Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences & Dept. of Chemistry & Biomolecular Sciences), 2007. / Thesis by publication. "August 2006" Bibliography: leaves 157-183.

No description available.

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.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences, 2005. / Supplementary material to figures contained on DVD only available with manuscript. Bibliography: p. 221-244.

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.

Cellulolytic enzymes capable of efficiently degrading crystalline cellulose are a complex mixture of endo- (endoglucanases) and exo-acting (cellobiohydrolases) enzymes. One approach to separating these enzymes is affinity chromatography. A new ligand, p-aminophenyl l-thio-β-D-cellobioside (APTC), is introduced for this purpose. The property of APTC in affinity chromatography is demonstrated using Trichoderma reesei cellulases. The behavior of these enzymes on APTC-affinity column was essentially equivalent to that reported for the same enzymes on p-aminobenzyl 1-thio-β- D-cellobioside (ABTC)-columns; ABTC being the traditional ligand for affinity chromatography of exocellulases. The primary advantage of the APTC ligand is its ease of preparation. The affinity between CBHs and APTC may be considerably affected by nonspecific interactions. In this study, the significance of nonspecific protein/matrix interactions in affinity chromatography of cellulolytic enzymes is evaluated. The role of pH, NaCl, coupling conditions and stationary phase functional groups (N-hydroxysuccinimide ester and cyanogen bromide) on the affinity purification of Trichoderma reesei CBHs has been systematically determined. The results suggest that the apparent discrepancies in existing methods for the affinity purification of CBHs are due to nonspecific interactions, i.e. ionic interactions, between the enzymes and the stationary phase matrix. Exocellulases can be classified into two classes, based on their hydrolytic specificities. Class I enzymes preferentially hydrolyze cellulose from the reducing end, while Class 11 enzymes preferentially hydrolyze cellulose from the nonreducing end. Trichoderma reesei CBH I is a class I enzyme and CBH 11 is a class II enzyme. CBH I and CBH II are both retained on the APTC-affinity column; showing that both CBH classes bind to immobilized APTC. To further understand the differences in the two CBH classes, the behavior of CBH I and CBH II on the APTC-affinity column was compared. The affinity of CBH I for immobilized APTC was found to decrease when glucose was present in the system. In contrast, glucose was found to increase the affinity of CBH 11 for immobilized APTC. An outcome of this difference is that in the presence of glucose CBH I can be selectively eluted from the column. Equilibrium binding studies with each enzyme clearly reflect that CBH II has a higher affinity for immobilized APTC than CBH I. / Graduation date: 1998

No description available.

Thèse d'exercice : Pharmacie : Université de Nantes : 2003. / Bibliogr. f. 71-75.

No description available.

The influence of substrate and enzyme concentration on the rate of saccharification of two defined, insoluble, cellulose substrates, Avicel and Solka-Floc, by the cellulase enzyme system of Trichoderma viride has been evaluated. Assays utilized enzyme concentrations ranging from 0.014 to 0.056 filter paper unit per mL and substrate concentrations up to 10% (w/v). Analysis by initial velocity methods found the maximum velocity of the enzyme to be nearly equivalent for the two substrates and the km for the two substrates to be of similar magnitude, i.e., 0.20% for Solka-Floc and 0.63% for Avicel (w/v). Studies utilizing relatively high substrate concentrations (greater than 15 times the Km) demonstrated that the enzyme exhibits very different apparent substrate inhibition properties for the two substrates. The rate of saccharification of Avicel at relatively high substrate concentrations was up to 35% lower than the maximum rate which was obtained at a lower substrate concentration. The Avicel concentration corresponding to the maximum rate of saccharification was dependent on enzyme concentration. In contrast to the results with Avicel, the enzyme did not exhibit substrate inhibition with the Solka-Floc substrate. Potential differences in the degree of substrate inhibition with different substrates, as reported in this paper, is particularly relevant to the experimental design of comparative studies. / Graduation date: 1990