Orientador: Rodrigo Fernando Costa Marques / Resumo: A biomassa lignocelulósica vem se destacando como uma matéria-prima essencial para a produção de muitos produtos químicos de interesse industrial em áreas como a produção de energia, alimentos, fármacos, agricultura, meio ambiente e assim por diante. Apesar disso, muitas aplicações vêm esbarrando em uma série de dificuldades encontradas nos processos de conversão enzimática, como instabilidade operação das enzimas, alto custo de produção e purificação, reações de inibição e problemas de recuperação e reciclo. Para contornar esses problemas, muitos métodos de imobilização enzimática têm surgido, entre os quais, destaca-se a obtenção de agregados enzimáticos reticulados magnéticos (MCLEAs). Esta classe de materiais é obtida a partir da reação de reticulação entre agregados físicos de enzimas e suportes magnéticos, o qual pode unir as importantes propriedades catalíticas dos agregados físicos (como resultado da manutenção da estrutura nativa da enzima) à capacidade de recuperação e reciclo do suporte magnético (devido suas propriedades magnéticas intrínsecas). Frente a isso, esse trabalho relata a síntese, caracterização e potencial aplicação de MCLEAs de enzimas celulases em processos de conversão de celulose. Dividido em três capítulos, primeiramente é apresentado um review sobre o estado da arte no que diz respeito a obtenção de produtos de valor agregado a partir da biomassa lignocelulósica utilizando MCLEAs. No segundo capítulo, diferentes MCLEAs foram preparados na presenç... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Lignocellulosic biomass has highlighted as an essential renewable raw material for production of many value-added chemicals of industrial interest in field as energy production, food, pharmaceutical, agriculture, environment and so on. Despite it, many applications have wrought with a series of difficulties in regarding enzymatic conversion processes, as enzyme operational instability, high production and purification cost, inhibition reactions, and issues of recovery and recycle. To overcome these issues, many enzyme immobilization methods have emerged, among which highlights the obtention of magnetic-cross linked enzyme aggregates (MCLEAs). This materials class is obtained from cross-linking reaction between enzyme physical aggregates and magnetic supports, which can gather the important catalytic properties of the physical aggregates (as a result of enzyme native structure maintenance) to recovery and recycle capacity of magnetic nanoparticles (as result of its intrinsic magnetic properties). Faced it, this work reports the synthesis, characterization and potential application of different cellulases MCLEAs in the cellulose enzymatic conversion process. Sectioned in three chapters, firstly is presented a review about the state of art in concern to obtention of value-added chemicals from lignocellulosic biomass using MCLEAs. In the second chapter, different MCLEAs were prepared in the presence of quitosana-coated magnetic nanoparticles with three different precipitation age... (Complete abstract click electronic access below) / Doutor

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Apple preharvest fruit drop frequently results in severe economic losses. Cultural control of preharvest drop has relied upon plant growth regulators (PGRs), but the loss of daminozide (Alar) and 2,4,5-TP has severely limited the choices of effective stop-drop compounds. A more complete understanding of factors involved in preharvest drop is therefore imperative. Experiments were conducted to provide information about cellulase activity in the abscission zone, effects of applied auxin and ethylene biosynthesis inhibition on drop, changing sensitivity to abscission induction during the season, and relationships among seed number, fruit weight, and day of drop. Observational studies were used to study effects of fruit maturity, canopy positions, and morphology of stem attachment on time of fruit drop as well as characterizing the natural timing of late-season fruit drop. Increased activity of cellulase, but not polygalacturonase, in the abscission zone was detected within 4 days of cutting fruit to induce abscission. Both aminoethoxyvinyl glycine (AVG) and naphthaleneacetic acid (NAA) applied 2 or 4 days after cutting delayed drop, but NAA delayed drop 1.6 days longer than did AVG. Fruit of "RedChief Delicious"(D) exhibited a significantly reduced sensitivity to abscission-inducing treatments from mid-June until early July compared to earlier orr later in the season. Application of plant growth regulators to cut fruit revealed a significant interaction of NAA treatment with AVG treatment such that NAA delayed drop when applied with AVG but not without AVG. Fallen fruit had lower starch and higher soluble solids than fruit on the tree on the day of collection. The highest fruit in the canopy fell an average of 4.4d earlier than the lowest fruit. Day of drop was not different for fruit from king blooms vs. side blooms within an inflorescence. There was a trend for fruit from first year wood to drop later than fruit from older wood on "Delicious", but not "Smoothee Golden Delicious" trees. There was no detectable effect of angle of orientation of the subtending spur on the limb, the pedicel:spur abscission zone, or fruit axis of symmetry on time of fruit drop. No difference was detected in time of fruit drop between East and West or North and South sides of the trees. No substantial variation in day of drop of individual fruit was explained by number of seed in the fruit. Daily drop was recorded for three cultivars ("RedChief Delicious", "Smoothee Golden Delicious", and "Commander York") for three years. Variance of average day of drop from year to year was 40.1, while variance among cultivars within a year was 51.8. Variance from tree to tree within each cultivar, within each year, was only 18.6. Multiple regression modeling to identify relationships between weather factors and daily fruit drop revealed that much of the variability in time of drop was due to factors other than the weather events modeled. The best regression models developed explained only 8% to 35% of the variability in time of drop. The most important weather factors were daily minimum temperatures and precipitation. Rain events of greater than 5.0 mm following a drier period appeared to cause increased drop of all three cultivars in one out of the three years investigated. / Ph. D.

L'activité cellulolytique est recherchée chez des champignons filamenteux microscopiques isolés de sols environnant les sources thermales des régions Guelma (Hammam Debagh) et de Mila (Hammam Grouz-Atmania et Hammam Safsaf-Teleghma). 88 souches fongiques sont isolées, appartenant à six genres différents : Aspergillus, Alternaria, Emericella, Fusarium, Penicillium et Trichoderma. Leur sélection (test au papier filtre et le test des plaques à trous), montre que seule la souche J2 possède une activité cellulolytique importante, comparable à celle de la souche T. reesei Rut C-30. Cet isolat appartient à l'espèce Trichoderma longibrachiatum Rifai. A 35°C, notre isolat ne montre pas de différences significatives des activités papier filtre et endoglucanase par rapport à T. reesei Rut C-30, mais l'activité β-glucosidase produite par notre isolat est deux fois plus que celle produite par cette dernière. Avec un taux d'ensemencement de 106spores/ml, la souche est en activité maximale. Un bon rendement en enzyme est obtenu avec des spores âgées de 6 jours. La souche D choisie du sous-clonage, est cultivée en fermenteur de 4 litres sur le milieu minéral Mandel Avicel 1%, et produit un maximum d'activité papier filtre 1.88UI/ml, d'activité endoglucanase 11.22UI/ml et d'activité β-glucosidase 0.64UI/ml après 128 heures, 144 heures et 120 heures d'incubation, respectivement. Les enzymes papier filtre et endoglucanase sont optimalement active à 60°C et 55°C, respectivement. Un pH optimum de 4.0 et 5.0 pour l'activité papier filtre, alors que l'activité endoglucanase à un pH optimum de 4.0. L'activité endoglucanase est thermostable, elle résiste à un traitement thermique pendant 5 heures à 70°C, 80% de son activité originale est maintenue. La demi-vie de l'activité papier filtre est de 3 heures à 60°C. Le substrat CMC améliore la stabilité thermique de ces enzymes. Ces enzymes sont stables à 50°C pendant 5 heures dans une gamme de pH de 3.0 à 6.0 et 4.0 à 6.0, respectivement. L'EDTA (5 mM), provoque une forte diminution des enzymes, alors que, le β-mercaptoethanol (5 mM) conduit à leur activation. Les cations divalents calcium (Ca2+) et zinc (Zn2+), provoquent une augmentation et une amélioration des activités enzymatiques en présence de l'EDTA. L'extrait enzymatique brut est capable d'hydrolyser les substrats cellulosiques insolubles, cette enzyme peut ainsi être classée comme un type endo et exo de la cellulase. Par ces caractéristiques, production de l'enzyme, thermostabilité et pH acide, notre souche sauvage Trichoderma longibrachiatum peut être attractive en industrie pour la production de la cellulase.Mots clés : Moisissures, isolement, écosystèmes extrêmes, cellulase, thermostabilité. / Filamentous fungi found in soils surrounding the hydrothermal stations of regions in the east of Algeria: Guelma (Hammam Debagh) and Mila (Hammam Grouz-Atmania, Hammam Safsaf-Teleghma), are screened for the presence of cellulase activity. 88 fungal strains were isolated and identified from six kinds: Aspergillus, Alternaria, Emericella, Fusarium, Penicillium and Trichoderma. Their selection (filter paper test and test of perforated plates) shows that only the strain J2 has a significant cellulolytic activity. This isolate belongs to the species of Trichoderma longibrachiatum Rifai. At 35°C, our isolate shows equivalent activities filter paper and endoglucanase than T. reesei. On the other hand the β-glucosidase activity of our isolate was until twice more important than T. reseei one. With an inoculum size of 106 spores/ml the strain produces the maximum enzyme activities. A good yield of enzyme is obtained with spores aged of six days. The strain D allowed from subcloning cultivated in 4 liter fermenter on Mandels medium with cellulose Avicel (1%) produces maximum activities of filter paper (1.88UI/ml), endoglucanase (11.22UI/ml) and β-glucosidase (0.66UI/ml) after 128 hours, 144 hours and 120 hours, respectively. The optimum temperatures were 55°C and 60°C for endoglucanase and FPA, respectively. The endoglucanase was optimally active at pH 4.0, and the FPA was optimal at pH 4.0 and 5.0. The endoglucanase was thermostable at 70°C after 5 hours incubation, preserved 80% of the original activity. The half-life of the filter paper activity appeared to be 3 hours at 60°C. These activities were stable at 50°C after 5 hours incubation in a pH range of 3.0 to 6.0 and 4.0 to 6.0, respectively. The EDTA (5mM) causes a significant diminution of the enzymes, while the β-mercaptoethanol (5mM) leading to their activation. Divalent cations calcium (Ca2+) and zinc (Zn2+) cause an increase and improvement of enzymes activities in the presence of EDTA (5mM). The crude enzyme extract is able to hydrolyse insoluble cellulosic substrates. This enzyme can be classified as an endo and exo type of the cellulase. These results suggested that the no-mutated strain Trichoderma longibrachiatum should be an attractive producer for cellulases production.Keywords: Fungi; isolation; extreme ecosystems; cellulase; thermostability.

Le génome de Mycobacterium tuberculosis, l’agent causal de la tuberculose, code pour une protéine ayant la capacité de se fixer sur la cellulose (Rv1987), une cellulase potentielle (Rv1090), et une cellulase pleinement active (Rv0062). Cette observation est surprenante, car la cellulose est un composant majeur des parois des cellules végétales, tandis que M. tuberculosis est un pathogène humain sans contact connu avec des plantes. Nous avons émis l’hypothèse que ces protéines pourraient jouer un rôle dans les interactions entre les mycobactéries du complexe M. tuberculosis avec les kystes d’amibes libres, dont la paroi contient également de la cellulose. Dans notre travail de thèse, nous avons cherché par une analyse in silico la présence de ces trois gènes chez toutes les bactéries ayant un génome complètement séquencé présentes dans la base de données CAZy (accessible en ligne à l’adresse www.cazy.org). Cette étude a montré que seulement 2,5% des bactéries codent pour les trois gènes simultanément. Parmi ces bacteries, nous avons ensuite confirmé expérimentalement par PCR et séquençage la présence des gènes Rv0062, Rv1090 et Rv1987 chez les mycobactéries du complexe M. tuberculosis. Nous avons ensuite vérifié la transcription de ces trois gènes chez la souche de référence M. tuberculosis H37Rv, puis produit dans Escherichia coli des protéines de fusion Rv1090 et Rv1987 et montré qu'elles étaient capables d'hydrolyser la cellulose (Rv1090) et de s’y fixer (Rv1987). De plus, nous avons mis en place un model expérimental d’interaction entre les mycobactéries du complexe M. tuberculosis et les amibes libres dans le but de comprendre le rôle des gènes Rv0062, Rv1090 et Rv1987. Dans un premier temps nous avons montré que M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii ainsi que Mycobacterium avium utilisé ici comme un controle positif étaient capables de survivre dans le cytoplasme des amibes libres telles que Acanthamoeba polyphaga. Ensuite, nous avons montré que M. tuberculosis et M. bovis mais pas M. canettii étaient capables de survivre à l’intérieur des kystes d’amibes. Enfin nous avons montré que M. tuberculosis, M. bovis et M. canettii étaient capables de survivre dans le sol pendant au moins 6 mois. Les données établies dans cette thèse soutiennent le rôle des cellulases dans la survie environnementale des mycobactéries du complexe M. tuberculosis, et ouvrent la voie à l’étude de cette phase méconnue dans le cycle de ces organismes / The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a protein with the ability to bind to cellulose (Rv1987), one potential cellulase (Rv1090), and one fully active cellulase (Rv0062). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. We hypothesized that these genes could play a role in the interactions between M. tuberculosis complex organisms and amoebal cysts, whose wall contains cellulose.In our thesis work, we have searched by in silico analysis for the presence of these three genes in all bacteria with complete sequenced genomes present in the CAZy database (available online at www.cazy. org). This study showed that only 2.5% of bacteria encode the three genes simultaneously. Among these bacteria we have confirmed experimentally by PCR and sequencing the presence of Rv0062, Rv1090 and Rv1987 in the M. tuberculosis complex organisms. We have checked the transcript of the three genes in the reference strain M. tuberculosis H37Rv and we subsequently produced Rv1090 and Rv1987 fusion proteins in Escherichia coli and demonstrated that they were indeed able to hydrolyze (Rv1090) and to bind (Rv1987) cellulose. In addition, we have developed an experimental model of interaction between M. tuberculosis organisms and the free-living amoebae in order to understand the role of Rv0062, Rv1090 and Rv1987 genes. Initially we have shown that M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii and Mycobacterium avium used here as a positive control were able to survive in the cytoplasm of the free-living amoeba such as Acanthamoeba polyphaga. We have further shown that M. tuberculosis and M. bovis but not M. canettii were able to survive within the amoebal cysts. Finally we have shown that M. tuberculosis, M. bovis and M. canettii were able to survive in soil for at least 6 months. The data obtained in this thesis support the role of cellulase in the survival of M. tuberculosis complex organisms in the environment and pave the way for the study of this unknown phase in the cycle of these organisms.

Parmi les ressources d'origines agricole et forestière utilisables aujourd'hui en tant que biomasse à destination énergétique, le miscanthus apparait comme l'une des espèces de graminées les plus prometteuses pour la production de bioéthanol de seconde génération grâce à son haut potentiel en biomasse. Ce procédé dit "2G" convertit la cellulose contenue dans ces biomasses lignocellulosiques en bioéthanol et ce via un procédé intégrant prétraitement physico-chimique, hydrolyse enzymatique et fermentation. Le principal objectif de ce projet de thèse visait à étudier l'impact de l'hétérogénéité tissulaire et structurale du miscanthus sur sa saccharification et s'est décliné en différents volets liés à l'étude de l'efficacité des prétraitements et à l'analyse des performances de différents cocktails enzymatiques de Trichoderma reesei. L'hydrolyse enzymatique est essentiellement limitée par la structure et la porosité des complexes pariétaux qui réduisent l'accessibilité de la cellulose aux cellulases. En plus des constituants hémicelluloses et lignines qui recouvrent la cellulose, les parois cellulaires du miscanthus sont riches en acides hydroxycinnamiques (pCA et FA) qui jouent un rôle important dans la cohésion du réseau pariétal complexe. L'application de prétraitements acide et alcalin sur le miscanthus a ainsi révélé une différence de réactivité en fonction des types cellulaires. Les parois secondaires du sclérenchyme sont plus facilement dégradées par les cellulases fongiques après prétraitement acide. L'étude de la distribution des composés phénoliques au niveau cellulaire par micro spectrophotométrie UV a rapporté une nette diminution de l'absorbance UV dans tous les tissus après chaque prétraitement. Ceci n'expliquant pas totalement les différences de réactivité observées, d'autres facteurs physicochimiques seraient donc impliqués. Une approche visant à évaluer la progression des cellulases au sein des parois par immunocytochimie a également été initiée mais elle s'est heurtée à des problématiques techniques liées à la nature des tissus et aux anticorps employés. Les performances en terme de conversion de la cellulose ont été évaluées avec des cocktails enzymatiques de T. reesei comprenant des activités (hemi-)cellulolytiques variables. Une meilleure efficacité du prétraitement par explosion à la vapeur a ainsi pu être montrée par réduction de la quantité d'enzymes mises en œuvre. Comme c'est le cas pour d'autres graminées, ces travaux ont permis de confirmer le rôle crucial de l'enzyme β-glucosidase, permettant de limiter l'inhibition par le cellobiose et améliorant la cinétique initiale de saccharification. L'amélioration du rendement d'hydrolyse par l'utilisation d'un sécrétome comprenant une bonne activité hémicellulolytique a pu être ensuite démontrée. L'utilisation de cocktails enzymatiques reconstitués à partir d'enzymes pures a enfin permis de définir un mélange "optimal" composé des quatre principales cellulases de T. reesei (CBH1, CBH2, EG1 et EG2) associées à une hémicellulase (XYN1). / Among agricultural and forest resources, the grass specie miscanthus has emerged as one of the most promising feedstock candidates for 2G-biofuel production due to its high biomass yield. The biofuels 2G-production process is based on cellulose conversion into bioethanol via physicochemical pretreatment, enzymatic hydrolysis and fermentation. The main objective of this Ph.D. project was to evaluate the effect of tissue and structure heterogeneity of miscanthus on its saccharification by evaluating pretreatment efficiency and analyzing the performance of different Trichoderma reesei cellulolytic cocktails.Enzymatic hydrolysis is mainly hindered by cell wall structure and porosity which limit cellulose accessibility to cellulase. In addition to hemicelluloses and lignin polymers, miscanthus cell walls, contain high amounts of hydroxycinnamic acids (pCA and FA) that play a significant role in cross-linking polymers into cohesive network. Applying acid and alkali pretreatments on miscanthus revealed a distinctive reactivity depending on cell types. Secondary cell walls of sclerenchyma appeared more digested by fungal cellulases after acid pretreatment. Addressing phenolics distribution (lignin and hydroxycinnamic acids) at cell level by UV micro spectrophotometry highlighted a significant decrease in UV absorbance after both pretreatments irrespective to cell type indicating that other physicochemical and structural features are involved in distinct cell wall reactivity. We have also attempted to evaluate cellulase progression into miscanthus cell walls by immunocytochemistry but we have had many technical problems due to the nature of miscanthus tissues and used antibodies. Cellulose conversion ability was then evaluated using enzymatic cocktails of T. reesei which vary in their (hemi-)cellulolytic activities. Higher efficiency of the steam explosion pretreatment was demonstrated by reducing enzymes loading. As reported previously on other grasses, β-glucosidase plays a crucial role by limiting the inhibiting effect of cellobiose and improving the initial saccharification step. We furthermore showed that the use of hemicellulases-improved cocktails allowed significant increase in saccharification yields. We finally identified an optimal reconstituted enzyme mixture composed of four major cellulases of T. reesei (CBH1, CBH2, EG1 and EG2) and the hemicellulase XYN-1.

R. cellulolyticum est une bactérie mésophile, anaérobie stricte et cellulolytique, qui sécrète des macro-complexes multienzymatiques (cellulosomes) très performants dans la dégradation des polysaccharides de la paroi végétale. Les Glycoside Hydrolases de famille 9 (GH9) sont toujours surreprésentées chez les bactéries à cellulosomes. Le génome de R. cellulolyticum code 13 GH9 dont 12 participent aux cellulosomes. Mon travail de thèse a consisté à étudier l’ensemble des GH9 de R. cellulolyticum, en déterminant leurs activités à l’état libre et en complexes, afin d’élucider leurs rôles dans la dégradation de la cellulose. Les GH9 ont chacune des activités et des spécificités de substrats différentes. Deux GH9 présentent des activités atypiques, puisque l’une d’elles est inactive et l’autre est une xyloglucanase. Les caractérisations en complexes ont souligné l’importance de la diversité des GH9 et ont montré qu’elles agissent en synergie dans la dégradation de la cellulose. De plus, l’élargissement du panel des GH9 de R. cellulolyticum par l’introduction d’une cellulase exogène de Lachnoclostridium phytofermentans a permis d’améliorer les capacités cellulolytiques de la clostridie. L’activité xyloglucanase d’une des GH9 m’a poussé à étudier le catabolisme du xyloglucane chez R. cellulolyticum. Ce travail a mis en exergue la présence d’un équipement spécialisé dans l’utilisation de ce sucre. Ainsi, après une dégradation du xyloglucane par les enzymes cellulosomales en xyloglucane dextrines, ces dernières sont importées dans le cytoplasme par un transporteur ABC spécifique puis hydrolysées séquentiellement par les enzymes cytoplasmiques en mono et disaccharides assimilables. / Ruminiclostridium cellulolyticum is a mesophilic and strictly anaerobic bacterium. It produces multienzymatic complexes called cellulosomes which efficiently degrade the plant cell wall polysaccharides. Family-9 Glycoside Hydrolases (GH9) are plethoric in cellulosome-producing bacteria. The genome of R. cellulolyticum thus encodes for 13 GH9 enzymes, 12 of them participate to the cellulosomes.My Ph. D. aimed at characterizing all GH9 enzymes from R. cellulolyticum, by determining their activities in a free and complexed states, in order to elucidate their role in cellulose degradation. All GH9 enzymes exhibit various activities and substrate specificities. Two of them have atypical activities, since one is inactive and one is a xyloglucanase. Results obtained when all GH9 are in complex highlighted the importance of GH9 diversity and revealed they act synergistically in cellulose depolymerization. Moreover, expanding the panel of GH9 enzymes by introducing an exogenous cellulase from Lachnoclostridium phytofermentans improved the cellulolytic capacities of R. cellulolyticum. The xyloglucanase activity of one GH9 enzyme prompted me to investigate the xyloglucan catabolism in R. cellulolyticum. This work uncovered the presence of a specialized equipment for xyloglucan utilization. After extracellular digestion of xyloglucan by cellulosomal enzymes, xyloglucan dextrins are imported into the cytoplasm via a specific ABC-transporter and sequentially hydrolyzed by cytoplasmic enzymes into fermentable mono and disaccharides.

Submitted by Lucienne Costa (lucienneferreira@ufcg.edu.br) on 2017-12-13T17:26:54Z No. of bitstreams: 1 CARLOS ALBERTO BISPO DE SOUSA - TESE (PPGEP) 2014.pdf: 2633408 bytes, checksum: 80087f7d1097f18a52b312e44cfc57af (MD5) / Made available in DSpace on 2017-12-13T17:26:54Z (GMT). No. of bitstreams: 1 CARLOS ALBERTO BISPO DE SOUSA - TESE (PPGEP) 2014.pdf: 2633408 bytes, checksum: 80087f7d1097f18a52b312e44cfc57af (MD5) Previous issue date: 2014-11-28 / Capes / A demanda por fontes de energia renovável tem crescido em todo o mundo. Nesse contexto, o bioetanol obtido a partir da hidrólise de materiais lignocelulósicos tem merecido destaque. Porém, a produção das enzimas celulolíticas usadas nesse processo é de custo elevado, e este é o principal empecilho para a obtenção do etanol celulósico em grande escala. O objetivo deste estudo foi produzir enzimas celulolíticas destinadas a produção de bioetanol, a partir da fermentação semissólida da biomassa da palma forrageira pelo fungo filamentoso Trichoderma reesei LCB 48. O estudo da fermentação revelou que a melhor condição foi atingida com umidade inicial de 90% e suplementação de 1% de fonte de nitrogênio. A atividade máxima foi alcançada em 110 horas de processo, com produção de 6,45 U/gds. O estudo de lixiviação das enzimas produzidas revelou como as melhores condições do processo: a relação solvente/substrato de 20g/ml, agitação de 50rpm e tempo de contato de 15 minutos, na qual obteve-se extratos brutos com 15,14 U/gds expressa em carboximetilcelulase (CMCase). As enzimas recuperadas na lixiviação exibiram atividade CMCase ótima em temperatura de 55°C e pH ótimo entre 4,0 e 5,0. Estudos de estabilidade mostraram que a enzima é desativada em valores de pH superiores a 6,5 e temperaturas superiores a 50°C. Ensaios de hidrólise utilisando a própria biomassa da palma e o resíduo da fermentação como material lignocelulósico apresentaram respectivamente produtividade máxima de de 334,4 mg/L.h e 308 mg/L.h de glicose em 4 horas de processo. Foi realizado um estudo de partição das enzimas obtidas utilizando sistemas aquosos bifásicos. O SAB composto por 18% Peg 4000 e 12% citrato de sódio em pH 5,0 resultou em um fator de purificação de 5,31 para a CMCase e 61,4 para celobiase. Para FPase o fator de purificação foi de 2,29 quando a concentração de citrato usada foi de 16%. A biomassa da palma mostrou-se viável tanto para a obtenção das enzimas celulases quanto para a obtenção de açúcares fermentescíveis para produção de bioetanol. Os SABs mostraram-se promissores como etapa inicial de um processo de recuperação e purificação das celulases produzidas. / The demand for renewable energy has grown worldwide. In this context, bioethanol obtained from the hydrolysis of lignocellulosic materials has been highlighted . However, the production of cellulolytic enzymes used in the hydrolysis process is costly, and this is the main obstacle for obtaining cellulosic ethanol on a large scale. This study was designed to produce cellulolytic enzymes production of bioethanol from biomass semisolid fermentation of forage cactus by the filamentous fungus Trichoderma reesei LCB 48. The study revealed that the best fermentation condition was achieved with 90 % humidity and supplementation 1% of the nitrogen source . The maximum activity was achieved in 110 hours of process, with production of 6.45 U/gds . The study of leaching of enzymes produced revealed as the best process conditions: solvent substrate ratio of 20mL/ g , 50 rpm of agitation and contact time of 15 minutes , which was obtained in crude extracts with 15.14 U/gds expressed in carboxymethylcellulase ( CMCase ) . The crude extract was stable for up to 20 days when stored at room temperature. The enzymes recovered in the leaching exhibited CMCase activity at optimal temperature of 55 ° C and optimum pH between 4.0 and 5.0 . Stability studies showed that the enzyme is deactivated at pH values above 6.5 and temperatures above 50 ° C. Hydrolysis assays of pear cactus biomass itself and the residue fermenting lignocellulosic material presented as maximum yield of 334.4 mg/Lh and 308 mg/Lh of glucose in 4 process hours respectively. A study ds enzymes obtained using aqueous two-phase partition systems was carried out . SAB composed of 18 % PEG 4000 and 12% sodium citrate at pH 5.0 resulted in a purification factor of 5.31 to 61.4 for CMCase and cellobiase. FPase for the purification factor was 2.29 when the concentration of citrate used was 16% . Biomass palm proved to be feasible for both obtaining the cellulase enzymes as to obtain fermentable sugars to bioethanol production. The SABs proved promising as an initial step in a process of recovery and purification of cellulases produced .

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-01-28Bitstream added on 2014-06-13T20:40:52Z : No. of bitstreams: 1 corabiadell_c_dr_rcla.pdf: 6736961 bytes, checksum: 2fe0d3faffabe9123c2196abfd1b12ec (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A diversidade do gênero Trichoderma no estado de São Paulo (BRASIL) foi analisada sob diferentes aspectos. Foi estabelecida uma coleção de 539 linhagens obtidas a partir de 52 pontos de coleta, distribuídos pelo estado, e uma coleção de referência com 30 linhagens. Durante o período de trabalho avaliou-se a viabilidade das culturas de acordo com os métodos de preservação utilizados Castellani e câmara fria. As avaliações do potencial de biocontrole pelas técnicas de pareamento e difusão de metabólitos inibidores não apresentaram uma correspondência clara com os testes em casa de vegetação. Para as observações microscópicas de culturas de lâminas foi desenvolvida uma técnica que permitiu visualizar mais facilmente o sistema de ramificação dos conidióforos. A avaliação celulolítica dos isolados permitiu identificar linhagens com atividade superior a da linhagem de referência, indicando alto potencial biotecnológico. A análise molecular dos isolados a partir do seqüenciamento da região ITS1-5,8S-ITS2 mostrou a ocorrência de 17 espécies distribuídas no estado, sobretudo nas áreas de mata e capoeira. As espécies mais freqüentes, T. harzianum, T. spirale e T. koningii ocorreram em quase todos os ecossistemas avaliados, enquanto a ocorrência da seção Longibrachiatum foi pouco expressiva. A técnica de RAPD se mostrou eficiente na identificação de linhagens redundantes indicando seu potencial de uso durante procedimentos de triagem. A aplicação de métodos de taxonomia numérica permitiu gerar fenogramas e gráficos multidimensionais coerentes com os dados morfológicos e moleculares em diversos casos. É sugerida a aplicação de métodos topológicos para a melhor descrição do sistema de ramificação e diferenciação de hifas e da teoria de sistemas não-lineares para a compreensão do seu comportamento morfológico in e ex vitro... / The diversity of the genus Trichoderma in São Paulo State (BRAZIL) was analised under different views. A collection of 539 strains was isolated from 52 different areas and a reference collection of 30 strains was established from different brazilian culture collections. The preservation techniques of Castellani and cold storage were evaluated during the course of the study. The biocontrol potential was evaluated through pairing cultures methodology and inhibitory metabolites production. There was no clear evidence of correspondence between the data obtained in vitro and under greenhouse conditions. For microscopic observation a new technique of slide culture was developed in order to visualise the branching system easily. Cellulolytic activitiy assay identified several high-producing cellulase strains comparing to the reference QM9414 indicating a biotechnological potential. The molecular analysis from the sequencing of ITS1-5,8S-ITS2 region indicated the ocurrency of 17 different species all over the state with predominance in forest and 'capoeira' ecossystems. The most frequently species were T.harzianum, T.spirale and T.koningii, ocurring in almost all environmental conditions sampled, while the presence of section Longibrachiatum was inexpressive. The RAPD technique in separate redundants strains was demonstrated indicating its potential for using in screening procedures. Numerical taxonomy methods produced phenograms and multidimensional graphics consistent with molecular and morphological data in some cases. The use of topological methods in describing the branching system and the non-linear system theory in the comprehension of its morphological behaviour in and ex vitro (anamorphic, teleomorphic and synanomorphic states) is suggested. For the first time the total evidence analysis was applied to the genus, showing consistent results with those reported in literature.