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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The aim of this study is to develop a metabolically engineered P. pastoris strain for production of an active extracellular thermostable glucose isomerase (GI) enzyme by using genetic engineering techniques. For this purpose, research program was performed in two sub-programs. In the first sub-program, xylA gene from Thermus thermophilus was amplified and inserted into pPICZ&alpha / -A expression vector. Thereafter, this pPICZ&alpha / -A::xylA vector was cloned into AOX1 locus in P. pastoris genome and expressed under alcohol oxidase promoter which is induced by methanol. After constructing the recombinant P. pastoris strains, the best producing strain was selected according to the specific enzyme activity assay and SDS-PAGE analyses in batch shaker-bioreactor experiments. The selected recombinant P. pastoris clone carrying xylA gene in its genome was named as eP20. Using recombinant P. pastoris eP20 clone, effects of salt and sorbitol concentration on the cell growth and recombinant GI activity were investigated. The data obtained from the experiments showed that the maximum cell and GI activity values were obtained in production medium that contained 30 g L-1 sorbitol, 4.35 g L-1 ammonium sulphate, 0.1 M potassium phosphate buffer (pH 6.0), 14.9 g L-1 MgSO4&bull / 7H2O, 1.17 g L-1 CaSO4 &bull / 2H2O, 1 ml L-1 chloramphenicol and 4.35 ml L-1 PTM1 / where, the maximum biomass and recombinant GI activity were calculated , respectively, as 6.3 g L-1 and 3.21 U L-1. Moreover, the research program related with the effect of initial sorbitol concentration shows that optimum initial sorbitol concentration, CS0 is 50 g L-1 that resulted a cell concentration and recombinant GI activity which are 7.32 g L-1 and 3.6 U L-1, respectively. In the second part of the M.Sc. of the study, a pilot scale bioreactor experiment in a working volume of 1 L was performed in controlled bioreactor system. The variations in the cell growth, recombinant GI activity, AOX activity, total protease activity and organic acid concentrations throughout the fermentation were analyzed whereas the specific growth rates, yield coefficients and specific consumption rates were also calculated. The results showed that a pH and oxygen controlled operation enabled an important increase in recombinant GI activity. In this context, recombinant GI activity was increased as 56.1-fold and resulted in 202.8 U L-1 at t=12 whereas the maximum biomass concentration was obtained as 85.2 g L-1 at t=36. In this study, an active thermostable recombinant GI enzyme was produced extracellularly by a yeast cell, i.e. recombinant P. pastoris, for the first time.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-02Bitstream added on 2014-06-13T19:43:44Z : No. of bitstreams: 1 martin_m_dr_rcla.pdf: 1037055 bytes, checksum: 944c98ccfd999b71ae555ffe401d2e45 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Organismos termofílicos produzem enzimas que, em geral, são mais termoestáveis que aquelas produzidas por mesofílicos. Além disso, essas enzimas geralmente apresentam várias características importantes sob o ponto de vista de aplicação industrial, como estabilidade em ampla faixa de pH e maior tolerância a solventes e outros desnaturantes proteicos. O tipo de processo fermentativo usado também pode influênciar a produção e as propriedades das enzimas obtidas. Foram isoladas várias cepas fúngicas termofílicas e pectinolíticas e entre elas, o fungo termofílico Thermomucor indicae-seudaticae N31 foi o que apresentou maior potencial de produção de poligalacturonase (PG) usando meios a base de resíduos agro-industriais. Em fermentação em estado sólido (FES) usando como meio de cultura uma mistura de farelo de trigo e bagaço de laranja (1:1) a 70% de umidade, o fungo produziu 14 U/mL em 48 horas e em fermentação submersa (FSm) com 1% farelo de trigo e 1% bagaço de laranja como substrato foram obtidas 13 U/mL em 96 horas de fermentação. A PG presente na solução enzimática bruta obtida de FSm foi mais termoestável do que aquela de FSS e mais estável em ampla faixa de pH. As exo-PGs produzidas em FSm e FSS foram purificadas até homegeneidade, com um fator de purificação de 8 e 2,5 vezes e um rendimento de 27,7% e 15,5% para FSm e FSS, respectivamente. A PG da FSm apresentou um massa molar de 38,9 kDa e a de FSS 37,1, kDa. O pH e temperatura ótimos foram de 5,5 e 4,5-5,0 e 55° e 60°C, para enzimas de FSm e FES, repectivamente. Ambas as PGs mostraram perfil de exo-PG, liberando ácido galacturônico por hidrólise de pectina com baixo grau de esterificação (DE). O km foi 590,9 e 661,6 e o Vmax de 4 e 4,9 umol min-1mg-1, para PG de FSm e FES, respectivamente. A termoestabilidade foi comprovada pela análise dos... / Thermophilic organisms produce enzymes that, in general, are more thermostable than the ones produced by mesophilic. Moreover, these enzymes generally exhibit many important characteristics under the perspective of industrial application, such as stability in a wide range of pH and more tolerance to solvents and other protein denaturating substances. The type of fermentative process can also influence the production and properties of the enzymes obtained. Numerous strains of thermophilic and pectinolytic fungi were isolated and among them, the Thermomucor indicae-seudaticae N31 was the one that presented the highest potential for polygalacturonase (PG) production using media containing agro industrial residues. In solid state fermentation (SSF) using a mixture of wheat bran and orange bagasse (1:1) as substrate, at 70% moisture, the fungus produced 14 U/mL in 48 hours and in submerged fermentation (SmF) with 1% wheat bran and 1% orange bagasse as substrate, 13 U/mL were obtained in 96 hours of fermentation. PG in the crude enzymatic extract obtained from SmF was more thermostable than the one produced in SSF and stable in a wider range of pH. The exo-PGs produced in SmF and SSF were purified to homogeneity, with a final purification of 8 and 2.5- fold and overall recovery of 27.7% and 15.5%, respectively. PG from SmF exhibited a molecular mass of 38.9 kDa and PG from SSF 37.1 kDa. Optimum pH and temperature were 5.5 and 4.5-5.0 and 55°C and 60°C for the enzymes from SmF and SSF, respectively. Both PGs exhibited exo-PG profile, releasing galacturonic acid through the hydrolysis of low esterification level pectin. Km was 590.9 and 661.6 and Vmax was 4 and 4.9 umol min-1mg-1, for PG from SmF and SSF, respectively. The thermostability was confirmed by analyzing the enzymes thermodynamic parameters with PG produced in SmF being the most thermostable exhibiting higher values... (Complete abstract click electronic access below)

Orientador: Eleni Gomes / Coorientador: Heloiza Ferreira Alves do Prado / Banca: Luis Henrique Souza Guimarães / Banca: Eleonora Cano Carmona / Banca: Lara Durães Sette / Banca: Valéria Marta Gomes de Lima / Resumo: Organismos termofílicos produzem enzimas que, em geral, são mais termoestáveis que aquelas produzidas por mesofílicos. Além disso, essas enzimas geralmente apresentam várias características importantes sob o ponto de vista de aplicação industrial, como estabilidade em ampla faixa de pH e maior tolerância a solventes e outros desnaturantes proteicos. O tipo de processo fermentativo usado também pode influênciar a produção e as propriedades das enzimas obtidas. Foram isoladas várias cepas fúngicas termofílicas e pectinolíticas e entre elas, o fungo termofílico Thermomucor indicae-seudaticae N31 foi o que apresentou maior potencial de produção de poligalacturonase (PG) usando meios a base de resíduos agro-industriais. Em fermentação em estado sólido (FES) usando como meio de cultura uma mistura de farelo de trigo e bagaço de laranja (1:1) a 70% de umidade, o fungo produziu 14 U/mL em 48 horas e em fermentação submersa (FSm) com 1% farelo de trigo e 1% bagaço de laranja como substrato foram obtidas 13 U/mL em 96 horas de fermentação. A PG presente na solução enzimática bruta obtida de FSm foi mais termoestável do que aquela de FSS e mais estável em ampla faixa de pH. As exo-PGs produzidas em FSm e FSS foram purificadas até homegeneidade, com um fator de purificação de 8 e 2,5 vezes e um rendimento de 27,7% e 15,5% para FSm e FSS, respectivamente. A PG da FSm apresentou um massa molar de 38,9 kDa e a de FSS 37,1, kDa. O pH e temperatura ótimos foram de 5,5 e 4,5-5,0 e 55° e 60°C, para enzimas de FSm e FES, repectivamente. Ambas as PGs mostraram perfil de exo-PG, liberando ácido galacturônico por hidrólise de pectina com baixo grau de esterificação (DE). O km foi 590,9 e 661,6 e o Vmax de 4 e 4,9 umol min-1mg-1, para PG de FSm e FES, respectivamente. A termoestabilidade foi comprovada pela análise dos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Thermophilic organisms produce enzymes that, in general, are more thermostable than the ones produced by mesophilic. Moreover, these enzymes generally exhibit many important characteristics under the perspective of industrial application, such as stability in a wide range of pH and more tolerance to solvents and other protein denaturating substances. The type of fermentative process can also influence the production and properties of the enzymes obtained. Numerous strains of thermophilic and pectinolytic fungi were isolated and among them, the Thermomucor indicae-seudaticae N31 was the one that presented the highest potential for polygalacturonase (PG) production using media containing agro industrial residues. In solid state fermentation (SSF) using a mixture of wheat bran and orange bagasse (1:1) as substrate, at 70% moisture, the fungus produced 14 U/mL in 48 hours and in submerged fermentation (SmF) with 1% wheat bran and 1% orange bagasse as substrate, 13 U/mL were obtained in 96 hours of fermentation. PG in the crude enzymatic extract obtained from SmF was more thermostable than the one produced in SSF and stable in a wider range of pH. The exo-PGs produced in SmF and SSF were purified to homogeneity, with a final purification of 8 and 2.5- fold and overall recovery of 27.7% and 15.5%, respectively. PG from SmF exhibited a molecular mass of 38.9 kDa and PG from SSF 37.1 kDa. Optimum pH and temperature were 5.5 and 4.5-5.0 and 55°C and 60°C for the enzymes from SmF and SSF, respectively. Both PGs exhibited exo-PG profile, releasing galacturonic acid through the hydrolysis of low esterification level pectin. Km was 590.9 and 661.6 and Vmax was 4 and 4.9 umol min-1mg-1, for PG from SmF and SSF, respectively. The thermostability was confirmed by analyzing the enzymes thermodynamic parameters with PG produced in SmF being the most thermostable exhibiting higher values... (Complete abstract click electronic access below) / Doutor

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Ce travail de thèse, en partenariat avec l’entreprise ALSTOM, s’inscrit dans une logique de remplacement de pièces industrielles en alliage métallique par des pièces composites pour l’allègement des structures. L’objectif est de contribuer à l’écriture de règles de dimensionnement permettant au partenaire industriel de certifier des pièces structurales annulaires réalisées en composite à matrice thermoplastique thermostable (TPTS) renforcée par des fibres de carbone pour des applications embarquées sur machine tournante. Il s’agit plus exactement de déterminer la durée de vieen fatigue de ces pièces, en particulier en présence d’endommagement, et lorsque celles -ci sont soumises aux chargements inertiels et thermiques de service.Au cours de ce travail de thèse, une méthodologie a été développée afin de répondre à cette demande. La stratégie a consisté à reproduire,à l’échelle d’éprouvettes de laboratoire, l’état de contrainte multi-axial et l’endommagement auxquels la structure industrielle est soumise, et ce en développant et optimisant un essai de traction sur des éprouvettes annulaires entaillées. Les essais multi axiaux plus classiques mettant en œuvre des sollicitations par pression interne présentent effectivement de nombreux problèmes techniques et sécuritaires lorsqu’ils doivent être mis en œuvre à chaud.Une fois la configuration d’essai sur anneau optimisé par simulation numérique, des essais ont été réalisés afin de confirmer l’apparition des endommagements escomptés à l’aide de techniques de contrôle non-destructif. Les essais cycliques réalisés dans cette configuration ont montré une excellente résistance du matériau en fatigue, en particulier pour un ratio de chargement R de 0.5, proche des conditions de service. Les résultats ont également soulignés le fort potentiel restant de ces structures, même après un grand nombre de cycles de chargement. / The objective of this thesis work, in partnership with ALSTOM Company, is to contribute to the writing of design rules in order to qualify and certify annular structures made of thermostable thermoplastic matrix composite reinforced by carbon fibre. These structures are used in rotating machines for embedded applications.This work proposes an innovative methodology to achieve this goal. It consists in reproducing, at the scale of a laboratory specimen, the multiaxial stress and damage states to which the industrial structure is subjected in-service byoptimizing a tensile test on annular notched specimen. More conventional multiaxial tests, based on internal pressureand tensile loading are particularly unsafe and difficult to be performedwhen implemented at elevated temperature.After the optimisation of the ring configuration by numerical simulation, experimental tests were performed to validatethe appearance of the expected damage under loading. Damage was characterized using non-destructive techniques suchas acoustic emission and infrared thermography. The cyclic tests achieved using this configuration showed high fatiguestrength of this material, in particular for a ratio R of 0.5 (equivalent to thein-service ratio). The results also highlight thegreat remaining strength and rigidity of these structures, even after a large number of cycles.

The aim of this study is to further our understanding of the forces that contribute to protein stability and to investigate how site-directed mutagenesis might be used for increasing protein stability. Eleven proteins ranging from 36 to 370 residues have been studied here. A 36-residue VHP and a 337-residue VlsE were used as model systems for studying the contribution of the hydrophobic effect on protein stability. Mutations were made in both proteins which replaced bulky hydrophobic side chains with smaller ones. All variants were less stable than their wild-type proteins. For VHP, the destabilizing effects of mutations were smaller when compared with similar mutations reported in the literature. For VlsE, a similarity was observed. This different behavior was investigated and reconciled by the difference in hydrophobicity and cavity modeling for both proteins. Therefore, the stabilizing mechanism of the hydrophobic effect appears to be similar for both proteins. Eight proteins were used as model systems for studying the effects of mutating non-proline and non-glycine residues to statistically favored proline and glycine residues in ?-turns. The results suggest that proline mutations generally increase protein stability, provided that the replaced residues are solvent exposed. The glycine mutations, however, only have a stabilizing effect when the wild-type residues have ?, ? angles in the L? region of Ramachandran plot. Nevertheless, this strategy still proves to be a simple and efficient way for increasing protein stability. Finally, using a combination of eight previously identified stabilizing mutations; we successfully designed two RNase Sa variants (7S, 8S) that have both much higher Tms and conformational stabilities than wild-type protein over the entire pH range studied. Further studies of the heat capacity change upon unfolding (?Cps) for both proteins and their variants suggest that residual structure may exist in the denatured state of the 8S variant. An analysis of stability curves for both variants suggests that they achieve their stabilization through different mechanisms, partly attributed to the different role of their denatured states. The 7S variants may have a more rigid denatured state and the 8S variant may have a compact denatured state in comparison with that of wild-type RNase Sa.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-25Bitstream added on 2014-06-13T20:24:21Z : No. of bitstreams: 1 pavezzi_fc_dr_rcla.pdf: 679532 bytes, checksum: a99630bf198f33da999b112631255c08 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A glucoamilase é uma enzima hidrolítica que catalisa a liberação sucessiva de β-D-glicose a partir do amido e oligossacarídeos relacionados. Neste trabalho foram estudadas as glucoamilases de Aspergillus awamori expressas em levedura Saccharomyces cerevisiae. Foram utilizadas duas linhagens alteradas denominadas M1 e M2, e uma linhagem selvagem (WT), utilizada como parâmetros na comparação dos resultados. As enzimas foram produzidas em fermentação submersa, e amidos de diferentes origens vegetais foram utilizados como uma fonte extra de carbono na produção das enzimas. O melhor substrato para a produção da glucoamilase selvagem e da mutante M2 foi o amido de batata com 8,2 e 6,6 U/mL, respectivamente. Para a linhagem M1 foi o amido de mandioca com atividade enzimática de 5,9 U/mL. O amido de milho mostrou ser um substrato menos indicado para a produção destas enzimas. Para a purificação foi preparada uma coluna de afinidade com resina sepharoseTM 6B epóxi ativada ligada a acarbose, onde diferentes concentrações do ligante foram avaliadas. A coluna apresentou boa eficiência no processo de purificação conforme análise por eletroforese SDS-PAGE, com massas moleculares estimada em 100 kDa. A temperatura ótima de atividade das enzimas M1 e M2 foi 65 °C, enquanto que a selvagem teve sua atividade máxima em 60 °C. O pH ótimo de atuação das enzimas foi 4,5. As glucoamilases mutantes apresentaram maior termoestabilidade que a glucoamilase selvagem durante o processo de termoinativação, destacando principalmente a glucoamilase M2. A meia vida a 70 °C foi de 8,1 minutos para a enzima mutante M2, 4,1 minutos para a M1 e 3,0 minutos para a enzima selvagem. A energia de ativação para a desnaturação (Ead) foi de 252,9 e 262,8 KJ mol-1 para as enzimas M1 e M2 respectivamente, e de 234,3 KJ mol-1 para a selvagem. A maior energia dos mutantes indica maior resistência... / Glucoamylase is a hydrolytic enzyme that catalyzes the consecutive liberation of β-D-glucose from starch and related oligosaccharides. In this work glucoamylases from Aspergillus awamori expressed in the yeast Saccharomyces cerevisiae were studied. Two mutant strains, denominated M1 and M2, were used and one wild strain (WS) was used as parameter to compare the results. The enzymes were produced in submerged fermentation and starches from different botanical origins were used as extra carbon source for enzyme production. The best substrate for the production of wild glucoamylase and of mutant M2 was potato starch with 8.2 and 6.6 U/mL, respectively. For strain M1 the best substrate was cassava starch with enzymatic activity of 5.9 U/mL. Corn starch revealed to be a less indicated starch for the production of these enzymes. For purification, an affinity column was prepared with activated SepharoseTM 6B epoxy linked to acarbose, and different concentrations of ligand were evaluated. The column exhibited good efficiency during the purification process according to SDS-PAGE analysis, with molecular masses estimated in 100 kDa. Optimum temperature for activities of M1 and M2 enzymes was 65°C, while the wild one exhibited maximum activity at 60°C. Optimum pH for enzyme action was 4.5. Mutant glucoamylases presented higher thermostability than wild glucoamylase during the thermoinactivation process, with M2 standing out. Half life at 70°C was of 8.1 minutes for mutant enzyme M2, 4.1 minutes for M1 and 3.0 minutes for wild enzyme. Activation energy for denaturation (Ead) was 252.9 and 262.8 KJ mol-1 for enzymes M1 and M2 respectively, and 234.3 KJ mol-1 for the wild one. The higher energy of the mutants indicates higher resistance of the protein structure, since more energy is required for the molecule to enter a transition and unfolding state. Thermodynamic parameter ΔG was higher... (Complete abstract click electronic access below)