were from white cultivars (Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained from micro-extraction from the finished wines showed that the terpenol content did increase, although this was not the only wine component influenced. Other flavour compounds also showed increases, especially the esters. This also played a role in the flavour increase in the wine. Future work would include optimizing the available results. This would entail the addition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome of the wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineering of a j3-glucosidase enzyme that is more active at low temperatures would also yield better results and release even more of the aroma of the wine. / AFRIKAANSE OPSOMMING: Monoterpeenalkohole (monoterpenole) speel 'n belangrike rol in die geur en aroma van druiwe en wyn. Dit is veral van toepassing op wyne van Muskaat-varieteite, maar hierdie geurkomponente is ook teenwoordig in ander nie-Muskaat druifsoorte, waar dit bydra tot die varieteitsqeur en aroma. Hierdie monoterpenole kom voor in druiwe as vry, vlugtige en aromatiese molekules, of as geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0-a-L-arabinofuranosiel-~-D-glukopiranosiede, 6- O-~-D-xilopiranosiel-~-D-glukopiranosiede (primverosiede), 6-0-~-D-glukopiranosiel-~-Dglukopiranosiede (gentiobiosiede), 6-0-a-L-ramno-pyranosiel-~-D-glukopiranosiede (rutinosiede), of 6-0-~-D-apiofuranosiel-~-D-glukopirano-siede van hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Hierdie geurvoorlopers word egter slegs tot In beperkte mate tydens die proses van fermentasie deur die endogene glikosidase ensieme gehidroliseer, aangesien hulle baie min aktiwiteit toon onder wynbereidingstoestande. Die monoterpenole kan op een van twee wyses van hul suikermolekules vrygestel word: 'n suurhidrolise, of ensiematiese hidrolise. Die ensiematiese hidroliseproses word baie goed begryp en behels twee opeenvolgende stappe: eerstens, afhangende van die aard van die voorloper, word die glikosidiese verbinding deur In a-L-arabinofuranosidase, In a-L-ramnosidase, In ~-D-xilosidase, of 'n ~-D-apiosidase gebreek. In die tweede stap word die monoterpeenalkohol deur In ~-glukosidase vrygestel. Hierdie ensiematiese afbraakproses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos wat met suurhidrolise die geval is nie. Omdat die endogene glikosidases van Vitis vinifera en die van die gis Saccharomyces cerevisiae baie lae aktiwiteit ten opsigte van die aromatiese voorlopers gedurende die hantering van die druiwesap en wynmaakprosesse toon, val die fokus al hoe meer op die inkorporering van eksogene ~-glukosidases in wyn en sappe. Gene wat vir ~- glukosidases en a-L-arabinofuranosidases kodeer, is al vanuit verskeie organismes gekloneer, insluitende bakteriee, fungi en giste. Die aktiwiteite en kenmerke van hierdie ensieme is egter nie altyd wenslik vir hul gebruik in wyn nie, aangesien dit In omgewing is met 'n lae pH, lae temperatuur, en hoe etanolvlakke en glukose-konsentrasies. In geneties veranderde wyngis van S. cerevisiae wat in staat is om glikosidases uit te druk wat onder hierdie kondisies aktief is, sal baie handig te pas kom in die verbetering van die geur en aroma van wyne, om daardeur die kompleksiteit en die waarde van die wyn te verhoog. Twee ~-glukosidasegene, BGL 1 en BGL2 vanaf die gis Saccharomycopsis fibuligera , is in twee afsonderlike Esccherichia coli-gis-pendelplasmiede gesubkloneer. In Dominante selekteerbare merkergeen (SMR1) is ook in hierdie plasmiede gekloneer. Hierdie plasmiede word onderskeidelik pBGL 1 (met die BGL 1-geen) en pBGL2 (bevattende die BGL2-geen) genoem. Hierdie twee plasmiede is hierna apart na twee rasse van S. cerevisiae getransformeer. Eerstens is 'n laboratoriumras, L1278, getransformeer om te bevestig dat effektiewe sekresie en uitdrukking van die proteTen plaasvind. Hierna is 'n industriele gisras, VIN13, getransformeer deur gebruik te maak van die selektiewe merker (bestandheid teen sulfometuron). Ensiem-bepalings met behulp van die sintetiese substraat p-nitrofeniel-p-O-glukopiranosied (pNPG) is gedoen om die aktiwiteit van die p-glukosidqses oor 'n aantal dae te bepaal, asook om die aktiwiteit by sekere temperature en pH-vlakke te meet. Die stabiliteit van die ensieme is ook bepaal. Hierdie rekombinante giste was in staat om pNPG effektief af te breek. Hulle het belowende resultate betreffende die pH-optima getoon, met 'n aansienlike hoeveelheid aktiwiteit by die pH-vlakke soos dit in die wynomgewing voorkom. Daar was ook 'n effense verhoging in die ensieme se aktiwiteite by laer temperature. Die rekombinante gisrasse is ook in kleinskaalse wynfermentasies gebruik. Drie verskillende wyne is gemaak, waarvan twee wit kultivars was (Chenin blanc en GewOrtztraminer) en een 'n rooi kultivar (Pinotage). Resultate wat deur die mikro-ekstraksie van die voltooide wyne verkry is, het getoon dat die terpenolinhoud wei verhoog het, alhoewel dit nie die enigste wynkomponente was wat beinvloed is nie. Ander geurkomponente het ook 'n verhoging in konsentrasie getoon, veral die esters. Hierdie verbindings het ook 'n rol in die verhoging van geur in die wyne gespeel. Toekomstige werk sal die beskikbare resultate verder optimaliseer. Dit sal insluit die byvoeging van nog 'n glikosidiese ensiem, soos a.-L-arabinofuranosidase, tot die genoom van die wyngis, om verdere afbraak van glikosidiese verbindings teweeg te bring. Die klonering of verandering van 'n p-glukosidase-ensiem met verhoogde aktiwiteit by laer temperature sal ook beter resultate toon en meer geur in die wyn kan vrystel.

Thesis (MEng) -- Stellenbosch University, 2014. / ENGLISH ABSTRACT: See PDF for abstract. / AFRIKKANSE OPSOMMING: Sien PDF vir die opsomming.

Thesis (PhD(Agric) (Viticulture and Oenology))--University of Stellenbosch, 2006. / The winemaking process involves different complex chemical and biochemical reactions, which include those of oxygen (O2). Oxygen can come into contact with the wine through various winemaking procedures and can be used by the winemaker to enhance the quality of red wine. In wine, the main substrates for oxidation are phenolic molecules, which form quinones. These can influence the sensory characteristics of the wine. O2 can be used in fresh must to remove oxidisable phenolic molecules through a process called hyper-oxidation and can also be added to fermenting must to enhance the fermentation performance of yeast. Controlled O2 additions during ageing can lead to the wine’s colour being increased and the astringency of the wine decreased. This is due to the formation of acetaldehyde from the oxidation of ethanol, which induces the polymerisation of tannin and anthocyanin molecules. The addition of too much O2 to wine can, however, lead to unwanted over-oxidation, with certain off-odours being formed. It can also enhance the growth of unwanted spoilage microorganisms, such as Brettanomyces and acetic acid bacteria. Although research on O2 in wine was started many years ago, many questions still remain. These include the general effect of O2 on the sensory and phenolic profile of red wine especially and the microbiology of wine during ageing. An effective way of measuring oxidation, especially in red wine must also be developed. In the first part of this study, the effects of O2 and sulfur dioxide (SO2) additions on a strain of Brettanomyces bruxellensis (also known as Dekkera bruxellensis) and Acetobacter pasteurianus were investigated. Epifluorescence microscopy and plating revealed that the A. pasteurianus strain went into a viable but non-culturable state in the wine after prolonged storage under relative anaerobic conditions. This state, however, could be negated with successive increases in culturability by the addition of O2, as would happen during the transfer of wine when air is introduced. The A. pasteurianus strain was also relatively resistant to SO2, but the B. bruxellensis strain was more sensitive to SO2. A short exposure time to molecular SO2 drastically decreased the culturability of the B. bruxellensis strain, but bound SO2 had no effect on the culturability or viability of either of the two types of microorganisms. Oxygen addition to the B. bruxellensis strain also led to a drastic increase in viability and culturability. It is thus clear that SO2 and O2 management in the cellar is of critical importance for the winemaker to produce wines that have not been spoiled by Brettanomyces or acetic acid bacteria. This study should contribute to the understanding of the factors responsible for the growth and survival of Brettanomyces and acetic acid bacteria in wine, but it should be kept in mind that only one strain of each microorganism was used. This should be expanded in future to include more strains that occur in wine. The second part of this study investigated the effect of micro-oxygenation on four different South African red wines. It was found that the micro-oxygenation led to an increase in the colour density and SO2 resistant pigments of the two wines in which micro-oxygenation was started just after the completion of malolactic fermentation. In one of these wines, a tasting panel preferred the micro-oxygenation treated wines to the control. In the other two red wines, in which the micro-oxygenation was started seven months after the completion of malolactic fermentation, very little colour increase was observed. One of these two wines was also matured in an oak barrel, where the change in phenolic composition was on par with the treated wines. A prolonged period of micro-oxygenation, however, led to this wine obtaining an oxidised, over-aged character. Micro-oxygenation and maturation in an oak barrel also enhanced the survival of acetic acid bacteria and Brettanomyces in this wine. Micro-oxygenation can hence be used by the wine producer on young red wines to enhance the quality of the wine, but should be applied with care in older red wines. Future research into micro-oxygenation should focus on whether it can simulate an oak barrel. More research into the effect of micro-oxygenation on the sensory profile of the wine is needed. As mentioned, the addition of O2 can lead to oxidative degradation of wine. The brown colour in wine is often used as an indication of oxidation, but oxidative aromas can be perceived before a drastic increase in the brown colour has been observed in red wine. The third part of this study was to assess the possible use of Fourier Transform Infrared Spectroscopy (FTIR) to measure the progression of oxidation in Pinotage red wines. Three wines were used in this study and clear separation between the control and aerated wines was observed by using Principle Component Analysis (PCA). Sensory analysis of these wines confirmed this observation, with a reduction especially in berry fruit and coffee characters and an increase first in potato skin and then acetaldehyde aroma characters as the oxidation progressed. PCA analysis also revealed that in certain wines the visible spectrum of light did not indicate the progression of oxidation as sensitively as with the use of FTIR. This also correlated with the inability of the panel to observe a drastic colour change. FTIR should be further investigated as a possible means of monitoring oxidation in wine and this study should be expanded to wines made from other cultivars as well.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: This study strives to improve two current industrial processes by making them more cost effective through the use of hydrolytic enzymes or microbial systems. The first process targeted is the industrial conversion of starch to ethanol. In the second process, hydrolytic enzymes are applied to the manufacturing of instant coffee. The engineering of microbial systems to convert starch to bio-ethanol in a one-step process may result in large cost reductions in current industrial processes. These reductions will be due to decreased heating energy requirements, as well as a decrease in money spent on the purchase of commercial enzymes for liquefaction and saccharification. In this study, a recombinant Saccharomyces cerevisiae strain was engineered to express the wild-type Aspergillus awamori glucoamylase (GA I) and α-amylase (AMYL III) as well as the Aspergillus oryzae glucoamylase (GLAA) as separately secreted polypeptides. The recombinant strain that secreted functional GA I and AMYL III was able to utilise raw corn starch as carbon source, and converted raw corn starch into bio-ethanol at a specific production rate of 0.037 grams per gram dry weight cells per hour. The ethanol yield of 0.40 gram ethanol per gram available sugar from starch translated to 71% of the theoretical maximum from starch as substrate. A promising raw starch converter was therefore generated. In the second part of this study, soluble solid yields were increased by hydrolysing spent coffee ground, which is the waste generated by the existing coffee process, with hydrolytic enzymes. Recombinant enzymes secreted from engineered Aspergillus strains (β-mannanase, β-endoglucanase 1, β-endo-glucanase 2, and β-xylanase 2), enzymes secreted from wild-type organisms (β-mannanases) and commercial enzyme cocktails displaying the necessary activities (β-mannanase, cellulase, and pectinase) were applied to coffee spent ground to hydrolyse the residual 42% mannan and 51% cellulose in the substrate. Hydrolysis experiments indicated that an enzyme cocktail containing mainly β-mannanase increased soluble solids extracted substantially, and a soluble solid yield of 23% was determined using the optimised enzyme extraction process. Soluble solid yield increases during the manufacturing of instant coffee will result in; (i) an increase in overall yield of instant coffee product, (ii) a decrease in amount of coffee beans important for the production of the product, and (iii) a reduction in the amount of waste product generated by the process. / AFRIKAANSE OPSOMMING: Hierdie studie poog om twee huidige industriële prosesse te verbeter deur die prosesse meer kosteeffektief met behulp van hidroltiese ensieme en mikrobiese sisteme te maak. Die eerste industrie wat geteiken word, is die omskakeling van rou stysel na etanol, en die tweede om hidrolities ensieme in die vervaardiging van kitskoffie te gebruik. Die skep van mikrobiese sisteme om rou-stysel in ’n ’een-stap’ proses om te skakel na bio-etanol sal groot koste besparing tot gevolg hê. Hierdie besparings sal te wyte wees aan die afname in verhittingsenergie wat tydens die omskakelingsproses benodig word, asook ’n afname in die koste verbonde aan die aankoop van duur kommersiële ensieme om die stysel na fermenteerbare suikers af te breek. In hierdie studie is ’n rekombinante Saccharomyces cerevisiae-gis gegenereer wat die glukoamilase (GA I) and α-amilase (AMYL III) van Aspergillus awamori, asook die glukoamilase van Aspergillus oryzae (GLAA) as aparte polipeptide uit te druk. Die rekombinante gis wat die funksionele GA I en AMYL III uitgeskei het, was in staat om op die rou-stysel as koolstofbron te groei, en het roustysel na bio-etanol teen ’n spesifieke tempo van 0.037 gram per gram droë gewig biomassa per uur omgeskakel. Die etanolopbrengs van 0.40 gram per gram beskikbare suiker vanaf stysel was gelykstaande aan 71% van die teoretiese maksimum vanaf stysel as substraat. ’n Belowende gis wat roustysel kan omskakel na bio-etnaol was dus geskep. In die tweede deel van hierdie studie is die opbrengs in oplosbare vastestowwe vermeerder deur die koffie-afval wat tydens die huidige industrieële proses genereer word, met hidrolitiese ensieme te behandel. Rekombinante ensieme afkomstig vanaf Aspergillus-rasse (β-mannanase, β-endoglukanase 1, β-endo-glukanase 2 en β-xilanase 2), ensieme deur wilde-tipe organismes uitgeskei (β-mannanase), asook kommersiële ensiempreparate wat die nodige ensiemaktiwiteite getoon het (β-mannanase, sellulase en pektinase) is gebruik om die oorblywende 42% mannaan en 51% sellulose in koffie-afval te hidroliseer. Hidrolise eksperimente het getoon dat ’n ensiempreparaat wat hoofsaaklik mannanase bevat, die oplosbare vastestofopbrengs grootliks kan verbeter, met ’n verhoogde opbrengs van 23% tydens geöptimiseerde ensiembehandelings. ’n Verhoogde opbrengs in oplosbare vastestowwe tydens die vervaardiging van kitskoffie sal die volgende tot gevolg hê: (i) ’n toename in totale opbrengs van kitskoffie produk, (ii) ’n afname in die hoeveelheid koffiebone wat vir die produksie ingevoer moet word, en (iii) ’n afname in die hoeveelheid afval wat tydens die vervaardigingsproses produseer word.

published_or_final_version / Industrial and Manufacturing Systems Engineering / Master / Master of Philosophy