Submitted in fulfillment of the requirements of the degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014. / Microbial enzymes are gaining worldwide attention due to their potential industrial applications. Microorganisms producing thermostable -xylanase and their associated hemicellulases have significant application in the paper and pulp, food, animal feed, and textile industries. The potential of partially purified xylanase from Thermomyces lanuginosus MC 134, Luminase PB 100, Luminase PB 200 (a commercial xylanase) and T. lanuginosus DSM 5826 (Sigma Aldrich) was evaluated in bleaching of bagasse pulp. The temperature and pH optima for all the enzymes were 60°C and pH 6, respectively. The temperature (50- 80°C) and pH (5-8) stability of the enzymes were also assessed. All the enzymes were relatively stable at 60°C and pH 6 for 180 min. T. lanuginosus MC 134 retained 80% of its activity at 60°C and pH 6 for 180 min and PB 200 retained 75% of its activity at 80°C for 180 min. T. lanuginosus MC 134 also exhibited good alkaline stability at pH 8. The commercial xylanases Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 (Sigma Aldrich) were purified to homogeneity using a gel filtration column packed with sephadex G-100 and characterized for Km and Vmax. However extracellular crude xylanases from T. lanuginosus MC 134 was purified to homogeneity using (N )2S04 precipitation and gel filtration column, packed with sephadex G-100. The purified xylanases exhibited a molecular mass of- 26 to 24 kDa, given range as determined by SDS page. The Km and Vmax values of Luminase PB 100, Luminase PB 200, T. lanuginosus MC 134, and T. lanuginosus DSM 5826, xylanases were determined by the Michaelis-Menten equation using birchwood xylan as the substrate. The Km value for Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 and T. lanuginosus MC 134 were, 8.1 mg/mL, 11.7 mg/mL and 14.3 mg/mL respectively. The Vmax for Luminase PB 100, Luminase PB 200, T lanuginosus DSM 5826 and T lanuginosus MC 134 were 232.6, 454.6 and 74.6 !Jl11ol/min/mg. Biobleaching conditions of the xylanases were also optimised and the release of reducing sugars and lignin derived compounds showed that an enzyme dosage of 50U/g of pulp was ideal for biobleaching at pH 6 and 60°C for 180 min. This brightness for T lanuginosus MC 134, Luminase PB 200, Luminase PB 100 was 45.5 ± 0.11%, 44.1 ± 0.007% and 42.7 ± 0.03% respectively at pH 6, compared to untreated samples. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. All the enzymes analysed exhibited similar trends in the release of lignin derived compounds and reducing sugars which indicated their potential in the pulp and paper industry. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M

As xilanases (EC 3.2.1.8), enzimas produzidas por diversos organismos, são capazes de hidrolisar as ligações -1,4 da cadeia principal da xilana, o mais abundante polissacarídeo hemicelulósico da natureza. O grande potencial biotecnológico das xilanases consiste na sua aplicação nas etapas de branqueamento do papel, nas quais a xilana é hidrolisada sob condição de temperatura elevada para facilitar a remoção da lignina (substância responsável pela coloração), diminuindo a quantidade de compostos clorados utilizados nestas etapas. A termoestabilidade e a especificidade pela xilana são as propriedades responsáveis pelo grande interesse biotecnológico e comercial que as xilanases têm atraído. As xilanases mesofílica, XBC, de temperatura ótima 55ºC (produzida pela bactéria Bacillus circulans) e termofílica, XTL, de temperatura ótima 70ºC (produxida pelo fungo Thermomyces lanuginosus) foram estudadas comparativamente por simulação de dinâmica. Os sistemas foram modelados pelo campo de força GROMOS-96(43A1) e as simulações realizadas pelo programa GROMACS 3.2. O objetivo do trabalho é relacionar as diferenças estruturais, energéticas e dinâmicas com as diferentes termostabilidades exibidas por estas enzimas. Os estudos por simulação sugerem claramente a existência de dois grandes tipos de regiões nas enzimas xilanases XBC e XTL: uma conservada e de grande estabilidade, que é o domínio palma, e a outra que pode sofrer grande movimentação, no caso o domínio polegar. Uma movimentação do tipo abre-fecha de dobradiça foi identificada. O monitoramento das ligações de hidrogênio inter/intramoleculares e pontes salinas ao longo do tempo e em função da temperatura permitem explicar clara e detalhadamente as diferentes termoestabilidades exibidas por duas proteínas da mesma família que compartilham de uma estrutura tridimensional altamente semelhante. Foi possível identificar 14 resíduos carregados que estão presentes na XTL e ausentes na XBC, tais resíduos devem ser considerados sítios potenciais de mutação na XBC. De uma maneira geral, tanto na XBC quanto na XTL, a presença do substrato não altera as características de cada domínio/região mas confere estabilidade para o domínio polegar. Nenhuma diferença clara na afinidade pelo substrato foi detectada pelas interações intermoleculares proteína-substrato. / The enzymes xylanases (EC 3.2.1.8) are produced by several microorganisms and used to hydrolyze the -1,4 bonds of the xylan main chain, the most abundant hemicellulose in nature. The great biotechnological potential of the xylanases is due to its application in the pulp-bleaching processes when the xylan is hydrolyzed under high temperature condition to optimize the lignin removal. This procedure presents the advantage to reduce the amount of chlorine chemicals used in the pulp-bleaching process. The required properties of a biotechnologically useful xylanase include thermostability and high affinity for xylan. The mesophilic, XBC, (from Bacillus circulans) and thermophilic, XTL, (from Thermomyces lanuginosus) xylanases were studied by molecular dynamics simulations. The primary structures of these enzymes are almost completely different while the tertiary structures are identical. The objective of the study is to get some insight on the factors that are responsible for the xylanase thermostability. The systems were modeled by the GROMOS96-(43A1) force field and the molecular dynamic simulations were performed by the GROMACS 3.2 package in the temperature range from 25 to 80ºC. The results obtained with both xylanases were compared. The existence of two kinds of regions was identified in XBC and XTL: the first one conserved and highly stable is formed by the so-called palm and fingers domains. The second region exhibits large movements: this is the thumb domain. A kind of open-close motion was identified that maybe can facilitate the access of the xylane to the active center. The inter/intramolecular hydrogen bonds and salt bridges allow to explain at great length the thermostability differences between the two enzymes. It was possible to identify 14 charged residues present in the XTL with no similar in the XBC: such residues must be considered outstanding mutation sites in XBC. In the presence of the substrate, the characteristics of each domain/region are not modified but the stability of the thumb domain is increased. No difference in the affinity for the substrate was detected between the xylanases and it can be suggested that the activation energies are similar. Two water molecules were found in the active site supporting the hydrolysis mechanisms proposed in the literature.

No description available.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-09-18T19:15:37Z No. of bitstreams: 2 Carolina C. de Queiroz Brito Cunha - 2012.pdf: 4678972 bytes, checksum: aa3da530028732bcbc87f49ba9ea6725 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Há problemas nos campos de palavras chaves e citação. Foi acrescentado da seguinte forma: Streptomyces - bagaço de cana. De acordo com as orientações seria: Streptomyces Bagaço de cana Foi acrescentado no campo de citação: Citação: Cunha, Carolina Cândida de Queiroz Brito - Caracterização de celulases e xilanases produzidas por Streptomyces sp. cultivado em resíduos lignocelulósicos - 2012 - 99 f. - Dissertação - Programa de Pós-graduação em Biologia (ICB) - Universidade Federal de Goiás - Goiânia, 2012. Deve-se usar a NBR6023, ex.: ALCÂNTARA, Guizelle Aparecida de. Caracterização farmacognostica e atividade antimicrobiana da folha e casca do caule da myrciarostratadc.(myrtaceae). 2012. 41 f. Dissertação (Mestrado em Ciências Farmacêuticas) - Universidade Federal de Goiás, Goiânia, 2012. on 2014-09-19T13:12:18Z (GMT) / Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-09-22T18:04:37Z No. of bitstreams: 2 Carolina C. de Queiroz Brito Cunha - 2012.pdf: 4678972 bytes, checksum: aa3da530028732bcbc87f49ba9ea6725 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-22T19:09:30Z (GMT) No. of bitstreams: 2 Carolina C. de Queiroz Brito Cunha - 2012.pdf: 4678972 bytes, checksum: aa3da530028732bcbc87f49ba9ea6725 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-22T19:09:30Z (GMT). No. of bitstreams: 2 Carolina C. de Queiroz Brito Cunha - 2012.pdf: 4678972 bytes, checksum: aa3da530028732bcbc87f49ba9ea6725 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-10-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / An actinomycete strain, isolated from cane sugar bagasse (CSB), identified as Streptomyces sp was selected for its ability to produce cellulases. The production of cellulases was analyzed by submerged fermentation by cultivation on minimal medium (MM) containing CSB, wheat bran (WB) or carboxymethylcellulose (CMC) as carbon source, and yeast extract (YE) as nitrogen source. The results show that WB was the best inducer of CMCases (2.0 U.mL-1). Aiming to analyze the production of cellulases and xylanases kinetics, the isolate was inoculated in minimal medium containing 0.5% (w/v) WB and maintained for 12 days at 45°C under constant agitation of 180 rpm. The highest yield of Avicelase was observed after 264 h of cultivation (5.646 Uml-1), after 144 h for CMCase (3.872 Uml-1), after 144 h for FPase (0.0947 Uml-1) and after 288 h for Xylanase (92.40 Uml- 1). Culture supernatants with maximum activity of Avicelase, CMCase, Fpase and Xylanase were analyzed for optima pH and temperature of the respective enzymes. The highest enzyme activities were detected at pH 7.0 at 35°C for Avicelase, pH 4.5/75°C for CMCase, pH 5.5/45°Cfor FPase and pH 5.5/70°C for Xylanase. The enzymes retained more than 70% of the initial activity after 2 h incubation at 50°C. The profile proteins analyzed by zymogram demonstrated a set of secreted cellulases (37, 21 and 17 kDa) and xylanases (39, 21, 18 and 17 kDa) when grown on FT for 144 h. The saccharification assay with CSB as substrate showed that the enzyme complex was able to release 19% of glucose and 62.9% of xylose. / Uma linhagem de Actinomiceto, isolada do bagaço de cana-de-açúcar (BCA), identificada como Streptomyces sp foi selecionada pela sua capacidade de produzir celulases. A produção de celulases foi analisada por fermentação submersa (FS) pelo cultivo do isolado em meio mínimo (MM) contendo BCA, farelo de trigo (FT) ou carboximetilcelulose (CMC) como fonte de carbono, e extrato de levedura (EL) como fonte de nitrogênio. Os resultados demostraram que o FT foi o melhor indutor da produção de CMCases (2,0 U.mL-1). Com o objetivo de analisar a cinética de produção de celulases e xilanases pelo isolado, este foi inoculado em meio mínimo contendo 0,5% (w/v) FT e mantido por 12 dias a 45°C sob agitação constante de 180 rpm. A maior produção de Avicelase foi observada após 264 h de cultivo (5,646 UmL-1), de CMCase após 144 h (3,872 UmL-1), de FPase após 144 h (0,0947 UmL-1) e de Xilanase após 288 h (92,40 UmL-1). Os sobrenantes de cultura com atividade máxima de Avicelase, CMCase, FPase e Xilanase foram analisados quanto ao pH e temperatura ótimos das respectivas enzimas. Os resultados obtidos demonstraram que a maior atividade de Avicelase foi detectada em pH 7,0 a 35°C; CMCase apresentou melhor atividade em pH 4,5 a 75°C; FPase apresentou melhor atividade em pH 5,5 a 45°C e Xilanase apresentou melhor atividade em pH 5,5 a 70°C. Quanto à termoestabilidade, as enzimas presentes mantiveram mais de 70% da atividade inicial após 2 h de incubação a 50°C. O perfil de proteínas analisado por zimograma demonstrou que o isolado secretou um conjunto de celulases (37, 21 e 17 KDa) e xilanases (39, 21, 18 e 17 KDa) quando cultivado em FT por 144 h. No ensaio de sacarificação de BCA o complexo enzimático foi capaz de liberar 19% de glicose e 62,9% de xilose.

Bibliography: leaves 127-166. This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes.

Xylanase production from a Bacillus subtilis gene cloned into a strain of Escherichia coli was measured. Although this gene was expressed in E. coli at several temperatures, efficient normal xylanase secretion did not occur, the observed protein release apparently depending on cell leakage or lysis. Screening for a better microbial protein secretor free of cellulase selected B. cereus #259. The strain had wild plasmids that were hard to eliminate using acridine orange and elevated temperature curing techniques. While still bearing 5 wild plasmids, attempts to transform B. cereus #259 were unsuccessful using conventional methods and electroporation. Another strain, B. cereus #518, found to be free of wild plasmids, was then used. A bidirectional vector shuttle plasmid (pMK3) was employed to carry the cloned gene into this B. cereus strain. Transformation was carried out by high voltage electroporation. Xylanase production by the new B. cereus clone was similar to that from E. coli, but was shown to be continuously and normally secreted. The xylanase gene products from the E. coli and B. cereus hosts were shown to function identically. Both xylanases improved the delignification of unbleached softwood and hardwood kraft pulps, thus reducing the Cl$ sb2$ required to achieve a given degree of bleaching, without altering the physical properties of the fibers. Using a target kappa number lignin content) of 5, xylanase pretreatment of aspen kraft pulp led to a 22% saving of chlorine. Adsorbable organic halogens in the bleachery effluent were also lowered by more than 50%.

Bibliography: leaves 127-166. / xvi, 166, [64] leaves, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Bibliography: leaves 127-166. / xvi, 166, [64] leaves, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Thesis (MScAgric) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass. These biopolymers are therefore an important renewable source of food, fuels and chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The individual molecules are held together by hydrogen bonds, forming largely crystalline fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide, coats and binds the cellulose microfibrils, preventing the cellulose from becoming too crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3- and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named according to the sugar type that forms the polymer backbone. Pectic substances contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side chains. Two groups of enzymes, cellulases and pectinases, are required for the microbial utilisation of crystalline cellulose and pectin. Cellulases are multicomponent complexes that are often composed of endoglucanases, exoglucanases and cellobiases. Cellobiose is the major end product of concerted endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying action of the saponifying enzymes, pectinesterase, releasing the methyl groups of the pectin molecule, and by hydrolase or lyase action of the depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a- 1.4-glycosidic linkages in the polygalacturonate chain. The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic beverage industry for fermentations of wine, beer and other alcoholic beverages for many years. However, it is unable to produce extracellular depolymerising enzymes that can efficiently degrade polysaccharides, which are the main cause of clarification and filtration problems. Enzyme preparations have been used in the alcoholic beverage industries to degrade haze-forming polysaccharides, thereby improving the filterability and quality of products such as beer and wine. An alternative would be to develop S. cerevisiae strains that produce extracellular polysaccharidases, enabling the yeast to degrade polysaccharides without the addition of commercial enzyme preparations. These strains can also be very useful in improving the quality of wine, as well as cutting the costs of the winemaking process. The objective of this study was to investigate the effects of two transformed S. cerevisiae strains on different wine grape varieties. The following genes have been cloned and characterised previously: the Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3- 1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t (designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE). Two yeast integrating plasmids were constructed, one containing the END1 and XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two plasmids were then integrated into the URA3 locus of two separate industrial wine yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast transformants in the absence of auxotrophic markers, the integrating plasmid containing the END1 and XYN4 gene cassettes was issued with the dominant selectable Geneticin G418-resistance {G f) marker. The integrating plasmid harbouring the PEH1-PELE gene cassette was issued with the dominant selectable sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4- glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase. These recombinant yeasts were capable of extracting more colour from grape skins of certain varieties, as well as leading to more freeflow wine as a result of the more effective degradation of glucans, xylans and pectins in the skins. They also led to decreased turbidity in the wine, making it more filterable. Future work will entail further investigation of the effects of these recombinant yeasts on different white and red wine grape varieties. Another objective of this study was to screen non-Saccharomyces wine yeasts for the production of extracellular hydrolytic enzymes. The reason for this part of the thesis was to determine the types of extracellular hydrolytic enzymes that are produced and to determine which genera produce which kinds of extracellular enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces, were screened for the production of extracellular pectinases, proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts were all isolated from grapes and clarified grape juice to ensure that they were yeasts found in must during the initial stages of fermentation. This information can be used to pave the way to pinpoint the specific effects in wine of these enzymes produced by the so-called wild yeasts associated with grape must. This information can also be used to transform Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces yeasts for the production of extracellular hydrolytic enzymes. However, future research will have to be done to determine the extent of the activity of these enzymes in wine fermentations and to obtain better knowledge of the physiological and metabolical features of non-Saccharomyces yeasts. / AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee. Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose, wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane, 1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings, onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas. Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en poligalakturonase), split die a-1,4-glukosidiese verbindings in die poligalakturonaatketting. Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate in die alkoholiese bedryf vir die degradasie van die probleem polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus polisakkariede self sonder die byvoeging van eksterne kommersiele ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases produseer, op verskillende wyndruifvarieteite na te vors. Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase- geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl) van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder (ADH2T), die gistriptofaansintase geen se termineerder (TRP5t) en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE). Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4- geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende (SMR1) merker is in die integreringsplasmied, wat die PEH1- PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-, XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase, pektaatliase en poligalaturonase. Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak. Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing gedoen moet word om die presiese effekte van hierdie rekombinante giste op verskillende rooi en wit druifvarieteite te bepaal. ‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word, asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase, sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme, wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van die problematiese polisakkariede in wyn te produseer. Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces wyngiste.

As celulases e as xilanases são enzimas que hidrolisam a celulose e a xilana, respectivamente, contidas nos resíduos lignocelulósicos. A possibilidade de aplicar estas enzimas na produção de etanol vem sendo objeto de diversos estudos. No entanto, ainda não há uma tecnologia economicamente viável para a produção deste biocombustível a partir da biomassa lignocelulósica. Entre os microrganismos que apresentam altos títulos para estas enzimas, incluem-se linhagens de Penicillium echinulatum; porém, ainda faltam dados de sua fisiologia e estudos da produção de enzimas em biorreator. Neste trabalho, empregou-se a linhagem mutante celulolítica desreprimida S1M29 de P. echinulatum e o meio de cultivo foi composto por celulose, sacarose, solução de sais, Tween 80, farelo trigo e farelo de soja. Avaliou-se o efeito de diferentes temperaturas e pHs na produção das enzimas. O efeito da concentração da celulose sobre as atividades enzimáticas foi avaliada em regime descontínuo (RD) e regime descontínuo alimentado (RDA). Verificou-se que a temperatura mais apropriada para a produção de celulases e xilanases é de 28ºC e dentre os valores de pHs avaliados, o pH 6,0 apresentou a maior produção das enzimas. O aumento da concentração da celulose no RD proporcionou maiores atividades para endoglicanases, porém o mesmo não foi obtido para xilanases. Para FPA (Filter Paper Activity), aumentos proporcionais nas atividades foram obtidos somente com concentrações de até 3% de celulose em RD, condição que também proporcionou as maiores atividades de - glicosidases. O RDA incrementou as atividades de FPA, endoglicanases e xilanases, mas não de -glicosidases. Estes resultados contribuem para a otimização de processos e para a produção econômica de enzimas por P. echinulatum, visando o desenvolvimento de tecnologias economicamente viáveis para produção de etanol a partir de materiais lignocelulósicos. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-11T13:29:22Z No. of bitstreams: 1 Dissertacao Laisa dos Reis.pdf: 1870988 bytes, checksum: 956ace97d10d44f22cb5eba7cea275d1 (MD5) / Made available in DSpace on 2014-06-11T13:29:23Z (GMT). No. of bitstreams: 1 Dissertacao Laisa dos Reis.pdf: 1870988 bytes, checksum: 956ace97d10d44f22cb5eba7cea275d1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cellulases and xylanases are enzymes that hydrolyze cellulose and xylan respectively, which are found in lignocellulosic residues. Although the applicability of these enzymes in the ethanol production has been the subject of several studies, an economically viable technology for the production of biofuel from lignocellulosic biomass is currently not available. Strains of Penicillium echinulatum are among the microorganisms that have high titers of these enzymes. However, data related to physiology and enzyme production in bioreactor for such strains are still missing. A cellulolytic mutant strain of P. echinulatum S1M29 and a culture medium composed of cellulose, sucrose, salt solution, Tween 80, wheat bran and soybean meal were used in this study. The effect of different temperatures and pHs during the enzymes production was evaluated. The effect of cellulose concentration in the enzymatic activity was evaluated in batch cultivation (BC) and fed-batch cultivation (FBC). It was found that the appropriate temperature for the production of cellulases and xylanases is 28°C, while the higher enzyme production occurred at pH 6.0. The high cellulose concentration in BC provided the greatest activities for endoglicanases, but the same result was not obtained for xylanases. For Filter Paper Activity (FPA), proportional increases in activity were obtained only with concentrations up to 3% of cellulose in BC, which is also linked to the highest activities for -glucosidases. FBC increased the activities of FPA, endoglucanases and xylanases, but it did not increase the -glucosidases activities. Such results contribute towards the optimization of enzyme production using P. echinulatum and the development of economically viable technologies for the production of ethanol from lignocellulosic materials.