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Hu, Gang. "Adsorpton and Activity of Cellulase Enzymes on Various of Cellulose Substrates." NCSU, 2009. http://www.lib.ncsu.edu/theses/available/etd-04222009-234535/.
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The objective of this research is to understand the interfacial behavior of cellulase enzymes and its effect on cellulose hydrolysis. This research began with an in-situ monitoring of cellulose hydrolysis using a piezoelectric based quartz crystal microbalance. The time-course kinetics was modeled using a dose response model. The adsorption indicated by the frequency drop followed a Langmuir model as cellulase enzyme increased. Another important part of this research is the development of a new cellulase activity assay based on the piezoelectric technique. This assay provides an easier and more user friendly method for cellulase enzyme activity measurement. It also helps to clarify an element of the interpretation of frequency drops after the injection of cellulase solutions in the hydrolysis of cellulose film, which has been neglected in previous research. Interfacial adsorption of cellulase protein was also investigated using the depletion method. The effects of substrate properties, primarily the crystallinity, which was characterized using X-ray diffraction, were investigated. The effect of surface area, which was measured using both laser light scattering and BET adsorption, on cellulase adsorption were also investigated. It was found that crystallinity played a more important role in cellulase adsorption than surface areas of cellulosic substrate. In characterization of cellulosic substrates, the water retention value (WRV) was also investigated. The results indicated that lower crystallintiy substrates have higher water retention ability. The cellulase adsorption, as well as desorption, was also studied by using sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The adsorption results followed the same trend as indicated by the depletion methods. The various isozymes demonstrated a uniform adsorption in proportion to their concentrations. Desorption appeared uniform. Higher pH was found to create higher desorption for a particular cellulase from a particular substrates. It was also found that cellulase from Trichoderma reesei had higher affinity to cellulosic substrates used in this work than the one from Aspergillus niger.
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Linder, Markus. "Structure-function relationships in fungal cellulose-binding domains /." Espoo, Finland : VTT, Technical Research Centre of Finland, 1996. http://www.vtt.fi/inf/pdf/publications/1996/P294.pdf.
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Imai, Makiko. "Analysis of interaction between cellulosic biomass and saccharification enzymes." Kyoto University, 2020. http://hdl.handle.net/2433/252998.
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Ubhayasekera, Wimal. "Structural studies of cellulose and chitin active enzymes /." Uppsala : Dept. of Molecular Biology, Swedish University of Agricultural Sciences, 2005. http://epsilon.slu.se/200518.pdf.
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Chakrabarti, Ajoy Chuni Carleton University Dissertation Biology. "One-step conversion of cellulose to fructose using co-immobilized cellulase, B-glucosidase and glucose isomerase." Ottawa, 1988.
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Reichstädter, Marek. "Imobilizace vybraných glykanohydroláz." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2015. http://www.nusl.cz/ntk/nusl-217152.
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The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.
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Kyriacou, Andreas. "Characterization and adsorption of the cellulase components from Trichoderma reesei." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75770.
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The cellulase enzyme system of the fungus Trichoderma reesei Rut C-30 was fractionated by DEAE ion exchange chromatography into four groups according to their substrate specificity. By analytical isoelectric focusing and activity stains it was revealed that fraction EGI is comprised of endoglucanases specific to cellulosic substrates, and that fractions EGII and EGIII are non-specific endoglucanases that hydrolyze cellulose as well as xylan substrates. The major protein fraction CBHI was shown to be a cellobiohydrolase. Turbidimetric measurement phase contrast microscopy and analysis of the products resulting from the hydrolysis of swollen cellulose demonstrated differences between endoglucanases and cellobiohydrolases. The enzyme component CBHII, previously described as a cellobiohydrolases was shown to be an endoglucanase.<br>The adsorption behavior of the four enzyme fractions was examined, with respect to pH, temperature and ionic strength. This was accomplished by using ($ sp3$H) radiolabeled cellulase fractions as tracers. The adsorption of the cellulases occurred within 60 minutes, and was described by a Langmuir type correlation. Increasing the adsorption temperature increased the saturation uptake of the endoglucanases but not of the cellobiohydrolases. Changes in pH and ionic strength affected both the degree and strength of adsorption of all the fractions, likely due to protein structure conformational changes.<br>Direct evidence of exchange between adsorbed and free enzymes was obtained for each component using ($ sp3$H) and ($ sp{14}$C) radiolabeled tracers. In simultaneous adsorption of enzyme pairs, CBHI was shown to predominate adsorption. Endoglucanase EGI was preferentially adsorbed over EGII and EGIII. Sequential adsorption studies have shown that interaction between enzyme components largely determine the degree of their adsorption. Evidence suggested both common and distinct adsorption sites exist, and that their occupation depends on which components are involved.<br>Light microscopy and monitoring of sugar production during cellulose hydrolysis indicated that conditions which limit predominance in adsorption by any one of the cellulase components, enhance synergism and increase degree of hydrolysis.
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Lucena, Guilherme Nunes. "Enzyme modified magnetic nanoparticles : an approach for biomass conversion processes /." Araraquara, 2020. http://hdl.handle.net/11449/192507.
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Orientador: Rodrigo Fernando Costa Marques<br>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)<br>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)<br>Doutor
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McKenzie, Belinda, and s9907915@student rmit edu au. "Heterologous expression of cellulase enzymes in transplastidic Nicotiana tabacum cv. Petit Havana." RMIT University. Applied Sciences, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080805.120923.
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Extensive research into enzyme-induced bio-conversion of lignocellulose to soluble sugars has been conducted and research continues in this area. Several approaches have been taken to attempt to alleviate the economic problems associated with utilisation of lignocellulose in fuel ethanol production. By expressing cellulase genes in planta, it is hoped that the cost of enzyme-mediated hydrolysis of cellulose to its soluble sugar monomers, will be reduced. Some accomplishments have been made in this area using nuclear genetic transformation (Abdeev et al., 2003; Abdeev et al., 2004; Austin-Phillips et al., 1999; Biswas et al., 2006; Dai et al., 2000a,b; Dai et al., 2005; Jin et al., 2003; Kawazu et al., 1999; Sakka et al., 2000; Ziegelhoffer et al., 1999; Ziegelhoffer et al., 2001; Ziegler et al., 2000), but more research is required to bring the levels of cellulase enzyme expression in plants to levels that will make the process economically competitive. Chloroplasts of N. tabacum were selected as a target for transformation for high level expression due to their extremely high rates of transcription and translation. These were transformed with two genes, the e1 gene from A. cellulolyticus, and the cbh1 gene from T. reesei. Further aims included the investigation of the effects of using different promoters, and the novel use of both nuclear and chloroplast-based expression in a single plant, on the level of protein production in the heterologous host. Heterologous expression of the cbh1 gene was not successful. This is thought to be due to toxicity of the protein in a prokaryotic environment. Future studies should focus on trying to avoid this toxicity by targeting of the chloroplast-expressed enzyme to specific tissues, such as the thylakoid membrane, for containment, creating a codon-optimised synthetic gene that better mimics the codon usage of the plant to be used for expression, or placing the expression under a reactive cascade that is only activated upon exposure to an external trigger. Heterologous expression of the full length gene for E1 from A. cellulolyticus was successful. Chloroplast homology vectors under the constitutive promoter Prrn, and the inducible promoter T7, were constructed and these were used to successfully transform N. tabacum cv. Petit Havana chloroplasts. Stable transgenic plants were produced and evaluated by a variety of means, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 3122 ± 466 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 0.35% ± 0.06 of the total soluble protein. Lastly, chloroplast transformation was combined with nuclear transformation to create novel dual-transgenic plants simultaneously expressing E1 from both the nuclear and chloroplast genomes. The combination of these technologies was very successful, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 35706 ± 955 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 4.78% ± 0.13 of the total soluble protein, and provides a new approach for increasing the accumulation levels of plant-produced cellulase enzymes.
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McCabe, Bernadette K., of Western Sydney Macarthur University, and Faculty of Business and Technology. "Production of cellulolytic enzymes using immobilised anaerobic fungi." THESIS_FBT_XXX_Mccabe_B.xml, 1998. http://handle.uws.edu.au:8081/1959.7/83.
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An investigation was made into the isolation and screening of highly cellulolytic anaerobic fungi and their production of cellulolytic enzymes using immobilised rhizomycelia. A total of 46 anaerobic fungi were isolated on cellulosic substrates from ruminant and non-ruminant herbivores. Primary screening of these isolates was performed using dye release from cellulose-azure which qualitatively detected cellulolytic activity. Twelve isolates were chosen on the basis of their maximum solubilisation rates of the labelled cellulose and then subjected to secondary screening which involved the quantification of enzyme activity. The enzyme mixtures were characterised by carboxymethylcellulase, xylanase, B-glucosidase, B-xylosidase and cellobiase assays, measured by the production of either reducing sugars, p-nitrophenol or glucose. All strains produced a number of enzymes that allowed them to hydrolyse straw and highest enzyme activity was measured in static cultures grown on 0.5% straw. A monocentric isolate, Piromyces strain KSX1 from a red kangaroo, and a cattle polycentric isolate, Orpinomyces strain 478P1, were selected for study of cellulolytic enzyme production on the basis of high fibre digestion capability and amenability toward encapsulation. The immobilised polycentric strain proved to be operationally superior to strain KSX1 as strain 478P1 did not produce any viable growth in the culture liquor. Studies into single batch cultures of free cells of strains KSX1 and 478P1 revealed that the maximum specific rate of B-glucosidase production occurred concomitantly with maximum specific growth rate suggesting that the immobilised fungus must grow for continuous enzyme production to occur. Although the physiology of cellulase synthesis in strains KSX1 and 478P1 was found to be growth-associated, immobilisation of the fungus offered the advantage of the repeat-batch use of cells with the accumulation of extracellular enzymes after each batch. Thus, operational gains were the key issues in assessing the potential application of immobilised anaerobic fungi in the production of cellulolytic enzymes. The repeat-batch system was operationally more efficient than the free cell batch cultures because immobilisation removed the need of reculturing the cells for every single batch.<br>Doctor of Philosophy (PhD)
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Oliveira, Simone Lopes do RÃgo de. "Optimization of the production of cellulase Melanoporia sp. submerged fermentation." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13646.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico<br>Cellulases are enzyme complex composed of endoglucanases, exoglucanases and β-glucosidases with several biotechnological applications. However, their production cost is a major obstacle for its industrial application. About 40% of the total cellulase production cost is related to the culture medium used for the microorganism growth. In this context, efficient processes for cellulolytic enzyme production are of technical and economical interest. Thus,
the present study aimed to optimize the production of cellulases by Melanoporia sp. using coconut shell powder as substrate in submerged fermentation. The influence of pH and temperature on the enzyme activity was evaluated by univariate experimental design. Then, the composition of the culture medium was sequentially optimized through Plaket
-Burman followed by Central Composite experimental
designs. The fermentation under optimized conditions was subsequently conducted in bioreactor to evaluate the influen
ce of pH control and aeration on enzyme production. The stability of the enzyme was evaluated for 6 and 8 months at 4 ÂC and - 20 ÂC, respectively. The ability of the
enzyme to hydrolyze coconut shell powder was evaluated at 65 ÂC and 80 ÂC using the crude enzyme extract produced by
Melanoporia sp. The enzyme activity was determined by the
quantification of reducing sugars using DNS method at pH 5.5 and 80 ÂC (optimum conditions).
The composition of the culture medium which provided the highest enzyme yield was: 5 g/L of
coconut shell; 15 g/L lactose; 3% tween 80; 1 g/L of KH2PO4
and 0.05 g/L FeSO4; pH 6.5 at 30ÂC for 72 hours. For batch enzyme production, the cult ure medium using non-delignified
substrate, with pH controlled at 6.5, without aeration
resulted in an increase of 90% in enzyme activity compared to the fermentation in a rotatory shaker. Under these conditions, the maximal enzyme production was obtained after 24 hours of fermentation. The crude enzyme extract
produced by Melanoporia sp. was able to hydrolyze cellulose (coconut shell powder) efficiently, presenting industrial potential for the degradation of lignocellulosic residues. Unlike most of the cellulases produced by Trichoderma
species, the strain reported as one of the best producers, the microorganism was capable of producing cellulases efficiently without the need of substrate
pretreatment. Another feature of this enzyme complex is its high stability in the crude broth at-20ÂC e 4 ÂC<br>Celulases sÃo um complexo enzimÃtico constituÃdo por endoglucanases, exoglucanases e β-glicosidases com diversas aplicaÃÃes biotecnolÃgicas. No entanto, o elevado custo de produÃÃo dessas enzimas à o principal obstÃculo para sua aplicaÃÃo industrial. Estima-se que cerca de 40% do custo total de produÃÃo de celulases esteja relacionado ao meio de cultura utilizado para o crescimento do micro-organismo. Nesse contexto, à de fundamental importÃncia o desenvolvimento de processos para a produÃÃo de enzimas do complexo celulolÃtico que se mostrem tÃcnico e economicamente viÃveis. Diante do exposto, o presente estudo teve como objetivo avaliar a produÃÃo de celulases produzidas por Melanoporia sp. utilizando o pà da casca de coco como substrato em fermentaÃÃo submersa. A influÃncia dos parÃmetros pH e temperatura na determinaÃÃo da atividade da enzima foi avaliada atravÃs de planejamento experimental univariado. Em seguida, a composiÃÃo do meio de cultura foi otimizada atravÃs dos planejamentos experimentais Plaket-Burman e Composto Central. A fermentaÃÃo em condiÃÃes otimizadas foi posteriormente conduzida em fermentador para avaliar a influÃncia do controle de pH e oxigÃnio na produÃÃo da enzima. A estabilidade da enzima foi avaliada por 6 e 8 meses nas temperaturas de 4 ÂC e -20 ÂC, respectivamente. A capacidade das enzimas em hidrolisar o pà da casca do coco foi avaliada nas temperaturas de 65 ÂC e 80 ÂC utilizando o extrato enzimÃtico bruto produzido por Melanoporia sp. A atividade da enzima foi determinada atravÃs da quantificaÃÃo de aÃÃcares redutores pelo mÃtodo de DNS. O pH e a temperatura de determinaÃÃo da atividade enzimÃtica foram pH 5,5 e 80 ÂC, respectivamente. A composiÃÃo do meio de cultura que proporcionou o maior rendimento de produÃÃo da enzima foi: 5 g/L de casca de coco; 15 g/L de lactose; 3% de tween 80; 1 g/L de KH2PO4 e 0,05 g/L de FeSO4; pH 6,5 a 30 ÂC em 72 horas. Para a produÃÃo da enzima em fermentador, o meio de cultura utilizando substrato nÃo deslignificado, com controle do pH em 6,5, sem aeraÃÃo proporcionou um aumento de 90% na atividade da enzima, comparado à fermentaÃÃo em shaker. Nessas condiÃÃes, a mÃxima produÃÃo da enzima foi obtida apÃs 24 horas de fermentaÃÃo. O extrato enzimÃtico bruto produzido por Melanoporia sp. exibiu capacidade de hidrolisar celulose presente na casca de coco com eficiÃncia, apresentando potencial industrial para a degradaÃÃo de resÃduos lignocelulÃsicos. Diferentemente da maior parte das celulases produzidas por espÃcies de Trichoderma, micro-organismo reportado como bom produtor de enzimas celulolÃticas, o micro-organismo utilizado neste trabalho à capaz de produzir celulases de forma eficiente, sem necessidade de prÃ-tratamento do substrato. Outra caracterÃstica diferencial desta enzima à sua elevada estabilidade nas temperaturas de -20 ÂC e 4 ÂC no caldo bruto.
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Han, Bin. "Regulation of the synthesis of extracellular protease and cellulase enzymes in Xanthomonas campestris." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316748.
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BOISSET, CLAIRE. "Modification de materiaux cellulosiques par des enzymes cellulolytiques." Université Joseph Fourier (Grenoble), 1996. http://www.theses.fr/1996GRE10225.
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La cellulose est le biopolymere le plus abondant sur terre, et l'etude de sa degradation presente une importance ecologique et industrielle croissante. Trois aspects fonctionnels et structuraux des cellulases ont ete abordes au cours de ce travail: l'adsorption, les interactions avec la cellulose cristalline et l'organisation des differents domaines constitutifs des cellulases. Les observations faites en microscopie electronique sur la morphologie de la degradation de fibres de coton, ont permis de mieux cerner le mode d'action d'une endoglucanase, eg v de humicola insolens, sur un substrat cellulosique industriel. Grace a un immunomarquage a l'or colloidal, nous avons pu visualiser les sites de fixation de eg v a la surface des fibres de coton. L'etude de la degradation enzymatique de microcristaux de cellulose de valonia natifs et modifies par trois cellulases de humicola insolens, met en evidence que l'ultrastructure du substrat est le facteur determinant de la reactivite enzymatique. Nos travaux demontrent qu'une diminution du degre de cristallinite et une augmentation de la surface accessible entraine un accroissement important de la reactivite du substrat. Les etudes en diffusion de la lumiere ont fourni des donnees precises sur les coefficients de diffusion de trois cellulases de humicola insolens ainsi qu'une estimation de la taille hydrodynamique de ces enzymes. De plus, cette technique represente un outil interessant pour analyser les differents variants produits lors d'une approche d'ingenierie des proteines. Elle permet, en effet, de suivre le comportement en solution d'un type de proteine en fonction de differents parametres tels que la temperature, le ph et la concentration en proteines
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Yesuf, Jemil N. "EVALUATION OF CELLULOLYTIC ENZYMES FROM A NEWLY ISOLATED BREVIBACILLUS SP. JXL; AND OPTIMIZATION OF COSLIF PRETREATMENT VARIABLES OF SWEET SORGHUM BAGASSE USING A RESPONSE SURFACE METHOD." OpenSIUC, 2012. https://opensiuc.lib.siu.edu/dissertations/515.
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The first part of the dissertation presented a potentially novel aerobic, thermophilic, and cellulolytic bacterium identified as Brevibacillus sp. Strain JXL which was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between the effects caused by these two simple sugars. After the end of the 24 hour period, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also observed when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and optimal pH range of 6-8. These cellulases also had high thermotolerance as demonstrated by retaining more than 50% activity after 1 h at 100°C. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose. In the second part of the dissertation, the effect of Cellulose- and Organic-Solvent based Lignocellulose Fractionation (COSLIF) (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng.2007, 97 (2), 214−223) pretreatment conditions on sweet sorghum bagasse (SSB) feedstock was studied using Response Surface Methodology (RSM). Batch experimental matrix was set up based on response surface method's central composite design in two factors to determine the effects of reaction time and temperature on the yield of simple sugars after a sequential pretreatment-enzyme hydrolysis process. Accordingly, changes in delignification, total reducing sugar (TRS) yield, glucan retention, digestibility and overall sugar yields resulting from various combinations of reaction times and temperatures were determined. The results suggested that both pretreatment temperature and reaction time were significant factors, although temperature was more so than reaction time. COSLIF pretreatment conditions of 50°C and 40 min were found to be the optimum pretreatment conditions for the saccharification of SSB. At the end of pretreatment and enzymatic hydrolysis, maximum values of 51.4% delignification, 85% overall glucose yield, and 44% overall xylose yield at an ACCELERASE®1500 loading of 0.25 mL/g sweet sorghum bagasse were achieved. Optimum ACCELERASE®1500 dosage of 0.1 mL/g of sweet sorghum bagasse was identified which resulted in an overall glucose yield of 82.2%±1.05. An effort has also been made to prescribe predictive models which represented the correlation between independent variables (reaction time and temperature), and dependent variables (delignification, and overall glucose yield) using RSM. The significance of the correlations and adequacy of these models were statistically tested for the selected objective functions. The outcomes suggested very competent and statistically adequate regression models which provided quantitative information both for delignification and overall glucose yield for the batch experiments studied.
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Nutt, Anu. "Hydrolytic and Oxidative Mechanisms Involved in Cellulose Degradation." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6888.
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Fleuri, Luciana Francisco. "Produção de [beta]-1,3 glucanases, proteases liticas e quitinases por microrganismos e aplicação na lise de leveduras." [s.n.], 2003. http://repositorio.unicamp.br/jspui/handle/REPOSIP/254349.
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Orientador : Helia Harumi Sato<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-08-03T11:29:00Z (GMT). No. of bitstreams: 1
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Previous issue date: 2003<br>Resumo: O presente trabalho visou o estudo da produção de b-1,3 glucanases, proteases líticas e quitinases pelas linhagens B1, B22, B26, FXX, Oerskovia sp. n04 e Celulomonas cartae nO191 em meios de cultura contendo diferentes indutores e aplicação na lise de leveduras. Entre as bactérias testadas as linhagens B26 e Cellulomonas cartae nº191 apresentaram maior produção de protease em meio de cultura TI composto de 8% de levedura seca instantânea; as linhagens Bl e C. cartae nº191 apresentaram maior produção de b-1,3 glucanase em meio de cultura m contendo 1% de parede celular de levedura extraída mecanicamente em Dyno- Mill; e a linhagem C. cartae nº191 apresentou maior produção de quitinase em meio de cultivo N contendo 1,5% de quitina neutralizada. Os sobrenadantes dos meios de cultura obtidos da fermentação das linhagens B1 e C. cartae nº191 cultivadas em meio de cultivo m mostraram maior atividade de lise de levedura. No estudo do fracionamento das enzimas líticas, a saturação do sobrenadante do meio de cultura com 60% de sulfato de amônio, foi adequada para a precipitação e obtenção de protease, b-1,3 glucanase e quitinase. As preparações de b-1,3 glucanase das linhagens B1 e C. cartae n °191 obtidas do sobrenadante do meio de cultura através de fracionamento com sulfato de amônio, apresentaram atividade de lise das leveduras Kluyveromyces lodderi, Saccharomyces cerevisiae (levedura de panificação Fleischmann), Saccharomyces cerevisiae (levedura de panificação Itaiquara) e sobre as linhagens "killer" Saccharomyces cerevisiae KL-88, Saccharomyces diastaticus NCYC 713, Saccharomyces cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587 e Hansenula mrakii NCYC 500. As linhagens K marxianus NCYC 587 e H. mrakii NCYC 500 mostraram-se mais sensíveis à ação das b-1,3 glucanases e as linhagens de levedura ltaiquara e C. glabrata NCYC 388 mostraram-se mais resistentes à ação das b-1,3 glucanases, quando comparada com a susceptibilidade das células da linhagem de S. cerevisiae KL-88 à preparação enzimática. A adição da preparação de quitinase, obtida do sobrenadante do meio de cultura através de ftacionamento com sulfato de amônio, da linhagem C. cartae nº191, em alguns casos aumentou a susceptibilidade das leveduras à lise celular. O pré-tratamento das suspensões das leveduras com as preparações de protease obtidas dos sobrenadantes dos meios de culturas através de fracionamento com sulfato de amônio, da linhagem C. cartae nº191, diminuiu a lise da leveduras principalmente quando utilizada em altas concentrações. A maior produção de b-1,3 glucanase da linhagem C. cartae nº191 em meio de cultura m ocorreu a 35°C após 48 horas de fermentação a 200 rpm, entretanto, atividade de b-1,3 glucanase muito próxima foi obtida na fermentação do microrganismo a 30°C durante 24 horas. A maior produção de protease da linhagem C. cartae nO191 em meio de cultura II ocorreu a 35°C após 30 horas de fermentação a 150 rpm, enquanto que a maior produção de quitinase da linhagem C. cartae nO191 em meio de cultura N ocorreu a 35°C após 72 horas de fermentação a 150 rpm. No estudo das superfícies de respostas e das curvas de contorno referentes ao planejamento fatorial completo, foi obtido maior atividade de lise da levedura S. cerevisiae KL-88 utilizando-se a preparação enzimática de _-1,3 glucanase em pH 6,5 e a 35°C. As células de leveduras obtidas após 10 horas de fermentação em frascos sem agitação mostraram-se mais susceptíveis à lise pela b-1,3 glucanase de C. cartae nº191. Foi obtido maior lise da levedura S. cerevisiae KL-88 quando a suspensão de células da levedura foi submetida ao tratamento com b-1,3 glucanase e cisteína 1mM. A enzima invertas e intracelular ou ligada à célula de S. cerevisiae KL-88 e K marxianus NCYC 587 foi extraída após tratamento da suspensão celular de levedura com b-1,3 glucanase da linhagem C. cartae nº191. O tratamento prévio das células de S. cerevisiae KL-88 e K marxianus NCYC 587 com a enzima b-1,3 glucanase da linhagem C. cartae nº191, aumentou a susceptibilidade das células de levedura à lise com ultra-som<br>Abstract: The aim of this work was the study of b-1,3 glucanases, proteases and chitinases production by B1, B22, B26, FXX, Oerskovia sp. nO4 and Cellulomonas cartae nº191 strains in culture media containing differents inductors as well as their application on yeast cell lysis.
The strains B26 and Cellulomonas cartae nO191 showed highest protease production using the culture medium II containing 8% of instant yeast. The strains Bl and C. cartae nº191 showed highest b-1,3 glucanase production using the culture medium III containing 1% of yeast cell wall obtained by Dyno-Mill. The strain C. cartae nº191 showed highest chitinase production using the culture medium IV containing 1.5% of chitin neutralized. The culture medium III supernatants obtained by fermentation of strains B1 and C. cartae nº191 demonstrated the biggest yeast cell lysis activity. The enzymes precipitation studies revealed that 60% ammonium sulphate was the best concentration for protease, b-1,3 glucanase and chitinase separation. The b-1,3 glucanase extract obtained by Bl and C. cartae nº191 strains demonstrated lysis activity against Kluyveromyces lodderi, Saccharomyces cerevisiae (yeast Fleischmann), Saccharomyces cerevisiae (yeast Itaiquara), Saccharomyces cerevisiae KL-88, Saccharomyces diastaticus NCYC 713, Saccharomyces cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587 and Hansenula mrakii NCYC 500. K. marxianus NCYC 587 and H mrakii NCYC 500 were more sensitive to b-1,3 glucanases action, whereas Itaiquara yeast and C. glabrata NCYC 388 were more resistant when compared with S. cerevisiae KL-88 susceptibility. The chitinase extract obtained by C. cartae nO191, in some cases increased the susceptibility of yeast to cellular lysis. The previous treatment of the yeast suspensions with protease from C. cartae nº191, decreased the yeasts cell lysis, mainly when used at high concentrations. The maximum b-1,3 glucanase production by C. cartae nº191, using culture medium III, occurred after 48 hours of fermentation at 35°C and 200 rpm. However, when the fermentation was perform after 24 hours of fermentation at 30ºC and 200 rpm, the b-1,3 glucanase activity obtained was almost the same. The maximum protease production by C. cartae nº191, using culture medium II, occurred after 30 hours of fermentation at 35ºC and 150 rpm, while the highest chitinase production by C. cartae nº191, using culture medium IV, occurred after 72 hours of fermentation at 35°C and 150 rpm. The experimental design study showed that the best conditions to S. cerevisiae KL-88 lysis by b-1,3 glucanase extract were pH 6,5 and 35°C. This study also demonstrated that the yeast cells were more susceptible to lysis after 10 hours cultivation in flasks without agitation. Lysis activity was increased when S. cerevisiae KL-88 cell suspension was treated b-1,3 glucanase and cystein 1mM. The enzyme invertase of S. cerevisiae KL-88 and K marxianus NCYC 587 was extracted after treatment of cell suspension with b-1,3 glucanase obtained from C. cartae nº191. The previous treatment of S. cerevisiae KL-88 and K marxianus NCYC 587 with b-1,3 glucanase, increased the susceptibility to lysis when ultrasonic treatment was used.<br>Mestrado<br>Mestre em Ciência de Alimentos
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Zampieri, Denise. "Expressão do complexo celulolítico em Penicillium echinulatum." reponame:Repositório Institucional da UCS, 2011. https://repositorio.ucs.br/handle/11338/1005.
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O Penicillium echinulatwn linhagem 9A02Sl é um fungo filamentoso que apresenta um sistema celulolítico com potencial para aplicaqão em processos de degradação de materiais lignocelulósicos para produção de etanol. O crescente interesse nesse combustível e a abundância de materiais lignocelulósicos que podem ser usados como matéria-prima fez aumentar o interesse no estudo de celulases. Neste estudo, a linhagem 9A02Sl de Penicillium echinulatum foi cultivada em cultivos submersos em frascos mantidos sob agitação recíproca, com variações quanto às fontes de carbono. Além de crescimento, foram avaliadas as produções de celulases, ~-glicosidases e xilanases e a expressão das enzimas através ale zimogramas em géis de poliacrilamida para detemlinação da massa molecular. Observou-se que o crescimento micelial provocou a redução do pH do meio de cultivo, e que não está relacionado a produção de enzimas. A celulose apresentou-se como indutora para todas as enzimas analisadas. A carboximetilcelulose mostrou-se uma eficiente fonte de carbono para a produção de atividade sobre papel filtro, endoglicanases e xilanases, apesar do baixo crescimento micelial. Celobiose, glicerol e glicose estimularam a produção de ~glicosidases. Uma banda de atividade endoglicanêlsica de 74 kDa foi detectada nos zimogramas de todos os caldos enzimáticas obtidos na presença d1e diferentes fontes de carbono, sugerindo esta seja uma enzima constitutiva. A expressão da ~-glicosidase oconeu ao fmal do cultivo (5° e 6° dias), sendo que em todos os cultivos avaliados houve a expressão de uma banda de 220 kDa, indicando tratar-se de uma enzima constitutiva. A expressão de outras bandas com diferentes massas moleculares sugerem que diferentes genes, fotmats multiméricas ou modificações pós-traducionais estão envolvidos no perfil destas enzimas em Peni'cillium echinulatum.<br>Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-09-22T17:54:12Z
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Dissertacao Denise Zampieri.pdf: 18671785 bytes, checksum: 0c85c4e913bd6377f21bfe2a1fe9e8ff (MD5)<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq<br>The st:rain of Penicillium echinulatum 9A02Sl is a filamentous fungus that presents a cellulolytic system with potential application in processes of degradation of lignocellulosic materiais for ethanol production. The growing interest in fhel and the abundance of lignocellulosic materiais that can be used as raw material has inc:reased the interest in cellulases. In this study, the strain P echinulatum 9A02Sl was grown in submerged cultivation in agitated flasks in presence of different carbon sources. In addition to growth, it was evaluated the production of cellula.ses, Pglucosidases and xylanases, and enzyme expres:sion in activity polyacrylamide gels in order to detemlinate the molecular ma.ss. The mycelial growth decreased pH o f the medium and this fact was not related to enzyme production. The cellulose was an inducer for all the enzymes ana.lyzed. The carboximetilcellulose was found to be an effi[cient carbon source for production o f filter paper a.ctivity, endogluca.nases and xylanases, despite the low mycelial growth. Cellobiose, glycerol and glucose stimulated the production of P-glucosidases. An endoglucanase band of 74 kDa was detected in zymograms o f all enzyme broths obtained in the presence o f different carbon sources, suggesting it is a constitutive enzyme. The expression of P-glucosidase occuned at the end of cultivation (5 and 6 days), and in all medium that was evaluated was observed a 250 kDa band, indica.ting that this is a. constitutive enzyme. The expression of other bands with different molecular mass suggest that different genes, multimeric fonns or post-translational modifications are involved in the expression ofthese enzymes in P echinulatum.
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Rouau, Xavier. "Production, purification et caractérisation de polyosidases du basidiomycète Dichomitus squalens." Paris 7, 1985. http://www.theses.fr/1985PA07F102.
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La production de cellulases de huit souches de basidiomycètes de pourriture blanche a été étudiée et comparée à celle des meilleurs organismes producteurs connus. Dichomitus squalens a été choisi pour la production d'endoglucannases. Celle-ci a été améliorée par l'addition de protéose peptone au milieu de culture. L'évolution du pH, de la concentration en protéines, et des activités enzymatiques a été suivie sur des cultures en fioles. Un mélange enzymatique initial a été préparé par précipitation au sulfate d'ammonium (80% de saturation) d'un surnageant de culture sur cellulose microcristalline de D. Squalens en fermenteur de 20 litres. Ce mélange a été fractionné par chromatofocalisation : 3 endoglucannases, 3 enzymes actives sur para-nitrophényl- β -D-cellobioside (pNPC) et 3 xylannases ont été identifiées, entre autres. Les 3 endoglucannases et 2 des enzymes actives sur pNPC ont été purifiées à homogénéité par chromatographie d'intéractions hydrophobes, chromatographie d'échange d'ions et perméation sur gel. Les propriétés physico-chimiques des enzymes purifiées ont été déterminées ainsi que leur mode d'action sur différents substrats. Les endoglucannases dégradent principalement la cellulose mais leurs spécificités diffèrent. Les enzymes actives sur pNPC ont été identifiées finalement à des xylannases montrant aussi une activité osidase.
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Liao, Hehuan. "High-Yield Cellulosic Hydrogen Production by Cell-Free Synthetic Cascade Enzymes: Minimal Bacterial Cellulase Cocktail and Thermostable Polyphosphate Glucokinase." Thesis, Virginia Tech, 2011. http://hdl.handle.net/10919/76997.
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Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes. Two of the biggest challenges for its commercialization are: effective release of fermentable sugars from pretreated biomass, and preparations of thermostable enzymes with low-cost.
The hydrolysis performance of 21 reconstituted bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase, family 9 Clostridium phytofermentans processive endoglucanase, and family 48 Clostridium phytofermentans cellobiohydrolase was investigated on microcrystalline cellulose (Avicel) and regenerated amorphous cellulose (RAC). The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was most important for high cellulose digestibility regardless of substrate type. These results suggested that the hydrolysis performance of reconstituted cellulase cocktail strongly depended on experimental conditions.
Thermobifida fusca YX was hypothesized to have a thermophilic polyphosphate glucokinase. T. fusca YX ORF Tfu_1811 encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was over expressed in Escherichia coli. By a simple one-step immobilization, the half-life time increased to 2 h, at 50 °C. These results suggest that this enzyme was the most thermostable PPGK reported.
My studies would provide important information for the on-going project: high-yield hydrogen production from cellulose by cell-free synthetic enzymatic pathway.<br>Master of Science
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Reye, John Timothy. "Enhanced enzymatic hydrolysis of cellulosic fibers by cationic polyelectrolytes." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/39633.
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A new method for enhancing rates of enzymatic hydrolysis for cellulosic fiber is presented. By adding a cationic polyelectrolyte to a cellulase/cellulose hydrolytic system, the polyelectrolyte binds to the cellulase and fiber forming flocs. The cellulase is bound by a patching mechanism. By using this technique, the rate of enzymatic hydrolysis can be enhanced. This thesis covered observations made about the cellulase/cationic polyelectrolyte/fiber interactions. A mechanism was proposed based on the experimental results.
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Humbert-Goffard, Anne. "Recherches sur les phénomènes enzymatiques intervenant lors de l'élevage des vins." Bordeaux 2, 2003. http://www.theses.fr/2003BOR21076.
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De nombreux phénomènes enzymatiques interviennent au cours de l'élevage des vins. Des expériences d'élevage en milieu modèle ont montré une libération de mannoprotéines et peptides pariétaux au cours de l'autolyse. La libération de ces derniers est favorisée par la présence de préparations enzymatiques industrielles. Les activités enzymatiques impliquées dans ce phénomène sont des protéases acides. Les peptides de bas poids moléculaire (0,5-3 kDa) libérés sont sapides et présents à des concentrations supérieures au seuil de perception. Neuf expérimentations d'élevage sur lies en vin rouge ont été réalisées au cours de ces travaux et quel que soit le protocole employé (quantité de lies, d'enzymes, moment de leur addition), l'analyse de la couleur, des polyphénols et la dégustation n'ont révélé aucune différence. L'absence d'effet des enzymes exogènes est en partie due à une inhibition totale des activités β-1,3-glucanase et protéases en moins de 30 jours. Une expérimentation en vin blanc réalisée en présence d'une quantité d'enzyme 10 fois supérieure à celle généralement utilisée a permis de mettre en évidence une libération plus importante de mannoprotéines et de peptides. Dans ces conditions particulières, l'addition de préparations à forte activité protéolytique contribue à modifier les caractéristiques organoleptiques de ce vin blanc. D'autre part, l'effet de préparations enzymatiques exogènes et d'activités purifiées issus d'A. Niger ou de T. Harzianum sur la clarification et la filtrabilité de vins blanc et rouge a été mesuré. Le rôle des pectinases et éventuellement d'autres activités secondaires issues d'A. Niger comme celui de la β-1,3-glucanase de T. Harzianum est prépondérant dans l'amélioration de ces phénomènes. La protéase ou d'autres activités secondaires issues de T. Harzianum sont également impliquées dans l'amélioration de la filtrabilité mais sans effet sur la sédimentation<br>Many enzymatic phenomena intervene during wine ageing. The autolysis experiments in synthetic medium showed the release of mannoproteins and parietal peptides. The release of the latter is enhanced by the presence of industrial enzymatic preparations. The enzymatic activities implied in this phenomenon are acid proteases. Peptides of low molecular weights (0,5-3 kDa) that are released have taste and are present at higher concentrations than their sensory treshold. Nine experiments of red wine aged on lees red were carried out during this work and whatever the protocol employed (quantity of lees or enzymes, positioning of their addition), the analysis of the color, polyphenols and tasting did not reveal any difference. The absence of effect of the exogenic enzymes is partly due to a total inhibition of the β-1,3-glucanase activities and proteases in less than 30 days. A white wine experimentation realized in the presence of a quantity of enzyme 10 times higher thanthat generally used made it possible to highlight a more important release of mannoproteins and peptides. Under these partcular conditions, the addition of preparations with strong proteolytic activity contributes to modify the organoleptic characteristics of this white wine. In addition, the effect of exogenic enzymatic preparations and purified activities from A. Niger or T. Harzianum on the clarification and the filterability of white and red wines was measured. The role of the pectinases and possibly of other secondary activities resulting from A. Niger or β -1,3-glucanase of T. Harzianum are dominating in the improvement of these phenomena. The protease of other secondary activities of T. Harzanium are also implied in the improvement of filterability but have no effects on sedimentation
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Reis, Laísa dos. "Estratégias para incremento das atividades de celulases e xilanases por penicillium echinulatum SIM29 em cultivo submerso." reponame:Repositório Institucional da UCS, 2017. https://repositorio.ucs.br/handle/11338/2926.
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Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-07-14T14:43:43Z
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Previous issue date: 2017-07-14<br>Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, FAPERGS.
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Thoresen, Mariska. "An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1017915.
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Väljamäe, Priit. "The kinetics of cellulose enzymatic hydrolysis : Implications of the synergism between enzymes." Doctoral thesis, Uppsala University, Department of Biochemistry, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3120.
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<p>The hydrolysis kinetics of bacterial cellulose and its derivatives by <i>Trichoderma reesei</i> cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases. </p><p>A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios. </p><p>The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.</p>
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Bertrand, Frédérique. "Extraction de l'huile de coco assistée par des enzymes." Aix-Marseille 3, 1994. http://www.theses.fr/1994AIX30058.
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Ce travail traite de l'extraction d'huile de noix de coco sur l'amande fraiche et de son optimisation. L'etude menee se deroule en 4 phases. La premiere de celles-ci s'interesse a la structure de l'albumen de noix de coco. L'observation microscopique met en evidence sa relative homogeneite et permet de visualiser l'huile sous forme de gouttelettes libres a l'interieur du cytoplasme. L'extraction chimique des composes de l'endosperme permet de verifier la nature cellulosique et hemicellulosique des constituants de la paroi. La seconde partie, consacree a l'enzymologie permet de montrer que la noix de coco peut realiser l'hydrolyse de ses propres parois grace a la presence de cellulases et beta-mannanases. La troisieme phase correspond a un rapide inventaire des contaminants bacteriens naturels de la noix de coco et leur action sur celle-ci. L'analyse des divers resultats obtenus permet donc de mettre au point un procede d'extraction conduisant a ameliorer fortement le rendement en huile sans en alterer la qualite. Des quantites interessantes d'huile vierge sont ainsi obtenues en utilisant des noix de coco fraiches ce qui supprime les problemes epineux de raffinage d'huile et de sechage de copra, source de perte d'huile. Ce procede simple, peu onereux, s'avere donc utilisable tant au niveau villageois qu'industriel
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Bey, Mathieu. "Etude d’une CDH et de glycosyl hydrolases de la famille 61 : Implication dans les processus de dégradation des lignocelluloses." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4719.
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En réponse aux préoccupations environnementales, les procédés industriels comme la production de bioéthanol de deuxième génération sont apparus. Basés sur la conversion enzymatique de la cellulose, ces processus font face à un problème majeur, la réticence de la biomasse lignocellulosique à l'hydrolyse. Afin de résoudre ce problème et celui lié aux coûts d'utilisation de cocktails de cellulases, les recherches se sont axées sur diverses méthodes permettant d'augmenter l'hydrolyse de la cellulose. Les champignons filamenteux sont connus pour être des dégradeurs naturels du bois et, par conséquent, sont utilisés dans de nombreuses applications biotechnologiques. Récemment, quelques études ont révélé l'importance d'enzymes fongiques telles que la CDH et les GH61 dans la dégradation oxydative de la lignocellulose. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose. L'utilisation de ces enzymes oxydatives offre de réelles voies d'amélioration de la production de bioéthanol et de compréhension de la dégradation in vivo des lignocelluloses par les champignons<br>In response to environmental concerns, industrial processes such as second generation bioethanol production have emerged. Based on enzymatic cellulose conversion, these processes are confronted with a major problem, the recalcitrance of lignocellulosic biomass. To solve the problem caused by substrate recalcitrance and high cost of cellulase cocktails, research has focused on various methods to enhance cellulose hydrolysis. Fungi are known to be natural degraders of wood and consequently are used in derived biotechnological applications. Recently, several studies have revealed the importance of fungal enzymes such as GH61 and CDH in the oxidative degradation of lignocellulose. During the work done on this thesis, we demonstrated implication of these oxidative enzymes in lignocellulose deconstruction to enhance hydrolysis performed by more classical cellulases. Utilization of oxidative enzymes offers a suitable way for bioethanol processing enhancement and comprehension of the in vivo lignocellulosic degradation by fungi
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Ritter, Carla Eliana Todero. "Efeito da adsorção e filtração na produção de celulases e xilanases." reponame:Repositório Institucional da UCS, 2015. https://repositorio.ucs.br/handle/11338/1020.
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Verbeke, Jonathan. "Vers l'optimisation du cocktail cellulolytique de trichoderma reesei par les protéines apparentées aux expansines." Aix-Marseille 1, 2009. http://theses.univ-amu.fr.lama.univ-amu.fr/2009AIX11064.pdf.
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La production industrielle de bioéthanol à partir de biomasse lignocellulosique nécessite l'amélioration de l'efficacité hydrolytique du cocktail enzymatique de Trichoderma reesei. Ce champignon filamenteux présente l'intérêt de sécréter des enzymes cellulolytiques en grande quantité mais l'analyse de son génome montre que la diversité de gènes codant ces enzymes est restreinte. C'est pourquoi, dans ce travail, une complémentation de ce cocktail par des enzymes auxiliaires a été envisagée. Récemment, la présence de la swollénine, une protéine apparentée aux expansines végétales, a été mise en évidence chez T. Reesei. Sa capacité à écarter les fibres de cellulose et l'induction de son gène parallèle à ceux des cellulases laisse penser que cette protéine peut avoir un rôle auxiliaire pendant la cellulolyse. L'étude in silico de séquences comportant des similarités avec les expansines chez les champignons a montré l'existence de plusieurs familles dont une est absente chez T. Reesei. Un représentant de cette famille, la protéine CELA d'Aspergillus fumigatus ainsi qu'une swollénine de cette espèce, SWOAfu, ont donc été choisies pour une expression hétérologue dans T. Reesei. De plus, des constructions chimériques destinées à rapprocher physiquement SWOAfu de la cellobiohydrolase CBH1 de T. Reesei ont été réalisées. Cette protéine de fusion, qui devrait permettre d'augmenter leur synergie a également été exprimée dans T. Reesei. Enfin, pour étudier une implication éventuelle des protéines Endoglucanase-45/Expansin-Like (EEL) de T. Reesei dans l'hydrolyse de la lignocellulose, des études transcriptionnelles ont été réalisées dans des conditions d’induction de cellulases. Une expression constitutive d'une d'entre elles, semblable à celle de l'endoglucanase Cel5b a pu être montrée. Après analyse de leur structure protéique et de leurs promoteurs, les fonctions potentielles des protéines EEL sont discutées<br>The industrial production of bioethanol from lignocellulosic biomass requires the increase of the hydrolytic efficiency of the enzymatic pool produced by Trichoderma reesei. This fungus is able to secrete large amounts of cellulolytic enzymes, but its genome shows a low diversity of genes encoding these enzymes. Therefore, in this work, a complementation of this cocktail with auxiliary proteins was envisaged. Recently, the presence of swollenin, a protein related to plant expansins, was brought to light in T. Reesei. Its capacity to loosen cellulose fibers and the induction of its gene parallel to cellulase genes suggests that this protein could have an auxiliary role in cellulose hydrolysis. A database search of sequences presenting similarities with plant expansins in fungi showed that different families exist, one of which is absent in T. Reesei. CELA from Aspergillus fumigatus, a protein belonging to this family, and a swollenin from this species, SWOAfu, were selected for heterologous expression in T. Reesei. Moreover, chimeric protein constructions were realised to approach the catalytic domains of SWOAfu and the cellobiohydrolase CBH1 from T. Reesei. This chimeric protein which should lead to an increase of their synergy was also expressed in T. Reesei. Finally, in order to investigate a potential implication of Endoglucanase-45/Expansin-Like (EEL) proteins of T. Reesei in the cellulolytic process, transcriptional studies were realised under conditions of cellulase induction. A constitutive expression, similarly to the endoglucanase Cel5b, was shown for one of them. Potential functions of the EEL proteins are discussed with regard to their protein and promoter structure
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Niranjane, Ajay Pundaiikrao, and ajay niranjane@gmail com. "Screening diverse cellulase enzymes from the white rot fungus Phlebia gigantea for high activity and large scale applications." RMIT University. Applied Sciences, 2006. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080513.150257.
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Cellulosic biomass is the major organic matter produced in the biosphere. The biodegradation of this cellulosic material is achieved by enzymatic activities of the cellulose degrading microorganisms. These organisms usually express a complex extracellular or a membrane bound cellulolytic system comprising combination of several cellulase enzymes. Cellulases are the group of hydrolytic enzymes capable of hydrolysing insoluble cellulose to glucose. Phlebia gigantea is an aggressive white rot basidiomycete with ability to tolerate resinous extracts on freshly cut wood and higher growth rate. This helps the fungus to colonise the sapwood preventing other fungi from becoming established. Early research on the cellulase system of this organism reported the presence of a cellulase system composed of P-glucosidase, endoglucanase and a cellobiohydrolase. Based on these unpublished studies, our aim was to obtain a complete sequence of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and isolate the cellulase gene resulted in an incomplete cDNA sequence of I 154 bp. To understand the cellulase system, expression and regulation of the cellulase enzymatic activity was examined for incubation of P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose and cellobiose. The pH, total protein and biomass production results indicated that the capacity of P. gigantea to degrade cellulose is dependent upon the nature of the carbon source and the regulation of the cellulase synthesis is repressed in the presence of simple sugars like glucose and xylose. The study employed the highly effective method of purification by affinity adsorption and purified cellulase complex in large quantity. Characterisation of the kinetic properties of this cellulase complex revealed that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50GC. The purified complex was comprised of multiple proteins and demonstrated significant CMCase and CBHase activity on zymogram analysis. The purified cellulase complex was characterised by 2D gel electrophoresis and by peptide mass finger printing using MALDI-TOF massspectrometry analysis. The 2D gel analysis of the purified cellulase complex showed 15 spots within the range of pI 3.5 to pI 7 and the molecular weight between 20KDa to 100KDa. Three protein spots were selected based on the IEF and SDS zymogram and identified using MALDI-TOF MS analysis. These proteins were identified based on the peptide mass data belonging to the 6-phospho-a-glucosidase, p-glucosidase and glycosyl hydrolase family 13 a-amylase or pullulanases, suggesting the divergent evolution of specific cellulase proteins. This study showed P. gigantea as a potential cellulase source and the cellulase complex secreted by the induction of substrate, comprises a variety of enzymes related to hydrolysis of cellulose biomass. It is evident from this and previous studies that P. gigantea cellulase complex comprises of a specific set of enzymes that possess the ability to degrade crystalline cellulose and is one of the first organisms to colonise freshly cut wood. Further studies on the cellulase system of this primary colonist may open up the prospects to utilise this organism as the potential onsite bioreactor agent, pre-treating the biomass and increasing the economic feasibility of the industrial bioenergy processes.
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Mba, Medie Felix. "Le rôle des cellulases dans les interactions entre les mycobactéries du complexe Mycobacterium tuberculosis et les amibes libres." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20695/document.
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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<br>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
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Leghlimi, Hind. "Cellulases de souches fongiques issues du sol d'un milieu extrême (sol proche de sources thermales). Sélection des souches et étude des caractéristiques des enzymes." Thesis, Reims, 2013. http://www.theses.fr/2013REIMS020/document.
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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<br>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
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Dalal, Pankaj. "Étude physiologique et cinétique de la production des enzymes cellulolytiques chez Trichoderma reesei en culture continue." Paris 11, 1989. http://www.theses.fr/1989PA112091.
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Cette étude vise à une meilleure connaissance de la production des enzymes cellulolytiques par le champignon cellulolytique Trichoderma reesei en culture submergée et à une meilleure compréhension des mécanismes de synthèse et de régulation de ces enzymes. La cinétique de la croissance et la production des enzymes cellulolytiques chez T. Reesei CL-847, mutant résistant à la répression catabolique et hyper producteur, ont été étudiées en culture continue limitée par le lactose, seule source de carbonne, et inducteur du système cellulolytique, et comparées à celles de la souche parentale T. Reesei QM9414. Le fonctionnement stable obtenu dans la conduite en culture continue a permis d'étudier particulièrement l'influence du glucose et du 2-désoxyglucose sur la production et l'activité des enzymes cellulolytiques. La chromafocalisation analytique est une méthode excellente pour la caractérisation des enzymes cellulolytiques. Nous l'avons utilisée pour caractériser les enzymes cellulolytiques excrétées dans des états physiologiques différents pendant la culture continue. Une meilleure compréhension de ces phénomènes génétiques et biochimiques liés à la production de cellulases pourrait permettre dans un proche avenir des développements importants à l'échelle industrielle.
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Hildén, Lars. "The characterization of wood and wood fibre ultrastructure using specific enzymes /." Uppsala : Dept. of Wood Science, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/s328.pdf.
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Guillemin, Sandrine Parmentier Michel. "Extraction aqueuse d'huile de colza assistée par hydrolyse enzymatique optimisation de la réaction, caractérisation de l'émulsion et étude de procédés de déstabilisation /." S. l. : S. n, 2006. http://www.scd.inpl-nancy.fr/theses/2006_GUILLEMIN_S.pdf.
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Swart, Shanna. "Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1017914.
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Bahceci, Humeyra. "Fatty Acid Methyl Ester Analysis Of Bacterial Isolates From Salt Lake, Turkey And Characterization Of Their Extracellular Enzymes." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/12605483/index.pdf.
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In this study, 11 bacterial isolates from Salt Lake,Turkey were identified by using fatty acid methyl ester (FAME) analysis. They were screened for production of industrially important enzymes xylanase, cellulase, &<br>#945<br>-amylase and protease. These enzymes were characterized in terms of enzyme activity, stability, optimum temperature and optimum pH.
One of the isolates was identified as Bacillus pumilus, and two of them were identified as Bacillus subtilis. Other isolates were determined to be Bacillus licheniformis.
All the isolates were determined to produce xylanase. Optimum temperatures and optimum pH values of xylanases were 50-55 °<br>C and pH 7.0-8.0. Xylanases were quite stable up to pH 8.0 and 70 °<br>C. Isolates were not significant cellulase
producers. Four of the isolates did not produce any cellulase enzyme and the rest produced negligible amounts of cellulase. Therefore, xylanases from the isolates were promising for pulp and paper industry, which requires cellulase free and stable xylanases.
All the isolates produced appreciable quantities of &<br>#945<br>-amylase. Optimum temperatures and optimum pH values of &<br>#945<br>-amylases 60-80 °<br>C and pH 7.0-8.0. &<br>#945<br>-Amylases were quite stable up to pH 9.0 and 80 °<br>C. &<br>#945<br>-Amylases from the isolates were promising for starch processing industry, which requires &<br>#945<br>-amylases stable at high temperatures and for detergent industry, which requires &<br>#945<br>-amylases stable at alkaline pH values.
Considerable protease productions were achieved by all the isolates. TTG 2 was the best protease producer with 271 U/ml. Optimum temperatures and optimum pH values of proteases were 50-60 °<br>C and pH 7.0-7.4. Proteases were quite stable up to pH 9.0 and 80 °<br>C. Proteases from the isolates were promising for detergent and leather industry, in which proteases must be stable at alkaline pH values.
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Coffman, Anthony M. "Production of Carbohydrases by Fungus Trichoderma Reesei Grown on Soy-based Media." University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1381761363.
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Welt, Thomas. "Enzymatic deinking effectiveness and mechanisms." Diss., Georgia Institute of Technology, 1996. http://hdl.handle.net/1853/7067.
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Zampieri, Denise. "Expressão gênica e atividades de celulases, ß-glicosidases, xilanases e swoleninas de Penicillium echinulatum S1M29." reponame:Repositório Institucional da UCS, 2015. https://repositorio.ucs.br/handle/11338/1087.
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A linhagem S1M29 de Penicillium echinulatum é um fungo filamentoso cujo sistema celulolítico tem potencial para aplicação em processos de degradação de materiais lignocelulósicos visando a obtenção de produtos com interesse biotecnológico, como o etanol de segunda geração. A abundância de biomassas lignocelulósicas associada à busca por alternativas aos combustíveis fósseis faz aumentar o interesse em estudos que envolvam a elucidação dos mecanismos de secreção de enzimas lignocelulolíticas. Neste estudo, o P. echinulatum S1M29 foi crescido em condições de indução para a produção de endoglicanases, celobiohidrolases, β-glicosidases, xilanases e swoleninas. Foram avaliados os perfis enzimáticos em géis de poliacrilamida e o padrão de expressão gênica para estas proteínas. Observou-se que a produção enzimática foi favorecida pela presença de celulose Celufloc E® e bagaço de cana-de-açúcar (BCA) no meio de cultivo em comparação à glicose, sendo que o uso de resíduo de biomassa proporcionou a obtenção dos melhores rendimentos. Resultado semelhante foi atingido na análise dos perfis de secreção enzimática em gel de poliacrilamida, sugerindo ainda, a presença de uma endoglicanase constitutiva de aproximadamente 80 kDa. O estudo de qRT-PCR revelou a presença de quatro genes para endoglicanases com padrão de expressão distintos, destacando-se um acúmulo de transcritos 9779,19 vezes superior à amostra calibradora do gene egl1 em 24 h de cultivo no meio formulado com celulose Celufloc E®. Este mesmo gene gerou 1257,58 vezes mais transcritos acumulados que a amostra calibradora em meio suplementado com BCA. Na avaliação da expressão relativa do gene para celobiohidrolase obteve-se expressão superior no meio formulado com BCA em 48 h em relação ao meio com celulose Celufloc E® em 24 h, correspondendo, respectivamente, a 6464,49 e 3093,26 vezes mais transcritos acumulados que a amostra calibradora. O gene para β-glicosidase expressou-se em meio formulado com BCA no início do cultivo, embora a atividade desta enzima tenha ocorrido ao final do cultivo. A expressão de xilanases foi mais significativa com o uso de celulose Celufloc E® no meio de cultivo. Já o gene para swolenina apresenta um perfil de expressão com valores semelhantes em comparação ao uso de celulose Celufloc E® e BCA no meio de cultivo, apesar da presença de BCA ter proporcionado uma expressão mais tardia. Para todos os genes avaliados foram verificados valores muito reduzidos de expressão quando a glicose foi utilizada como fonte de carbono para o P. echinulatum. Observou-se ainda uma expressão coordenada de genes codificadores de endoglicanases, celobiohidrolase, β-glicosidase e swolenina.<br>Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2016-01-27T12:06:29Z
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Tese Denise Zampieri.pdf: 4586084 bytes, checksum: be5eb9fbb555f957a4e737205334ac6d (MD5)<br>Made available in DSpace on 2016-01-27T12:06:30Z (GMT). No. of bitstreams: 1
Tese Denise Zampieri.pdf: 4586084 bytes, checksum: be5eb9fbb555f957a4e737205334ac6d (MD5)<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq.<br>The strain S1M29 of Penicillium echinulatum is a filamentous fungus that presents a cellulolytic system with potential for application in the degradation process of lignocellulosic materials to obtain products of biotechnological interest, such as second-generation ethanol. The availability of lignocellulosic biomass associated with the search for alternatives to fossil fuels increases the interest in studies that involve understanding the secretion mechanisms of lignocellulolytic enzymes. In this study, P. echinulatum S1M29 was grown under inducing conditions for the production of endoglucanases, cellobiohydrolases, β-glucosidases, xylanases and swollenins. The enzyme secretion profiles were evaluated in polyacrylamide gels and the pattern of gene expression for these proteins was also assessed. It was observed that enzyme production was enhanced in the presence of cellulose Celufloc E® and sugar cane bagasse (SCB) in the culture medium compared to glucose, with the use of biomass residue providing the best yields. A similar result was obtained analyzing enzyme secretion profiles in polyacrylamide gels, suggesting the presence of constitutive endoglucanases of approximately 80 kDa. Studies with qRT-PCR revealed the presence of four genes encoding endoglucanases with distinct expression patterns, standing out an accumulation of transcripts from egl1 gene 9779.19 times higher than the calibrator sample in 24 h of growth in medium formulated with cellulose Celufloc E®. The same gene generated 1257.58 more accumulated transcripts than the reference sample in medium supplemented with SCB. Evaluation of gene expression for cellobiohydrolase yielded higher expression levels in the medium formulated with SCB in 48 h, in comparison to the medium containing cellulose Celufloc E® in 24 h, corresponding, respectively, to 6464.49 and 3093.26 more accumulated transcripts than the reference sample. The β-glucosidase gene was expressed in medium formulated with SCB at the beginning of growth, as opposed to that seen for activity of this enzyme, which occurred at the end of cultivation. The xylanase expression was more significant with the use of cellulose Celufloc E® in the culture medium. On the other hand, the gene for swollenin presents an expression profile with similar values compared to using cellulose Celufloc E® and SCB in the culture medium, although the presence of SCB has provided a later expression. Very low values of gene expression have been found for all genes evaluated when glucose was used as carbon source for P. echinulatum. A coordinated expression of endoglucanases, cellobiohydrolase, β-glucosidase and swollenin was was also verified.
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Fortman, Joshua A. "Techno-economic comparison of hot water and dilute acid pretreatment for biochemical production of ethanol from corn stover and evaluation of alternative scenarios to purchasing cellulase enzymes." [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1470016.
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Bastien-Uluis, Geraldine. "Découverte de nouvelles enzymes de dégradation des polysaccharides végétaux par métagénomique fonctionnelle." Thesis, Toulouse, INSA, 2012. http://www.theses.fr/2012ISAT0005.
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Une approche de métagénomique fonctionnelle a été mise en œuvre afin d’étudier les arsenaux enzymatiques produits par les microbiotes intestinaux de termites phytophages et d’identifier de nouvelles enzymes impliquées dans l’hydrolyse des polysaccharides végétaux, notamment des hétéroxylanes. Le criblage à haut débit des banques métagénomiques constituées à partir de trois espèces de termites sur une gamme de substrats chromogéniques a permis d’identifier plusieurs centaines de clones à activité dépolymérisante (glucanase, xylanase, mannanase, arabinanase), ainsi que des clones exprimant des activités auxiliaires (α-L-arabinofuranosidases, β-D-xylosidases, cellobiose hydrolases). Un total de 42 clones métagénomiques a été séquencé, générant 1,5 Mpb d’ADN assemblé en 58 séquences contigües d’une taille moyenne de 37,8 Kbp. 63 nouvelles Glycoside Hydrolases (GH) ont été identifiées. Ces dernières représentent 19 familles de la classification CAZy, dont les familles GH3, GH8, GH10, GH11, GH43 et GH51. Enfin, huit nouvelles enzymes des familles GH43 et GH51 ont été produites chez E. coli et leurs propriétés biochimiques ont été étudiées. Ces enzymes présentent des activités α-L-arabinofuranosidase, β-D-xylosidase ou L-arabinanase<br>A functional metagenomics approach was used to reveal the enzymatic diversity present in the guts of biomass-feeding termites and to identify enzymes involved in the degradation of biomass components, notably heteroxylans. High-throughput screening of metagenomic libraries, created using three different termite species, was performed using a variety of chromogenic substrates. This allowed the discovery of hundreds of clones expressing targeted biomass-degrading activities (e.g. depolymerases such as glucanase, xylanase, mannanase arabinanase and auxiliary activities such as α-L-arabinofuranosidases, β-D-xylosidases and cellobiohydrolases). A total of 42 clones were selected for a DNA sequence analysis, thus generating 1.5 Mbp that were assembled into 58 contiguous sequences. 63 new Glycoside Hydrolases (GH) belonging to 19 different families of the CAZy classification were identified, including ones from families GH3, GH8, GH10, GH11, GH43 and GH51. Finally, eight new enzymes, from families GH43 and GH51, were produced in E. coli and their biochemical properties were studied. These enzymes display α-L-arabinofuranosidase, β-D-xylosidase or arabinanase activities
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Cochet-Giraud, Nelly. "Les Cellulases de Trichoderma reesei production et application /." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37604010n.
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Díaz, Rodriguez Oscar Mauricio. "Otimização da produção de celulases a partir de substratos alternativos." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266799.
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Orientadores: Rubens Maciel Filho, Célia Maria Araujo Galvão<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química<br>Made available in DSpace on 2018-08-19T04:00:44Z (GMT). No. of bitstreams: 1
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Previous issue date: 2011<br>Resumo: O objetivo deste trabalho foi otimizar a produção de celulases, a partir de substratos alternativos, entre eles o bagaço de cana de açúcar, no processo de fermentação em estado sólido, utilizando o microrganismo Aspergillus niger. Na primeira etapa do trabalho foi realizada a identificação dos componentes do meio de cultura que mais favorecem o aumento da produtividade das celulases, entre eles foram avaliados: substrato (bagaço de cana in natura ou pré-tratado por explosão a vapor); fonte de carbono (lactose e extrato de soja) e a fonte de nitrogênio (peptona e extrato de levedura). Na segunda etapa do trabalho, a partir destas variáveis foi realizada a otimização da produção de celulases de acordo com um delineamento composto central rotacional (DCCR). Para isso foi utilizado um planejamento fatorial completo 2 , incluindo 6 pontos axiais e 4 repetições no ponto central. Finalmente, utilizando a técnica de regressão linear múltipla foi realizada a modelagem matemática para produção de celulases. A produção de celulases foi determinada baseada na atividade enzimática do complexo produzido nos ensaios: celulase total (FPAse); carboximetilcelulase (CMCase) e p- glucosidase. Os resultados mostraram que a máxima produção de celulaces, para um período de fermentação de 4 dias foi: 0,774 UI/g.s.s de FPAse; 14,41 UI/g.s.s de CMCase e 26,37 UI/g.s.s de p-glucosidase. As condições nas quais a máxima produção foi atingida foram: concentração de extrato de soja de 11 g/L; concentração de extrato de levedura de 1,5 g/L e porcentagem de umidade 80%. Esses resultados são bastante promissores levando em conta a identificação do extrato de soja como indutor da produção de celulases e do extrato de levedura, componentes que seriam de fácil e baixo custo de disponibilização no Brasil, que impactariam na redução global do custo de produção de celulases<br>Abstract: The objective of this work was the optimization of cellulases production, from alternatives substrates, such as sugarcane bagasse, in a process of solid state fermentation by Aspergillus niger. In the first project stage was identified the main components of culture medium: substrate (sugarcane bagasse in natura or pretreated by steam explosion); carbon source (lactose and soybean extract) and the source of nitrogen (peptone and yeast extract). In the second stage of the work, from that variables was optimized cellulases production, according to a central composite rotational design (DCCR). It used a full factorial 2 , including six axial points and 4 replications at the central point. Finally, it using the multiple linear regression was carried out the mathematical models for cellulases production. Thus, we evaluated the influence of selected variables on the production of cellulases quantified as three types of enzymatic activity: total cellulase (FPAse); carboximetilcelulase (CMCase) e p- glucosidase. The maximum production of cellulases was: 0,774 IU/g.d.s of FPAse; 14,41 IU/g.d.s of CMCase and 26,37 IU/g.d.s of p- glucosidase, for a period of 4 days of fermentation. And, the conditions in which maximum production was reached were: concentration of soybean extract 11 g/L, yeast extract concentration of 1,5 g/L and 80% moisture content. These results are very promising, considering the identification of the soybean extract as a inductor of cellulases production, and the least extract, these items are purchased at a low cost in Brazil, that impact in the reduction of global cost of cellulases production<br>Mestrado<br>Desenvolvimento de Processos Biotecnologicos<br>Mestre em Engenharia Química
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Pavasovic, Ana. "Evaluation of the nutritional requirements of redclaw crayfish, Cherax quadricarinatus." Queensland University of Technology, 2008. http://eprints.qut.edu.au/16615/.
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Aquaculture represents a sustainable alternative to natural fisheries for provision of high quality, animal protein. Crustaceans make a significant contribution to global aquaculture production, of which decapods are the most economically important group. Among freshwater crayfish, the genus Cherax includes several species that have emerged as important culture species. A suite of favourable biological attributes, including fast growth and an omnivorous feeding habit, have contributed to establishment of successful culture of Cherax quadricarinatus (redclaw) in many countries. Aspects of redclaw production, however, remain relatively undeveloped, in particular feed formulation. To better understand the digestive processes and nutritional requirements of redclaw, this study examined the relationship between diet composition and digestive enzyme activity, growth performance and diet digestibility coefficients. The extent to which redclaw can efficiently utilise complex polysaccharides, such as cellulose, has been speculated on by authors who reported endogenous cellulase activity in this species. I evaluated the use of insoluble α-cellulose by redclaw, demonstrated that high dietary levels (30%) can significantly reduce the specific activity of selected digestive enzymes (amylase and cellulase), while also lowering apparent digestibility coefficients. Inclusion of α-cellulose above 12% also significantly reduced survival rate, specific growth rate and feeding efficiency in this organism which corresponds with low tolerance for insoluble fibre by other decapods. Even though redclaw possess endogenous cellulases, they appear to have only a limited capacity to utilise insoluble fibre in their diets. Further, I assessed the impact of different nutrient profiles on digestive enzyme activity, growth and tail muscle composition in redclaw. Purified diets containing varying levels of dietary protein significantly affected activity of digestive enzymes (protease, amylase and cellulase) and the composition of the tail muscle tissue. Redclaw have a relatively low protein requirement, which was reflected here, as little significant difference was observed in growth rates and the feed conversion ratio was only significantly affected by the lowest protein diet. Manipulation of the non-protein energy component in purified diets (protein to lipid ratio) had no effect on growth performance indices in redclaw. Digestive enzyme activity (protease) was however, strongly influenced by both the amount of protein and lipid in the diet and a significant correlation was observed between protease activity and growth performance indices. The findings here, provide preliminary data for consideration of digestive enzymes such as proteases as potential growth indicators for freshwater crayfish. These enzymes are already recognised as reliable biological indicators for comparison of digestive efficiency and potential growth rate in fish. The relationship between diet composition and digestive enzyme expression observed here, stress the need for further empirical evaluation of specific ingredients in artificial diets for redclaw. A range of single cell, plant and animal-based, agricultural products were assessed for their potential use in diets formulated for redclaw. Analysis of dietary supplements revealed that apparent digestibility of crude protein was generally higher for diets containing plant-based ingredients. A similar outcome was observed for digestibility coefficients of test ingredients. Ingredient type also had a significant effect on digestive enzyme activity. Importantly, a significant correlation was observed for enzyme activity and apparent digestibility coefficients. It appears that redclaw have the capacity to utilise nutrients from a broad range of dietary ingredients successfully including animal, single cell and in particular, plant matter in their diet. Taken together, the results presented here demonstrate that digestive enzyme activities in redclaw are significantly influenced by diet composition. I show clearly that the ability of redclaw to utilise various nutrients (measured as digestibility coefficients) is highly correlated with digestive enzyme activity. Finally, protease activity demonstrated a potential for use as an indicator of redclaw growth performance. The data presented here will contribute to development of better and cheaper feed formulations for use in redclaw aquaculture and have broader applications to freshwater crustacean culture. In particular, the potential for use of plant-based ingredients in aqua-feeds for redclaw will contribute to a more economically and environmentally sustainable redclaw culture.
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Peri, Suma Lee Yoon Y. "Kinetic investigation and modeling of cellulase enzyme using non-crystalline cellulose and cello-oligosaccharides." Auburn, Ala., 2006. http://repo.lib.auburn.edu/2006%20Summer/Theses/PERI_SUMA_47.pdf.
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Guillemin, Sandrine. "Extraction aqueuse d'huile de colza assistée par hydrolyse enzymatique : optimisation de la réaction, caractérisation de l'émulsion et étude de procédés de déstabilisation." Thesis, Vandoeuvre-les-Nancy, INPL, 2006. http://www.theses.fr/2006INPL073N/document.
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En réponse aux attentes actuelles des consommateurs pour des produits de haute qualité nutritionnelle et environnementale, et face aux impératifs industriels conduisant à minimiser les risques et l’impact environnemental lors de l’extraction à l'hexane de l’huile de la graine de colza, l’étude de l’extraction aqueuse avec assistance enzymatique de cette huile a été reprise avec 2 objectifs principaux : déterminer les enzymes et mélanges d'enzymes adaptés à la meilleure déstructuration du tissu adipeux végétal, et d'autre part étudier les propriétés et la déstabilisation de l'émulsion formée. De l'optimisation de ces 2 séquences du process dépendent les rendements finaux en huile de l'extraction et les propriétés du tourteau, qui constituent les clés économiques de l'émergence de cette nouvelle technologie. Pour cela, après caractérisation physicochimique des constituants de la graine, protéases et polysaccharides hydrolases ont été testées seules ou en combinaison afin d’optimiser le rendement en huile libre et en huile contenue dans l’émulsion engendrée lors de l’extraction et obtenue par centrifugation. Après caractérisation de l’émulsion (rhéologie, diffusion statique de la lumière, pH, conductivité), des tests de déstabilisation physicochimiques ou thermo-mécaniques ont été mis en œuvre afin de séparer les constituants de l’émulsion formée, et obtenir ainsi la libération de l'huile. Les tests réalisés ont permis de retenir trois procédés de déstabilisation: l’addition de talcs, l’inversion de phase par addition d’huile exogène en présence de NaCl dans la phase aqueuse, et les cycles de congélation/décongélation. Afin d’apporter les premiers éléments de l’optimisation de ce dernier procédé, une étude par planification expérimentale a été mise en œuvre<br>Consumer's concerns about the quality and environmental impact of the products as well as the industrial requirements regarding the risk assessment and the environmental and health repercussions of the solvent extraction of rapeseed oil using hexane led us to work on the optimisation of the aqueous enzymatic extraction of this oil. The study has been carried out to determine the best combination of enzymes able to achieve the disruption of the vegetal adipose tissue, and to characterize the emulsion obtained after centrifugation. The final objective was to maximize the yields of the oil extraction and to obtain adequate nutritional properties of the cake. After the physicochemical characterization of the rapeseed raw material, several proteases and polysaccharide hydrolases have been tested individually and in combination in order to optimize the removal of free oil and the emulsion oil yield occurring during the aqueous extraction process. The physicochemical properties of the emulsion have been determined: rheological properties, pH, conductivity, spectroscopy by Short Angles Light Scattering). Thereafter some physicochemical and thermo-mechanical treatments have been carried out to destabilize the oil-in-water emulsion obtained after the centrifugation, which contained a large part of the total oil of the reaction mixture. Three destabilization processes appeared particularly interesting to increase the free oil removal from the emulsion: talc addition before centrifugation, phase inversion by addition of exogenous oil in presence of NaCl in the aqueous phase, and freezing/thawing cycles. Finally, an optimisation trial of the freezing/thawing process using a Doehlert experimental design has been done as an example
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Jourdier, Etienne. "Modélisation et optimisation de la production de cellulases par Trichoderma reesei pour les bioraffineries lignocellulosiques." Thesis, Clermont-Ferrand 2, 2012. http://www.theses.fr/2012CLF22264.
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Dans le contexte énergétique et climatique mondial, le coût élevé des enzymes Cellulolytiques (cellulases) freine le développement des bioraffineries lignocellulosiques, pour produire des biocarburants et composés chimiques à partir d'une matière première végétale renouvelable. L'objectif de ce travail est de caractériser et de modéliser le métabolisme du micro-organisme Trichoderma reesei, afin d'optimiser le protocole industriel de production de cellulases. Cette étude a été réalisée sur des milieux modèles représentatifs de ceux attendus à l'échelle industrielle. Tout d'abord, la stoechiométrie des réactions de croissance et de production a été établie, puis une étude cinétique a été menée pour mesurer précisément le comportement du micro-organisme à forte induction de la production de cellulases. Le modèle résultant a été utilisé pour optimiser le protocole industriel de production. Ensuite, l'intégration de cette étape dans une bioraffinerie lignocellulosique a été étudiée, avec l'effet sur le métabolisme i) des mélanges de sucres disponibles, ii) des composés inhibiteurs issus de la dégradation de la lignocellulose, et iii) du changement d'échelle. Ces travaux ont fait progresser de façon substantielle les connaissances du métabolisme de T. reesei en ce qui concerne la production de cellulases, et les modèles développés sont des outils d'aide rationnelle à la définition d'un procédé de production de cellulases intégré dans une bioraffinerie lignocellulosique<br>In the global energetic and climatic context, the high cost of the cellulolytic enzymes (cellulases) postpones the development of lignocellulosic biorefineries, dedicated to produce biofuels and chemical compounds from renewable vegetable feedstocks. The aim of this work was to measure and model the metabolism of the micro-organism Trichoderma reesei, in order to optimize the industrial protocol for the production of cellulase. This study was carried out using synthetic media representative of industrial ones. First, the stoichiometries of growth and protein production reactions were determined. Then, a kinetic study was conducted to precisely measure the specific rates of T. reesei at high induction of cellulase production. The resulting model was used to optimize the industrial production protocol. Finally the integration of this step in a lignocellulosic biorefinery was studied by determining the impacts on the metabolism of i) available sugar mixtures, ii) inhibitory compounds from lignocellulosic biomass degradation, and iii) scale-up. These results significantly contributed to improve the knowledge of T. reesei metabolism on cellulase production. The developed models are rational tools for the optimization of a cellulase production protocol suited to lignocellulosic biorefineries
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Maheshwari, Sweta. "Caractérisation biochimique et cellulaire des enzymes clés du métabolisme des phospholipides chez Plasmodium falciparum." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20004.
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Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique<br>Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach
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Liu, Wen. "BREAKDOWN OF HARD-DEGRADABLE POLYSACCHARIDES IN WETLANDS." Kyoto University, 2016. http://hdl.handle.net/2433/215584.
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Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第19758号<br>農博第2154号<br>新制||農||1039(附属図書館)<br>学位論文||H28||N4974(農学部図書室)<br>32794<br>京都大学大学院農学研究科応用生物科学専攻<br>(主査)教授 佐藤 健司, 教授 山下 洋, 准教授 豊原 治彦<br>学位規則第4条第1項該当
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Baker, Mark Andrew 1974. "Purification and characterisation of the plasma membrane NADH:oxidoreductase." Monash University, Dept. of Biochemistry and Molecular Biology, 2002. http://arrow.monash.edu.au/hdl/1959.1/8440.
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