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Miller, Laurie. "Studies on CBH1 : a cellobiohydrolase of Sclerotinia sclerotiorum." Thesis, University of Sheffield, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364190.
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Zulu, Joseph. "Cloning and regulation of the cellobiohydrolase I gene from Pleurotus sajor-caju." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285568.
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Lamour, Jarryd. "Expression of stress-tolerance related genes in Saccharomyces cerevisiae producing heterologous cellobiohydrolase." University of the Western Cape, 2017. http://hdl.handle.net/11394/5885.
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Magister Scientiae - MSc (Biotechnology)Cellulose is the most abundant naturally occurring renewable biopolymer on earth and a major structural component in plant cell walls, making it an ideal source of renewable energy. Consolidated bioprocessing (CBP) is a cost effective method of converting cellulose to liquid fuels such as ethanol. For CBP to be achieved an organism needs to be able hydrolyze cellulose and produce high yields of ethanol. The yeast Saccharomyces cerevisiae is an ideal CBP candidate, however wild type strains do not produce cellulases and these activities need to be engineered into yeast. In addition, the generally low secretion titers achieved by this yeast will have to be overcome.
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Keawsompong, Suttipun. "Cloning of a cellobiohydrolase II gene and its expression in Pleurotus sajor-caju." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368231.
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Voltatodio, Maria Luiza. "Caracterização bioquímica e biofísica da Celobiohidrolase II do fungo Trichoderma harzianum IOC3844 produzida por expressão homóloga." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-19102012-090550/.
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O esgotamento das reservas, especialmente do petróleo mais fino, aliado à crescente demanda energética e à necessidade inadiável de reduzir as emissões de carbono para a atmosfera, sinalizam para a necessidade da busca de novas fontes de energia renováveis e limpas. As preocupações com o aquecimento global têm feito crescer o interesse mundial pelos biocombustíveis. O novo conceito de biocombustíveis de segunda geração corresponde à produção de etanol combustível a partir de biomassa lignocelulósica como matéria-prima. No entanto, para tornar possível a utilização da biomassa é necessária a conversão das moléculas constituintes da parede celular em açúcares fermentáveis. A tecnologia mais promissora para a conversão dessa biomassa lignocelulósica à etanol combustível é com base na hidrólise enzimática da celulose usando celulases. Alguns microrganismos como o fungo Trichoderma SSP. secretam um eficiente complexo enzimático de celulases. Tendo as celobiohidrolases, elevada importância na hidrólise primária da celulose, o objetivo desse trabalho foi realizar a caracterização bioquímica e biofísica a celobiohidrolase II (CBHII) do complexo de celulases do fungo filamentoso Trichoderma harzianum IOC 3844. A enzima depois de purificada mostrou uma melhor atividade contra o substrato pNPC a 60°C em pH 4,8. Estudos de eletroforese capilar mostraram apenas moléculas com uma unidade de glicose para um substrato simples inicial contendo 5 glicoses. Análises de dicroísmo circular mostraram um padrão de estrutura secundária predominante em alfa hélice, e na análise da estrutura terciária, o espectro de emissão da CBHII mostrou um comprimento de onda de fluorescência máxima a 333nm em pH5,0, indicando que os triptofanos estão parcialmente expostos ao solvente. Ensaios utilizando a técnica de espalhamento de luz a baixo ângulo, permitiram a geração de um modelo tridimensional o qual mostrou-se domínios globulares unidos por um linker, e as posições relativas entre eles, demonstrando grande similaridade com enzimas CBHII já descritas na literatura, e sendo assim, de grande interesse biotecnológico para hidrólises de biomassas.The depletion of reserves, especially of refined oil , with increased energy demands and the urgent need to reduce the carbon emissions on the atmosphere, signals the necessity to search for new sources of energy renewable and clean. Concerns about global warming have led to an increased world interest in biofuels. The new concept of second generation biofuels corresponds to fuel ethanol production from biomass lignocellulosic feedstock. However, to make possible the use of biomass is necessary the conversion of cell-wall molecules into fermentable sugars. The most promising technology for the conversion of lignocellulosic biomass to ethanol fuel is based on the enzymatic degradation of cellulose using cellulase. Some microorganisms such Trichoderma ssp. secretes an efficient enzymatic complex of cellulase. Since the cellobiohydrolases are highly importance in the primary hydrolysis of cellulose, the objective of this study was to perform the biochemical and biophysical characterization of cellobiohydrolase II (CBHII) present into the cellulase complex from the Trichoderma harzianum IOC 3844. The enzyme showed its better activity against pNPC at 60°C and pH 4,8. Capillary electrophoresis showed only glucose molecules as the final product of C5 oligosaccharide hydrolysis. Circular dichroism analysis showed a pattern of secondary structure mainly composed of alpha helix, and the tertiary structure analysis by the emission spectrum of the CBHII showed a wavelength of maximum fluorescence at 33nm at pH 5, indicating that the tryptophans are exposed to solvent. The three dimensional model generated by SAXS showed a structure with two globular domains joined by a linker, and the relative positions among them exhibited great similarity with CBHII described on the literature, and thus, presenting a great biotechnological interest for hydrolysis of biomass.
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Tokunaga, Yuki. "Interaction analysis between lignin and carbohydrate-binding module of cellobiohydrolase I from Trichoderma reesei." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263699.
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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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Koivula, Anu. "Structure-function studies of two polysacchride-degrading enzymes : Bacillus stearothermophilus [alpha]-amylase and Trichoderma reese cellobiohydrolase II /." Espoo : Technical Research Centre of Finland, 1996. http://www.vtt.fi/inf/pdf/publications/1996/P277.pdf.
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Texier, Helene. "Ingénierie des xylanases de Penicillium funiculosum IMI 378536 : amélioration de la robustesse de l'activité xylanolytique dans la préparation commerciale Rovabio Excel™." Thesis, Toulouse, INSA, 2012. http://www.theses.fr/2012ISAT0048.
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Le Rovabio Excel ™ est un cocktail enzymatique complexe sécrété par le champignon filamenteux Penicillium funiculosum. La société ADISSEO commercialise cet additif alimentaire destiné à la nutrition animale car les principales enzymes qui le constituent dégradent les polymères contenus dans les céréales, tels que les polysaccharides non amylacés. Ainsi, le Rovabio Excel™ permet d’améliorer la digestibilité et d’augmenter la valeur nutritionnelle des matières premières agricoles en réduisant la viscosité du bol alimentaire des animaux. Dans le but d’augmenter sa compétitivité, ADISSEO a fait conduire des études sur cette solution pour la caractériser biochimiquement et optimiser son potentiel xylanolytique.Ces travaux de thèse s’inscrivent dans ces projets industriels et ont poursuivi deux objectifs distincts. Le premier correspondait à l’augmentation de la thermostabilité de la protéine XynB du Rovabio Excel™, pour lui permettre de résister à la granulation. Le second concernait XynA, la protéine majoritaire de la solution multienzymatique, qui a été caractérisée biochimiquement. Les premiers résultats de caractérisation biochimique de XynA ont montré que la protéine était 100 fois plus active sur β-1,4-glucane que sur xylane. Des tests complémentaires sur pNP-cellobiose et pNP-β-D-Lactopyranose ont révélé que XynA était 5,2 fois plus active sur pNP-cellobiose et possédait une activité « exo ». Enfin, l’analyse des produits d’hydrolyse d’oligosaccharides composés de 2 à 5 unités de glucose a confirmé que la protéine XynA était une cellobiohydrolase de type I, très sensible à l’inhibition par le cellobiose (IC50 - C2 = 17,7 µM). L’étude la thermostabilité de XynB a confirmé que cette protéine n’était pas naturellement thermostable. Les résultats des travaux d’ingénierie avec l’ajout d’un pont disulfure pour rigidifier la structure 3D de la protéine n’ont pas été probants. En revanche, la création de protéines chimères à partir de protéines plus thermostables (TfxA de Thermomonospora fusca et XynII de Trichoderma reesei) a permis d’améliorer la stabilité thermodynamique de XynB avec des Tm augmentés de plus de 10°CThe Rovabio Excel™ is a complex enzymatic cocktail secreted by the filamentous fungus Penicillium funiculosum. The ADISSEO company sells it as food additive for animal feed because the main enzymes degrade polymers contained in grains, such as non-starch polysaccharides. Thus, the Rovabio Excel™ improves the digestibility and increases the nutritional value of agricultural raw materials by reducing the viscosity of the diet of animals. In order to increase its competitiveness, ADISSEO did conduct studies on this solution to characterize it biochemically and maximize its xylanolytic potential.This thesis takes part of this industrial project and have pursued two distinct objectives. The first corresponds to the increase in the thermostability of the protein XynB from the Rovabio Excel™, to enable it to resist at the granulation process. The second was XynA, the major protein of the multienzyme solution, which was characterized biochemically.Initial results of biochemical characterization of XynA showed that the protein was 100 times more active on β-1,4-glucan on xylan. Additional tests on pNP-cellobiose and pNP-β-D-Lactopyranose revealed that XynA was 5.2 times more active on pNP-cellobiose and possess an "exo-acting" activity. Finally, the analysis of products from oligosaccharides hydrolysis, composed of 2 to 5 units of glucose, confirmed that the protein XynA was a type I cellobiohydrolase, very sensitive to inhibition by cellobiose (IC50-C2 = 17.7 µM).The thermostability of XynB study has confirmed that this protein was not thermostable naturally. The results of the engineering work with the addition of a disulfide bridge to rigidify the 3D structure of the protein were not conclusive. However, the creation of chimeric proteins with more thermostable proteins (TfxA from Thermomonospora fusca and XynII from Trichoderma reesei) has improved the thermodynamic stability of XynB with Tm increased by more than 10°C
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Sibanda, Ntsako. "Evaluation of high recombinant protein secretion phenotype of saccharomyces cerevisiae segregant." Thesis, University of Limpopo, 2016. http://hdl.handle.net/10386/1803.
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Thesis (MSc. (Biochemistry)) --University of Limpopo, 2016The ever increasing cost of fossil-based fuels and the accompanying concerns about their impact on the environment is driving research towards clean and renewable sources of energy. Bioethanol has the potential to be a replacement for liquid transportation fuels. In addition to its near zero nett carbon dioxide emissions, bio-ethanol has a high energy to weight ratio and can easily be stored in high volumes. To produce bioethanol at economically competitive prices, the major cost in the production process needs to be addressed. The addition of enzymes to hydrolyse the lignocellulosic fraction of the agricultural waste to simple sugars is considered to be the major contributor to high production cost. A consolidated bioprocess (CBP) which ideally combines all the steps that are currently accomplished in different reactors by different microorganisms into a single process step would be a more economically feasible solution. In this study the potential of yeast hybridization with a CBP approach was used. In order to evaluate the reduction or elimination of the addition of cellulolytic and hemi-cellulolytic enzymes to the ethanol production process. High cellobiohydrolase I secreting progeny from hybridization of an industrial bioethanol yeast strain, S. cerevisiae M0341, and a laboratory strain S. cerevisiae Y294 were isolated. In order to determine if this characteristic was specific to cellobiohydrolase I secretion, these strains were evaluated for their ability to secrete other relevant recombinant hydrolase enzymes for CBP-based ethanol production. A total of seven S. cerevisiae strains were chosen from a progeny pool of 28 supersecreting hybrids and reconstructed to create two parental strains; S. cerevisiae M0341 and S. cerevisiae Y294, together with their hybrid segregants strains H3M1, H3M28, H3H29, H3K27 and H3O23. Three episomal plasmids namely pNS201, pNS202 and pNS203 were constructed; these plasmids together with two already available plasmids, namely pRDH166 and pRDH182 contained genes for different reporter enzymes, namely β-glucosidase I, xylanase II, endoglucanase lll, cellobiohydrolase l and α-glucuronidase. To allow for selection of the episomal plasmids, homologous recombination was used to replace the functional URA3 gene of selected strains, with the non-functional ura3 allele from the Y294 strain. Enzyme activity was used as an indicator of the amount of enzyme secreted. Fermentation studies in a bioreactor were used to determine the metabolic burden imposed on the segregants expressing the cellobiohydrolase at high levels. In addition all segregants were tested for resistance to inhibitors commonly found in pre-treated lignocellulosic material. The M28_Cel7A was found to be the best secretor of Cel7A (Cellobiohydrolase l); however it seems as though this phenomenon imposes a significant metabolic burden on the yeast. The supersecreting hybrid strains cannot tolerate lignocellulosic inhibitors at concentrations commonly produced during pretreatment
The National Research Foundation - Renewable Energy Scholarship (NRF-RSES)
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Colussi, Francieli. "Caracterização bioquímica, biofísica e estrutural da Celobiohidrolase I de Trichoderma harzianum envolvida na hidrólise da biomassa lignocelulósica." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-07122012-145352/.
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Devido à sua importante atividade celulolítica, o fungo Trichoderma harzianum possui um grande potencial de aplicação na hidrólise da biomassa. No entanto, as celulases deste fungo filamentoso ainda não foram caracterizadas em profundidade. A celobiohidrolase I (CBHI) é a principal enzima celulolítica produzida por Trichoderma sp. e atualmente é uma das celulases mais investigadas para aplicações de biocombustíveis. A CBHI hidrolisa celulose cristalina à unidades solúveis de celobiose, o que a torna uma enzima chave para a produção de açúcares fermentáveis a partir da biomassa. O objetivo deste trabalho foi purificar e caracterizar a CBHI de Trichoderma harzianum (ThCBHI) bioquímica, biofísica e estruturalmente. Primeiramente foi estabelecido um protocolo de purificação eficiente da proteína a partir da expressão homóloga no fungo. A caracterização bioquímica ThCBHI mostrou que a proteína possui uma massa molecular de 66 kDa, pI de 5,23 e o pH e a temperatura de atividade ótima foram 5,0 e 50 ºC, respectivamente. A influência do pH e temperatura sobre as estruturas secundárias e terciárias e atividade enzimática da ThCBHI foram analisados por espectroscopia de CD, fluorescência e SAXS, e os resultados mostraram que as perturbações de pH e de temperatura afetam a estabilidade por dois mecanismos diferentes. As variações de pH podem modificar a protonação dos resíduos, afetando diretamente sua atividade, levando a desestabilização estrutural apenas em limites extremos de pH, como pH 9,0. A temperatura, por outro lado, tem uma influência direta sobre enovelamento e compactação da enzima, fazendo com que na temperatura em torno de 60 ºC ocorra perda da estrutura secundária, e terciária. Quando as análises foram realizadas na presença do produto de reação e também inibidor competitivo, celobiose, a estabilidade térmica da ThCBHI aumentou significativamente de 61,5 para 65,9 ºC. Os estudos estruturais e simulações de dinâmica molecular mostraram que a flexibilidade do resíduo Tyr260, em comparação com a Tyr247 do homólogo de T. reesei CBHI (TrCBHI), é aumentada devido às cadeias laterais curtas adjacentes de Val216 e Ala384 criando uma abertura adicional na face lateral do túnel catalítico. A ThCBHI também apresenta um loop encurtado na entrada do túnel de interação com a celulose, o que tem sido descrito como o responsável por interagir com o substrato de TrCBHI. Estas características estruturais podem explicar por que a ThCBHI apresenta maior valor de kcat e menor inibição pelo produto em comparação com TrCBHI.Trichoderma harzianum is a fungus that has a considerable potential in biomass hydrolysis application due to its elevated cellulolytic activity. Cellulases from Trichoderma reesei have been widely used as model in studies of cellulose breakdown. However, cellulases from Trichoderma harzianum are less-studied enzymes which have not been characterized biophysically and biochemically as yet. CBHI, a cellobiohydrolase I, is the major cellulolytic enzyme produced by Trichoderma sp. and is currently one of the most investigated cellulases for biofuel applications. CBHI hydrolyzes crystalline cellulose to soluble cellobiose units, which turns it into a key enzyme for producing fermentable sugars from biomass. The aim of this work was to purify and characterize the CBHI of Trichoderma harzianum (ThCBHI). We established an efficient purification protocol of ThCBHI, from the homologous expression. The biochemical characterization of ThCBHI showed that the protein has a molecular mass of 66 kDa, a pI of 5,23, and the optimum pH and temperature for its activity are 5,0 and 50 ºC, respectively. The effect of pH and temperature on secondary and tertiary structure and enzymatic activity of ThCBHI were analyzed by CD and Fluorescence spectroscopy and showed that they affect protein stability by two distinct mechanisms. Variations of pH modify protonation of the residues, affecting directly its activity, leading to structural destabilization only at extreme pH values, such as pH 9, 0. On the other hand, temperature has direct influence on mobility, fold and compactness of the folding enzyme, at temperatures above 60 ºC, there is loss of secondary and tertiary structure. When the assays were conducted in the presence of the cellobiose, a competitive inhibitor, thermal stability of ThCBHI was significantly increased to 61,5 to 65,9 ºC. Structural studies and molecular dynamics simulations showed that the flexibility of Tyr260, in comparison to the Tyr247 from the homologous T. reesei CBHI, is enhanced due to the short side chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. In addition, CBHI of T. harzianum has a shortened loop at the entrance of the cellulose-binding tunnel, which has been described to interact with the substrate in T. reesei CBHI. These structural features might explain why T. harzianum enzyme displays higher kcat value and lower product inhibition on both glucosides and lactosides substrates in comparison to T. reesei CBHI.
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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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The hydrolysis kinetics of bacterial cellulose and its derivatives by Trichoderma reesei 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.
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.
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.
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Harang, Valérie. "Aspects of Optimisation of Separation of Drugs by Chemometrics." Doctoral thesis, Uppsala University, Analytical Pharmaceutical Chemistry, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3738.
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Statistical experimental designs have been used for method development and optimisation of separation. Two reversed phase HPLC methods were optimised. Parameters such as the pH, the amount of tetrabutylammonium (TBA; co-ion) and the gradient slope (acetonitrile) were investigated and optimised for separation of erythromycin A and eight related compounds. In the second method, a statistical experimental design was used, where the amounts of acetonitrile and octane sulphonate (OSA; counter ion) and the buffer concentration were studied, and generation of an α-plot with chromatogram simulations optimised the separation of six analytes.
The partial filling technique was used in capillary electrophoresis to introduce the chiral selector Cel7A. The effect of the pH, the ionic strength and the amount of acetonitrile on the separation and the peak shape of R- and S-propranolol were investigated.
Microemulsion electrokinetic chromatography (MEEKC) is a technique similar to micellar electrokinetic chromatography (MEKC), except that the microemulsion has a core of tiny droplets of oil inside the micelles. A large number of factors can be varied when using this technique. A screening design using the amounts of sodium dodecyl sulphate (SDS), Brij 35, 1-butanol and 2-propanol, the buffer concentration and the temperature as factors revealed that the amounts of SDS and 2-propanol were the most important factors for migration time and selectivity manipulation of eight different compounds varying in charge and hydrophobicity. SDS and 2-propanol in the MEEKC method were further investigated in a three-level full factorial design analysing 29 different compounds sorted into five different groups. Different optimisation strategies were evaluated such as generating response surface plots of the selectivity/resolution of the most critical pair of peaks, employing chromatographic functions, simplex optimisation in MODDE and 3D resolution maps in DryLab™.
Molecular descriptors were fitted in a PLS model to retention data from the three-level full factorial design of the MEEKC system. Two different test sets were used to study the predictive ability of the training set. It was concluded that 86 – 89% of the retention data could be predicted correctly for new molecules (80 – 120% of the experimental values) with different settings of SDS and 2-propanol.
Statistical experimental designs and chemometrics are valuable tools for the development and optimisation of analytical methods. The same chemometric strategies can be employed for all types of separation techniques.
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Harjunpää, Vesa. "Enzymes hydrolysing wood polysaccharides : a progress curve study of oligosaccharide hydrolysis by two cellobiohydrolases and three ß-mannanases /." Espoo : Technical Research Centre of Finland, 1998. http://www.vtt.fi/inf/pdf/publications/1998/P372.pdf.
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Lima, Leonardo Henrique França de. "Estudos por modelagem e dinâmica molecular integradas a técnicas físicas para biomoléculas em solução - interação de receptores nucleares a elementos responsivos no DNA e dinâmica inter-domínios da celobiohidrolase I." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-06122011-084130/.
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Movimentos coletivos prestam um papel fundamental na dinâmica e energética de biomoléculas em solução. Estes movimentos permitem o acoplamento de regiões significativamente distantes, apresentando considerável influência, por exemplo, no alosterismo para a formação de complexos macromoleculares e no funcionamento integrado de proteínas multidomínios como \"máquinas moleculares\". Neste trabalho de doutoramento, serão apresentados os resultados referentes à aplicação conjunta de técnicas experimentais biofísicas, de modelagem estrutural e de dinâmica molecular no estudo de dois sistemas para os quais estes movimentos coletivos demonstram considerável importância funcional. Para a interação do receptor nuclear do ácido 9-cis-retinóico com seu elemento responsivo específico no DNA (HRE), a comparação de estudos de dinâmica molecular com ensaios de afinidade por anisotropia de fluorescência sugere que a resistência inicial para a associação do monômero, seguida da acentuada colaboratividade na associação do dímero é regida por um impedimento da associação do domínio de ligação ao DNA (DBD) para o primeiro à sequência responsiva devido, em última análise, a uma não complementaridade dos modos coletivos mútuos. Este impedimento para a associação monomérica inicial é mais acentuado para o monômero 5\' (para o qual a menor especificidade de ligação à seqüência específica já é bem documentada), devido aos efeitos conjuntos de um \"defeito\" natural no empacotamento de bases da seqüência responsiva, que se manifesta mais significativamente na interface entre o meio-sítio 5\' e a seqüência espaçadora, e dos modos vibracionais entre os dois sítios decorrentes de seu faseamento relativo na topologia do DNA na seqüência responsiva, caracterizando um mecanismo \"chave e fechadura\" para a interação obrigatoriamente simultânea dos dois monômeros ao DNA. No segundo caso, um estudo integrado utilizando a técnica experimental de espalhamento de raios X a baixos ângulos e uma abordagem de modelagem estrutural baseada em dinâmica molecular foi realizado para a celobiohidrolase I de Trichoderma harziannum. Este estudo permitiu tanto a elaboração de um modelo estrutural de maior resolução para esta enzima de alto potencial biotecnológico como a constatação dos possíveis mecanismos moleculares a partir dos quais as glicosilações no peptídeo conector impõem restrições à orientação e modos vibracionais entre seus dois domínios de forma condizente com sua ação concertada na interação e no deslize da enzima sobre a superfície celulósica, ambos de fundamental importância para a processividade da enzima na hidrólise do substrato microcristalino.Collective motions play a fundamental role in solution biomolecule dynamics and energetics. These movements can couple very distant regions in the protein structures affection, for instance, allosteric mechanisms, the establishment of macromolecular complexes, and on the integrated function of multidomain proteins as molecullar machines. In this thesis, we present results concerning to the joint use of experimental biophysical techniques, structural modeling and molecular dynamics simulations on the study of two systems for which these collective motions have substantial importance. First, we study the interaction of the nuclear retinoid X receptor with its specific DNA hormone response element (HRE) using a combination of molecular dynamics simulations and affinity assays performed by using fluorescence anisotropy. We find out that collective motions mediate the low binding affinity of monomers and the high cooperative binding of HRE dimers. The lower binding affinity of the monomer is more prominent for 5´ monomers. This occur due to an natural ineffective stacking of the last base pair step at the 5´-half-site and to the phasing of the two binding half-sites in the DNA topology, that impose a collective motions that tends to occlude the 5´ binding site. This behavior, in turn, is concurrent with the well known 3´ polarity and the decreased binding specificity to the 5´ half site for the hRXRα monomer. This same pattern impose a lock-and-key mechanisms dependent on the binding of the full dimer. Second, an integrated Small angle X ray scattering and molecular dynamics based structural modeling was used to comprehend the interdomain motions of cellobiohydrolase I of Trichoderma harziannum. We manage to build a refined model for this enzime, with important biotechnological potential. We also provide insights into molecular mechanisms of linker and glycosylation imposed restraints on the orientation and vibrational modes of the full-length enzyme, supporting a mechanism of sliding of on the cellulose surface. This mechanism is fundamental for the high processivity on the hydrolysis of microcrystalline cellulose.
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Hui, Joseph P. M. "Characterization of protein glycoforms in cellobiohydrolases and endoglucanases from Trichoderma reesei RUT-C30 and mutant strains using capillary isoelectric focusing and mass spectrometry." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0015/MQ57123.pdf.
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WU, Chia-Hua, and 吳佳樺. "Functional Expression of a Cellobiohydrolase Gene from Pleurotus citrinopileatus." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/hq495n.
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碩士國立屏東科技大學
生物科技系所
105
Cellulose, one of a main component of lignocellulosic, cellulases hydrolyze lignocellulose into sugar molecules. Cellulase are a crucial component of various industrial processes, such as in juice extraction and animal feed additives, as detergent enzyme, in cotton and paper manufacturing, etc. Utilization of cellulosic biomass for the production of renewable liquid biofueis like ethanol, or other fermentative products of sugar. In this study, previous studies is used to clone the exocellulase (celA) gene from an edible mushroom Pleurotus citrinopileatus. celA cDNA is successfully expressed in the yeast Pichia pastoris, and the secreted recombinant celA is purified as a soluble protein with a size of 66 kDa from the culture fluid. Use pET28a、pET29a experession vectors for soluble recombinant proteins with molecular weights of 57.883 kDa、56.145 kDa、55.72 kDa、53.982 kDa, respectively. The optimum temperature and pH of purified pET28a-celA-2/BL21(DE3) recombinant proteins are 40℃ and 4.0, respectively. The study can convert cellulose to useful products.
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Huang, Bo-Kuan, and 黃博冠. "Molecular Cloning and Analysis of a Cellobiohydrolase Gene from Pleurotus citrinopileatus." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/95361241437022902373.
Full textAbstract:
碩士國立屏東科技大學
生物科技系所
102
Cellulose, a component of lignocellulosic raw material, is the most abundant carbohydrate polymer in nature. The tremendous amount of fiber wastes produced from agriculture will lead to not only serious environmental problems but also a waste of nature resources caused by inappropriate treatment. Cellulose is a polymer of β (1→4)-linked glucose which is hardly soluble in water due to the inter-molecular hydrogen bond, and consequently difficult to be digested by most organisms. The development of fibrolytic enzymes can be employed in the cellulose waste treatment and other agri-industrial applications such as detergents, feed additives, cellulose degradation in pulp fibers and clarification of fruit juice, etc. The carbohydrate products from fibrolysis can be also used as the energy source for microbial fermentation for the production of bioethanol. In this study, RT-PCR is used to clone the exocellulase (celA) gene from an edible mushroom Pleurotus citrinopileatus. The cDNA sequence of celA is 1,572 bp in length and contains a 1,515 bp open reading frame encoding a CelA protein precursor of 505 amino acid residues. celA cDNA is successfully expressed in the yeast Pichia pastoris, and the secreted recombinant CelA is purified as a soluble protein with a size of 66 kDa from the culture fluid. The optimum conditions of enzymatic activity are determined for further exploiting the capacity of yeast to convert cellulose to useful products.
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Chu, Chih-Yuan, and 朱致遠. "Molecular cloning of a cellobiohydrolase from Piromyces rhizinflatus and heterologous expression." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/28405025856078805951.
Full textAbstract:
碩士國立臺灣大學
動物科學技術學研究所
99
Abstract Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a β-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. The cDNA sequence of cbhYW23-2 contained an open reading frame of 1,557 bp encoding a protein of 519 amino acids with a predicted molecular weight of 57 kDa. A putative conserved domain of glycosyl hydrolase (GH) family 6 was observed at the C-terminus and a putative conserved domain of cellulose binding domain (CBD) family 1 was observed at the N-terminus of CbhYW23-2. These two domains were separated by an Asn-rich linker. To examine the enzyme activities, CbhYW23-2 was expressed in E. coli as recombinant fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis were then employed for the planned statistical optimization of the β-glucanase activities of CbhYW23-2. The optimal conditions for the highest β-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. At this condition, the specific activity of CbhYW23-2 was 2411.1±197.9 U/mg. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the highly activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications. Then cbhYW23-2 was constructed into the expression vector of S. cerevisiae. CbhYW23-2 was displayed on the cell surface of S. cerevisiae via a-agglutinin. The localization of a-agglutinin-CbhYW23-2 fusion protein on the cell surface was confirmed by analysis of β-glucanase activity assay, enzyme diffusion method, western blot, and immunofluorescence microscopy. In summary, the results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. The a-agglutinin-CbhYW23-2 fusion protein was expressed on the cell surface of S. cerevisiae successfully and still remained the β-glucanase activity.
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Tsai, Chih-Cheng, and 蔡志誠. "Heterologous expression and characterization of a cellobiohydrolase cloned from rumen fungi." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/54660476329124840908.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
100
Cellulase catalyzes the hydrolysis of glycosidic bonds in cellulose. It can be applied in several industries, including food processing, feed additives, textile industry, pulp and paper, and bioenergy. However, efficiency of degrading lignocellulose by cellulase is still low. Thus, it is necessary to explore for cellulase of high efficiency or activity. The complete hydrolysis of cellulose requires the coordinated actions of at least three types of cellulases, i.e. endocellulase, cellobiohydrolase (or exocellulase), and β-glucosidase. Many studies have pointed out that rumen fungi contain highly-active cellulases which deserve to be investigated. The objective of this study was to characterize a cellulase, designated as CelA5, cloned from rumen fungi Orpinomyces sp. Y102, aiming to evaluate the potential of CelA5 in industrial application. CelA5 was expressed in Escherichia coli as a fusion protein, with maltose-binding protein (MBP) as the fusion tag. MBP-CelA5 was purified and characterized. The optimal reaction conditions of MBP-CelA5 were 50℃ and pH 7.0. It was stable in 30 ℃ - 50 ℃, pH 5.0 - 11.0, and lost about 50% activity in 0.5 M NaCl. Moreover, the activity of MBP-CelA5 was undetectable in 0.2% SDS, but MBP-CelA5 remained about 50% activity in 0.2 - 2% of Tween-20. Substrate-specificity assays and product analysis suggested that CelA5 is a cellobiohydrolase. The Vmax, Km and kcat of CelA5 were 3900±180 µmole min-1 mg-1, 3.63±0.41 mg/ml and 6739.77±312.39 sec-1 respectively, which suggested that CelA5 is a high-activity enzyme when compared with other cellobiohydrolase. In filter-paper-digestion tests, CelA5 was able to degrade filter paper in conjunction with endocellulase and β-glucosidase. Thus, CelA5 is considered a potential cellulase for industrial application.
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Chang, Pi-Chu, and 張碧珠. "Expression of Trichoderma koningii G-39 cellobiohydrolase I gene in Saccharomyces cerevisiae." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/37993419323637093366.
Full textAbstract:
碩士國立清華大學
生命科學系
87
Cellobiohydrolase I (CBH I) is one of cellulose hydrolysis enzymes (cellulases). CBH I catalyzes the exocellulolytic cleavage of β-1,4-glucosidic bonds in cellulose and release cellobiose. For a long time, Cellulases have been used in commercial processes for the partial hydrolysis and softening of crude cellulose in food and cotton. Saccharomyces cerevisiae is an ideal organism for production of commercially important products from cellulose, a relatively cheap carbon source. However, S. cerevisiae can't directly utilize cellulose as carbon source. The aim of this study is to allow a stable expression of CBH I gene in the S. cerevisiae. Recombinant plasmid named pRUC1 was constructed in this investigation. pRUC1 contains CBH I cDNA gene from Trichoderma koningii G-39, portion of S. cerevisiae ribosomal DNA (rDNA), S. cerevisiae ura3 gene with a partially deleted promoter, and bacterial cloning vector pSP72. The rDNA of recombinant plasmid was used to target the homologous recombination in yeast chromosome, while marker gene with a partially deleted promoter renders high copy number of integration of transformed plasmid under selection pressure. pRUC1 was transformed into S. cerevisiae. A part of transformants were able to form clear zone on the plate containing 0.5% soluble carboxymethylcellulose (CMC), indicating the secretion of CBH I by the yeast. Southern hybridization analysis revealed that plasmids were integrated into S. cerevisiae chromosome as tandem repeats and dispersed copies. The Ura+ phenotype of transformants was stably maintained in the nonselective medium for 50 generations.
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Tung, Wey Ta, and 魏大同. "Molecular biological studies of cellobiohydrolase I gene from Trichoderma koningii G-39." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/30656054366158793005.
Full textAbstract:
博士國立清華大學
生命科學研究所
81
Cellobiohydrolase I (CBH I) from a filamentous fungus Trichoderma koningii G-39 has been purified by SP- Sephadex C-50 and DEAE-Sephadex A-50 ion-exchange chromatographies. It showed a molecular weight of 68 k and pI of 4.0 upon sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. This enzyme has a specific activity of 1.4 unit/mg using p-nitrophenyl-b-D-cellobioside as a substrate and prefers to interact toward crystalline substrates. It exhibited both exocellobiohydrolytic and cellulose binding activities, with different chemical and physical characteristics. CBH I encoding gene cbh1 has been cloned, including a 1.4 kb cDNA and 7.0 kb genomic DNA. The sequence analysis reveals that part of the cloned sequence encodes a 513 amino acids-protein. The coding region is split by two introns with lengths of 67 and 63 base pairs. Nucleotide sequence alignment to the putative promoter region of cbh1 reveals significant homology to the carbon catabolite repressor binding sites of Aspergillus nidulans CREA and Saccharomyces cerevisiae MIG1 promoters which respond to a wide- domain regulatory system of glucose repression. In addition, several transcription factor binding site-like sequences are identified in 5'' flanking region of cbh1 as well. The electrokaryotype of T. koningii is obtained by pulsed field gel electrophoresis (PFGE). The fungal chromosomes were separated to five bands. The lowest band showed non-proportional fluorescent intensity, it is possible that at least two similar size chromosomes comigrated on this band. Accordingly, the fungal genome is thought to be constituted of five or more chromosomes. The sizes of chromosomes are estimated as 4.4, 4.7, 5.7, 6.7 and 7.2 Mb, respectively, and the genome size is calculated to be about 28.7 Mb. The results of PFGE followed by Southern hybridization indicates that cbh1 and xyl2, a gene coding for an endoxylanase, are both mapped at the lowest band.
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Yi, Yang Shi, and 楊士儀. "Glucose Effect On The Biosynthesis Of The Trichoderma koningii G-39 Cellobiohydrolase I." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/33413671868784914597.
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Chen, Chien-Chi, and 陳倩琪. "Molecular studies of glucose repression of cellobiohydrolase I gene in Trichoderma koningii G-39." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/31941189318363791247.
Full textAbstract:
博士國立清華大學
生命科學系
87
Cellobiohydrolase 1 (CBH1) is the major component of the Trichoderma cellulase system, amounting to more than 60 % of the total excreted proteins. As the production of CBH1 is inducible by various compounds and repressible by easily metabolizable carbon source like glucose, CBH1 synthesis thus appears to be controlled by its promoter in a complicated and variable mechanism. In order to investigate the mechanism of carbon catabolite repression in the filamentous fungus T. koningii G-39, the regulatory protein GRPT was obtained from the glucose-grown mycelia and purified from a DNA-ligand affinity column. The estimated molecular weight is comparable to the previous published Cre1, which is equivalent to the Aspergillus nidulans CREA and identified to be involved in the glucose repression. From the data of gel retardation, GRPT possess binding activity to the DNA fragments carrying putative consensus sequences. In vitro DNase I footprinting protection experiments reveal that binding of GRPT to the sequence 5-(G/C)TGGGG-3’, which matches well with the consensus sequences for MIG1/CREA repressors. We propose that the mediations by carbon catabolite repression in A. nidulans and Saccharomyces cerevisiae are operative in the cbh1 gene expression of T. koningii G-39. Moreover, reporter gene lacZ was expressed in S. cerevisiae to study the function of cbh1 promoter in vivo. Removal of the GRPT binding sites led the reporter -galactosidase activity of yeast in 2 % glucose to a level similar to that observed in a derepressing carbon source (5 % glycerol). The results indicated that glucose inhibition affects the —galactosidase activity more than galactose induction does. The data presented suggest that the five identified cis-acting elements, in cooperation with the repressor, GRPT, mediate the regulation of cbh1 transcription.
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Quirk, Amanda. "Atomic Force Microscopy Study of Endoglucanases and Cellobiohydrolases on Native Cellulose Films." Thesis, 2012. http://hdl.handle.net/10214/3468.
Full textAbstract:
Atomic force microscopy was used to image the action of cellulolytic enzymes in situ on never-dried native cellulose films. Cellomonas fimi, CenA was used as a model enzyme for proof of concept experiments and for the identification of different enzyme action on different cellulose structures. Inactive and active Trichoderma reesei enzymes EGI and CBHI were studied to disentangle the action of the cellulose binding domain from the catalytic domain.
A novel procedure, volume analysis, was developed to quantify changes in cellulose fibers as a result of this action. Volume analysis was used to compare fibers in different experiments (with different structural features and enzymes) regardless of where the change in the fiber occurred. The site-specific nature of cellulose-enzyme interactions is accessible using this analysis technique. Additionally, the reported volume change reflects a change in mass that is of interest for industrial purposes.
From inactive CBHI action there was no distinguishable change between enzyme action on defect or crystalline regions of the cellulose fiber. From the active enzyme results a quantifiable degradation event was measured. Digestion was initially quick then after one hour the volume plateaued. The crystalline cellulose region plateaued at -20 ± 1% and the defect region at -31 ± 2%.
The inactive EGI enzyme was found to have significant non-hydrolytic action on insoluble cellulose fibers. There was more significant swelling effect on the defect than the crystalline regions of the cellulose fiber. From the active EGI results a quantifiable degradation event was measured followed by swelling events. Degradation was initially quick with the total mass loss occurring within the first hour of the experiment. The volume then increased as the enzyme induced swelling of the fiber structure. The extent of degradation and swelling is structure limited with more disordered regions showing larger decreases in volume and predominantly crystalline regions showing mainly swelling events.
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Reiter, Kyle. "Analysis of Enzymatic Degradation of Cellulose Microfibrils by Quantitative Surface Plasmon Resonance Imaging." Thesis, 2012. http://hdl.handle.net/10214/4895.
Full textAbstract:
Cellulose is the most plentiful biopolymer on the planet, and as such, is a large potential energy source. Converting cellulose into ethanol first requires the disruption of the crystallinity of cellulose fibers and subsequent hydrolysis into glucose. The glucose can then be fermented, producing ethanol. The conversion of cellulose fibers to glucose is an energy intensive and costly step, which is a barrier to industrial production of cellulosic ethanol. The use of enzymes to facilitate this conversion is a promising approach. In the present study, the action of individual enzymes and combinations of enzymes from the Hypocrea jecorina secretome on bacterial cellulose fibers has been studied, to better understand their individual and synergistic action.
I have used a custom Surface Plasmon Resonance imaging (SPRi) device to measure changes in the thickness of cellulose fiber coverage of a thioglucose-functionalized gold substrate upon exposure to enzymes. The cellulose fibers were deposited using a Langmuir-Blodgett technique, resulting in non-uniform cellulose coverage of the substrate. By defining local Regions of Interest (ROIs) of the cellulose-covered gold film, and by measuring the SPR curves at elevated temperature for the ROIs as a function of time, we are able to determine the rate and extent of degradation of the cellulose fibers within individual ROIs. We have fit the change in SPR angle over time after exposure to enzyme to an exponential decay function that allows us to determine the average time constant of action of these enzymes on the deposited cellulose fibers.
We have used the above procedure to measure the average time constants of action and the average degradation fraction (the change in average thickness divided by the initial average thickness) of cellulose fibers exposed to CBH-1, CBH-2, and EG-1, as well as combinations of these enzymes. We have measured an increase in the average degradation fraction and a decrease in the average time constants of action for cellulose fibers exposed to 23 μg/mL CBH-2 compared to fibers exposed to the same concentration of CBH-1. Additionally, for concurrent exposure of CBH-1 and EG-1 (with individual concentrations of 23 μg/mL), as well as concurrent introduction of CBH-1, CBH-2 and EG-1, we observed increases in the average degradation fraction and decreases in average time constants relative to the values measured for the individual enzymes. These measurements allow us to determine the relative activity of these enzymes and they demonstrate cooperativity and complementarity of action of the different enzymes.
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Wu, Indira. "Engineering Thermostable Fungal Cellobiohydrolases." Thesis, 2013. https://thesis.library.caltech.edu/7657/1/Indira_Wu_thesis_doublesided.pdf.
Full textAbstract:
Meeting the world's growing energy demands while protecting our fragile environment is a challenging issue. Second generation biofuels are liquid fuels like long-chain alcohols produced from lignocellulosic biomass. To reduce the cost of biofuel production, we engineered fungal family 6 cellobiohydrolases (Cel6A) for enhanced thermostability using random mutagenesis and recombination of beneficial mutations. During long-time hydrolysis, engineered thermostable cellulases hydrolyze more sugars than wild-type Cel6A as single enzymes and binary mixtures at their respective optimum temperatures. Engineered thermostable cellulases exhibit synergy in binary mixtures similar to wild-type cellulases, demonstrating the utility of engineering individual cellulases to produce novel thermostable mixtures. Crystal structures of the engineered thermostable cellulases indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by proline substitutions. At high temperature, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A and wild-type Cel6A. The mechanism of thermal inactivation in this cellulase family is consistent with disulfide bond degradation and thiol-disulfide exchange. Enhancing the thermostability of Cel6A also increases tolerance to pretreatment chemicals, demonstrated by the strong correlation between thermostability and tolerance to 1-ethyl-3-methylimidazolium acetate. Several semi-rational protein engineering approaches – on the basis of consensus sequence analysis, proline stabilization, FoldX energy calculation, and high B-factors – were evaluated to further enhance the thermostability of Cel6A.
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Sangseethong, Kunruedee. "Immobilized cellooligosaccharides in the study of trichoderma reesei cellobiohydrolases." Thesis, 1999. http://hdl.handle.net/1957/27212.
Full textAbstract:
A novel type of model substrates, i.e. immobilized p-aminophenyl-β-D-cellooligosaccharides,
was developed and used in the study of exocellulases. The
two major cellobiohydrolases from Trichoderma reesei, CBH I and CBH II were
used as representative enzymes. p-Aminophenyl derivatives of cellobiose (PAPG₂),
cellotriose (PAPG₃), and cellotetraose (PAPG₄) were synthesized from the reaction
of p-nitrophenol and peracetylated glycosyl bromide of the corresponding
cellooligosaccharides under the phase-transfer catalyzed conditions, followed by
deacetylation and catalytic hydrogenation. p-Aminophenyl cellooligosaccharides
were then tethered via their amino functional groups to N-hydroxy succinimide-activated
agarose. The ability of CBH I and CBH II to associate with and catalyze
the hydrolysis of reducing end tethered cellooligosaccharides was tested. CBH I
catalyzed the hydrolysis of free PAPG₂ but CBH II did not. Both CBH I and CBH
II reversibly bound, but did not hydrolyze, immobilized PAPG₂. Hence, the
immobilized PAPG₂ was tested for the affinity chromatographic application. PAPG₂ is shown to be an effective ligand for the chromato graphic fractionation of
cellobiohydrolases (CBHs). The PAPG₂-derivatized agarose specifically retained
the CBH component of relatively complex cellulase mixtures. The purity of the
resulting CBH preparation was comparable to that of corresponding enzyme
preparations obtained using more traditional thioglycoside-based affinity ligands.
The application of PAPG₂ as an affinity ligand suggests that the immobilized
reducing end-blocked ligand associate with the T. reesei CBHs in a catalytically
nonproductive mode.
The catalytic activity for the hydrolysis of free and immobilized arylcellodextrins
by the CBH I and CBH II were determined. CBH II attacked free and
immobilized PAPG₃ and PAPG₄ in a typical exo manner in which cellobiose is a
major hydrolytic product released from the nonreducing end. The rate of hydrolysis
increases with increasing chain length suggesting the extended binding sites (at
least 4 binding sites). Like CBH II, CBH I preferentially cleaved immobilized
PAPG₃ and PAPG₄ at a second glycosidic linkage from the nonreducing end; the
rate of hydrolysis increases as a function of chain length. However, it attacked free
aryl-cellodextrins in a random manner. The rate of hydrolysis increases only from
PAPG₂ to PAPG₃ and significantly drops in PAPG₄. This suggests that CBH I
interacts with free and immobilized substrates in different modes.Graduation date: 1999
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山根, 隆., 信司 飯島, 能雅 佐藤, 勲. 田中, 安雄 畑, 賢作 濱田, 繁春 原田, et al. "解糖系酵素の構造と機能." 1993. http://hdl.handle.net/2237/12974.
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Žifčáková, Lucia. "Charakterizace společenstva hub, podílejícího se na rozkladu opadu v jehličnatých lesích Národního parku Šumava." Master's thesis, 2012. http://www.nusl.cz/ntk/nusl-310481.
Full textAbstract:
Understanding of carbon cycling in coniferous forests that represent a large carbon sink is crucial for our understanding of natural processes under global climate change. Recognition of fungi as fundamental decomposers can contribute to this understanding. Fungi are able to decompose numbers of substrates and possess a variety of enzymes to do so In this study I present litter decomposing fungi in mountain spruce forest from national park Šumava. The aim of my thesis was to follow succession and community changes of fungi from the early stages of decomposition of Picea abies needles until degradation of organic matter in the organic horizon of the soil. This aim was accomplished partly by recording the extracellular enzyme production of fungi in different stages of decomposition from needles attached to the twigs of a fallen tree to a litter material in later stages of decomposition on the soil surface. In addition to testing of fungi on their natural substrata - needle litter, enzyme activities were also measured in laboratory agar cultures, which allow comparison of diverse fungi with different origins. Enzyme activities were aimed at enzymes decomposing cellulose and compounds found in litter. Although ecology of endophytic and saprothrophic fungi suggest differences in enzyme production, these...