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.

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

Graduation date: 1994

In this study the enzymatic activity of adsorbed Thermomonospora fusca E��� and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. In particular, cellulases were allowed to contact hydrophobic polystyrene surfaces under conditions of different solution concentrations, and adsorption times. Each of these variables is known to have a potential effect on enzyme structure and activity at an interface. Enzymatic activity was measured after partial elution of the adsorbed layer with both protein-free buffer and the surfactant, dodecyltrimethylammonium bromide. For E��� at high concentration (0.5 mg/ml), adsorbed enzyme activity decreased about 20% in increasing adsorption time from 0.25 h to 24 h. At low concentration (0.001 mg/ml), adsorbed enzyme activity decreased by one order of magnitude during a 24 h period. CBHI layers lost activity only after a sufficiently long contact time with the surface, and this effect was not strongly dependent on enzyme concentrations in solution. These findings were explained with reference to structural changes undergone by adsorbed enzyme as a function of time and available interfacial area. / Graduation date: 2000

Graduation date: 1999

There are generally four factors recognized as delimiting in the study of lignocelluloses for fuel ethanol production, viz., the source of the cellulase-system and its quality characteristics for cellulose hydrolysis, the substrate and pretreatment method, the process for cellulase production and bioreactor design, and the ability of yeast to ferment mixed hexose and pentose sugars. Wheat straw (WS) and T. reesei mutants were used in the study to evaluate the production of cellulase-systems. Hydrolysis of cellulose revealed the superiority of mild NaOH pretreatment over steam explosion for cellulase production with T. reesei MCG 80 and QMY-1. Response surface models were capable of predicting that NaOH could be used for the pretreatment of WS at 4% (w/w) without urea in the fermentation medium to yield optimum filter paper activity (FPA) of 9.9 IU/mL (247 IU/g WS) and beta-glucosidase activity ($ beta$GA) of 6.4 IU/mL (159 IU/g WS) under solid-state fermentation (SSF) conditions. Multiple regression analysis with multiple coefficients of correlation, R, between 0.957 and 0.99 from the experimental data showed close agreement between the cellulase activities (FPA and $ beta$GA) from the experiments and predicted values. / The superiority of SSF over liquid-state fermentation (LSF) in the production of cellulase-systems was also established, and a prototype pan-bioreactor showed good potential for upgrading cellulase production under SSF conditions. The economics of fuel ethanol production was considered in the optimization model that sought to establish threshold cellulase loadings needed to achieve maximum cellulose hydrolysis for fermentation. High substrate concentrations of up to 7.5% were hydrolyzed with cellulase loadings of 24-30 IU/g and fermented by Pichia stipitis to achieve 90-100% conversion into ethanol. / Crude unextracted cellulase yielded over 90% hydrolysis of delignified wheat straw and proved to be better than extracted cellulase and commercial cellulases for the hydrolysis of pure cellulose and pretreated wheat straw. Studies were also conducted to demonstrate the importance of the ratio of $ beta$GA- to FPA in cellulose hydrolysis which showed that ratios closer to one (1), produced more sugars and lowered the cellobiose content in the hydrolysates. It was also shown that the source of the cellulase is important in eliminating the accumulation of cellobiose during hydrolysis as was demonstrated with cellulase from mixed cultures of T. reesei and Aspergillus phoenicis. Higher $ beta$GA from the latter were implicated since A. phoenicis is a good $ beta$-glucosidase producer. / Delignified wheat straw at 5% concentration when subjected to separate hydrolysis and fermentation and simultaneous hydrolysis and fermentation resulted in similar volumetric productivities (g/L/h) of ethanol.

Thesis (doctoral)--University of Helsinki, 2002. / Includes bibliographical references. Also available on the World Wide Web.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-12T14:01:35Z No. of bitstreams: 2 DISSERTAÇÃO Márcia Josefa da Silva.pdf: 1750850 bytes, checksum: d36504435c0ecdaffe970fd5045e35c0 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T14:01:35Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Márcia Josefa da Silva.pdf: 1750850 bytes, checksum: d36504435c0ecdaffe970fd5045e35c0 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-02-28 / CAPES / A biomassa lignocelulósica destaca-se como matéria-prima alternativa para a produção de combustíveis e outros produtos. Devido à alta complexidade desse material, é necessária uma hidrólise enzimática eficiente com a utilização de um pool enzimático adequado. O objetivo deste trabalho foi estudar o perfil de produção de enzimas celulolíticas e xilanolítica por Trichoderma reesei RUT C-30 em meios com diferentes capacidades de indução. A produção de enzimas foi realizada em biorreator de bancada (Bioflo 110) com 1,3 ou 3 L de volume de trabalho, nas seguintes condições: 500 rpm, 28° C, 2 vvm e pH 5,0. As fontes de carbono investigadas foram: lactose, xilana, pectina, celulose microcristalina, melaço, biomassa de palma forrageira e hidrolisado hemicelulósico. O hidrolisado foi obtido por tratamento hidrotérmico de bagaço de cana-de-açúcar em reator descontínuo de 20 L (Regmed AU/20), com volume de trabalho de 10 L e carga de sólidos de 5% (m/v), a 185°C, por 16 minutos. Em substratos solúveis, a determinação da concentração celular foi realizada por peso seco e as concentrações dos substratos foram obtidas por cromatografia líquida de alta eficiência. Ao final dos cultivos, foram isoladas proteínas extracelulares, que servirão para futura análise do secretoma de T. reesei RUT C-30. Em meio de lactose, os valores de atividades enzimáticas obtidos com 54 horas de cultivo foram: FPase (1,43 UI mL-1), CMCase (15,67 UI mL-1), xilanase (11,91 UI mL-1) e β-glicosidase (0,24 UI mL-1). A velocidade máxima específica de crescimento, μmax, e o coeficiente de rendimento de biomassa no substrato, Yx/s, foram 0,06 h-1 e 0,38 g g-1, respectivamente. Em meio de melaço, μmax e Yx/s foram 0,26 h-1 e 0,52 g g-1, respectivamente, e as atividades enzimáticas insignificantes. No hidrolisado, o crescimento do micro-organismo foi inibido devido à presença de compostos inibidores produzidos no tratamento hidrotérmico. Os valores de atividades enzimáticas com 54 horas foram: FPase (0,06 UI mL-1), CMCase (0,24 UI mL-1), xilanase (1,40 UI mL-1) e β-glicosidase nula. Em meio com celulose, também foram obtidos baixos valores de atividades enzimáticas, porém, a xilanase apresentou valor de 1,52 UI mL-1 com 50 horas de cultivo. A xilanase foi a enzima mais evidente nos cultivos com xilana, atingindo valor máximo de 11,93 UI mL-1, ao final do cultivo. Em meio com pectina: FPase, CMCase, xilanase e β-glicosidase foram: 0,01 UI mL-1, 1,25 UI mL-1, 2,83 UI mL-1 e 0,10 UI mL-1, respectivamente. Nos cultivos em meio à base de palma, observaram-se os seguintes valores com 50 horas: FPase (0,29 UI mL-1), CMCase (3,30 UI mL-1), xilanase (6,21 UI mL-1) e β-glicosidase (0,09 UI mL-1). Entre as fontes investigadas, a lactose é o melhor substrato para a indução de todas as enzimas estudadas, enquanto o melaço favorece o crescimento rápido do micro-organismo. O hidrolisado hemicelulósico e a palma forrageira são potenciais meios para a produção de enzimas, em particular xilanases. Para a utilização do hidrolisado, no entanto, será necessária a sua detoxificação ou, alternativamente, a obtenção de linhagens resistentes aos inibidores por engenharia metabólica e/ou engenharia evolutiva.

No description available.

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