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1
Gal, Laurent. "Etude du cellulosome de Clostridium cellulolyticum et de l'un de ses composants : la cellulase CelG." Aix-Marseille 1, 1997. http://www.theses.fr/1997AIX11071.
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Le cellulosome de la bacterie cellulolytique clostridium cellulolyticum atcc 35319 a ete etudie. C'est un complexe de 600 kda, en moyenne, qui degrade efficacement la cellulose cristalline. Il est constitue de plusieurs proteines de masse moleculaire relative variant entre 170 et 30 kda. L'utilisation du minicipc1 biotinyle a permis de montrer qu'au moins treize d'entre elles possedaient une dockerine. Parmi celles-ci, les proteines cela, celc, cele, celf et celg, dont les genes sont clones et sequences, ont ete identifiees. Afin de rechercher de nouveaux genes cel codant pour des proteines possedant une dockerine, une marche sur le chromosome en aval de cele a ete entreprise. Elle a revele l'existence de trois nouveaux genes impliques dans la cellulolyse : cipx, celh et celj. - cipx est une proteine tres originale constituee d'un domaine de type linker et d'une cohesine. - celh et celj, tout comme celg, possedent un domaine catalytique de la famille 9 de la classification des glycosyl-hydrolases, suivi d'un domaine de type cbd de la famille iii et enfin d'une dockerine c-terminale. Pour tenter de comprendre le role joue par ce dernier type de proteines dans la cellulolyse, la caracterisation biochimique de celg a ete entreprise. Le gene celg a ete surexprime chez escherichia coli et la proteine recombinante correspondante a ete purifiee. Celg est une endoglucanase qui degrade efficacement la cmc et le glucane d'orge. Elle est egalement active sur la cellulose amorphe et degrade les cellodextrines ayant un degre de polymerisation superieur ou egal a trois. Contrairement aux autres endoglucanases caracterisees jusqu'a present chez c. Cellulolyticum, celg degrade les celluloses cristallines, et en particulier la bmcc de maniere tres efficace. Pour etudier l'influence relative de chacun des domaines de celg dans ce mecanisme, differentes constructions genetiques, permettant la synthese du domaine catalytique seul ou du domaine de type cbd fusionne derriere la glutathion-s-transferase, ont ete realisees. Dans tous les cas, les proteines recombinantes ne presentent ni activite catalytique ni proprietes d'adhesion a la cellulose. L'ensemble de ces resultats suggere qu'a la difference de la plupart des cellulases, les differents domaines constitutifs de celg ne sont pas independants. La presence d'au moins deux autres proteines homologues a celg (celh et celj) au sein du systeme cellulolytique de c. Cellulolyticum suggere que ces proteines jouent un role fondamental dans les mecanismes de degradation de la cellulose chez cet organisme.
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Thomas, D. J. "Microbial cellulase systems." Thesis, Swansea University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639202.
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The work presented studies the cellulolytic system of Trichoderma koningii with particular reference to its ability to produce "short fibres" in the early stages of cellulose degradation. The culture filtrate of this organism was shown to produce short fibres from both filter paper (Whatman No.1) and cotton (Texas, non-dewaxed). The optimum conditions for production were identified and an assay system developed to measure this activity. Assay using filter paper was rapid and sensitive in determining short fibre producing activity, all results were subsequently confirmed on the more resistant substrate (cotton). The cellulase system was separated using an ion exchanger with a non-carbohydrate matrix and affinity chromatography on cellulose. Initial separation on ion exchange yielded the main cellobiohydrolase (CBH 1). Another fraction from this column separated on cellulose columns gave purified fractions of β-glucosidase, CM-cellulase and the short fibres forming activity (D2Cc). Only this latter fraction produced short fibres and synergised with CM-cellulase and β-glucosidase to increase short fibre production. Short fibres produced by D2Cc were more susceptible to subsequent hydrolysis by culture filtrate or CBH 1, degraded (bacterial) cellulose showed no physical changes on action of D2Cc but subsequent hydrolysis by CBH 1 or culture filtrate was increased. The main product of D2Cc was cellobiose but some cellotriose was detected from filter paper. D2Cc was inactive against cellobiose and cellotriose, both were potent inhibitors of D2Cc activity. Cellobiose was also an inhibitor of CBH 1 but cellotriose was not. D2Cc was shown to reduce the DP of bacterial cellulose. D2Cc and CBH 1 synergised in hydrolysing degraded cellulose, filter paper and cotton. The suggested role for this enzyme component is that it produces short fibres in concert with CM-cellulase which are then attacked by CBH 1 to produce cellobiose which is utilized by the organism.
3
Tchunden, Jeannette. "Cellulolyse Anaérobie Mésophile : étude de l'amélioration de la production de cellulases par Cl. cellulolyticum ATCC 35319." Nancy 1, 1990. http://docnum.univ-lorraine.fr/public/SCD_T_1990_0044_TCHUNDEN.pdf.
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Cl. Cellulolyticum est une bactérie cellulolytique mésophile isolée au laboratoire à partir d'herbes en décomposition. Cette bactérie est capable de dégrader la cellulose en une seule étape en acides organiques et en éthanol. Mais l'activité cellulolytique de la souche reste faible. Pour arriver à une possibilité d'utilisation industrielle de la bactérie, les performances cellulolytiques de la souche doivent être améliorées. L'amélioration de la production de cellulases par cl. Cellulolyticum passe à la fois par l'amélioration de ses conditions de culture et l'amélioration de la souche. Ainsi nous avons montré qu'une culture en fermenteur avec régulation de ph à 7,2 par une solution d'ammoniac 4n conduit à une plus forte production de cellulases. Par l'emploi du rayonnement ultraviolet comme agent mutagène et de cellobiose comme crible de sélection, nous avons isolé le mutant 42 qui produit deux fois plus de cellulases que la souche parent. Nous avons également montré que la cellulose avicel est meilleure inductrice de l'endo et de l'exoglucanase aussi bien chez cl. Cellulolyticum que chez le mutant. L'étude de l'association de cl. Cellulolyticum et du mutant 42 a permis de produire 4,7 fois plus d'activité xylanasique.
4
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.
5
Qian, Chen. "Adsorption of Xyloglucan onto Cellulose and Cellulase onto Self-assembled Monolayers." Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/42496.
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Adsorption of xyloglucan (XG) onto thin desulfated nanocrystalline cellulose (DNC) films was studied by surface plasmon resonance spectroscopy (SPR), quartz crystal microbalance with dissipation monitoring (QCM-D), and atomic force microscopy (AFM) measurements. These studies were compared to adsorption studies of XG onto thin sulfated nanocrystalline cellulose (SNC) films and regenerated cellulose (RC) films performed by others. Collectively, these studies show the accessible surface area is the key factor for the differences in surface concentrations observed for XG adsorbed onto the three cellulose surfaces. XG penetrated into the porous nanocrystalline cellulose films. In contrast, XG was confined to the surfaces of the smooth, non-porous RC films. Surprisingly surface charge and cellulose morphology played a limited role on XG adsorption.
The effect of the non-ionic surfactant Tween 80 on the adsorption of cellulase onto alkane thiol self-assembled monolayers (SAMs) on gold was also studied. Methyl (-CH3), hydroxyl
(-OH) and carboxyl (-COOH) terminated SAMs were prepared. Adsorption of cellulase onto untreated and Tween 80-treated SAMs were monitored by SPR, QCM-D and AFM. The results indicated cellulase adsorption onto SAM-CH3 and SAM-COOH were driven by strong hydrophobic and electrostatic interactions, however, hydrogen bonding between cellulase and SAM-OH was weak. Tween 80 effectively hindered the adsorption of cellulase onto hydrophobic SAM-CH3 substrates. In contrast, it had almost no effect on the adsorption of cellulase onto SAM-OH and SAM-COOH substrates because of its reversible adsorption on these substrates.Master of Science
6
Du, Plessis Lisa. "Co-expression of cellulase genes in Saccharomyces cerevisiae for cellulose degradation." Thesis, Link to the online version, 2008. http://hdl.handle.net/10019/1818.
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Zhu, Zhiguang. "Investigating biomass saccharification for the production of cellulosic ethanol." Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/32189.
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The production of second generation biofuels -- cellulosic ethanol from renewable lignocellulosic biomass has the potential to lead the bioindustrial revolution necessary to the transition from a fossil fuel-based economy to a sustainable carbohydrate economy. Effective release of fermentable sugars through biomass pretreatment followed by enzymatic hydrolysis is among the most costly steps for emerging cellulosic ethanol biorefineries.
In this project, two pretreatment methods (dilute acid, DA, and cellulose solvent- and organic solvent-lignocellulose fractionation, COSLIF) for corn stover were compared. It was found that glucan digestibility of the corn stover pretreated by COSLIF was much higher, along with faster hydrolysis rate, than that by DA- pretreated. This difference was more significant at a low enzyme loading. Quantitative measurements of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were established to find out the cause. The COSLIF-pretreated corn stover had a CAC nearly twice that of the DA-pretreated biomass. Further supported by qualitative scanning electron microscopy images, these results suggested that COSLIF treatment disrupted microfibrillar structures within biomass while DA treatment mainly removed hemicelluloses, resulting in a much less substrate accessibility of the latter than of the former. It also concluded that enhancing substrate accessibility was the key to an efficient bioconversion of lignocellulose.
A simple method for determining the adsorbed cellulase on cellulosic materials or pretreated lignocellulose was established for better understanding of cellulase adsorption and desorption. This method involved hydrolysis of adsorbed cellulase in the presence of 10 M of NaOH at 121oC for 20 min, followed by the ninhydrin assay for the amino acids released from the hydrolyzed cellulase. The major lignocellulosic components (i.e. cellulose, hemicellulose, and lignin) did not interfere with the ninhydrin assay. A number of cellulase desorption methods were investigated, including pH adjustment, detergents, high salt solution, and polyhydric alcohols. The pH adjustment to 13.0 and the elution by 72% ethylene glycol at a neutral pH were among the most efficient approaches for desorbing the adsorbed cellulase. For the recycling of active cellulase, a modest pH adjustment to 10.0 may be a low-cost method to desorb active cellulase. More than 90% of cellulase for hydrolysis of the pretreated corn stover could be recycled by washing at pH 10.0.
This study provided an in-depth understanding of biomass saccharification for the production of cellulosic ethanol for cellulose hydrolysis and cellulase adsorption and desorption. It will be of great importance for developing better lignocellulose pretreatment technologies and improving cellulose hydrolysis by engineered cellulases.Master of Science
8
Mayende, Lungisa. "Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion." Thesis, Rhodes University, 2007. http://hdl.handle.net/10962/d1004032.
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Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
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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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Roussos, Sevastianos. "Croissance de Trichoderma harzianum par fermentation en milieu solide : physiologie, sporulation et production de cellulases /." Paris : Ed. de l'ORSTOM, 1987. http://catalogue.bnf.fr/ark:/12148/cb349545941.
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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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Lever, Mitchell. "Cellulose to ethanol conversion with on-site cellulase production using solid-state fermentation." Thesis, Lever, Mitchell (2009) Cellulose to ethanol conversion with on-site cellulase production using solid-state fermentation. PhD thesis, Murdoch University, 2009. https://researchrepository.murdoch.edu.au/id/eprint/32795/.
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The economics and environmental sustainability of enzymatic lignocellulose-to-ethanol conversion processes are adversely affected by the use of purchased cellulase preparations. Commercial cellulase preparations lack the microorganisms that produce them and thus cannot be cultured on-site, resulting in a significant ongoing expense. Commercial cellulase production is energy intensive and a significant contributor to the overall environmental impact of the cellulose to ethanol conversion process...
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Cerclier, Carole. "Films multicouches à base de polymères végétauxβ : élaboration et application à la détection d'activités enzymatiques." Nantes, 2010. http://archive.bu.univ-nantes.fr/pollux/show.action?id=19992f55-8948-4aa3-b33f-69e1bae3bb65.
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La cellulose et le xyloglucane constituent un réseau majeur de la paroi des cellules végétales. Ces molécules sont liées par des interactions de Van der Waals et des liaisons hydrogène que nous avons mis à profit pour l’élaboration de films multicouches nanométriques. En fonction de leur épaisseur et de leur indice de réfraction, les films nanométriques peuvent présenter des couleurs qui sont dues à des phénomènes d’interférences. De tels films composés de nanocristaux de cellulose et de xyloglucane peuvent être valoriser comme détecteurs d’activité enzymatique de cellulases. En effet, l’action d’une enzyme hydrolytique peut être détectée par une diminution de l’épaisseur du film qui résulte en un changement de couleur. Les modes de croissance de deux types de films ont été étudiés afin de déterminer les conditions opératoires les plus appropriées pour l’obtention de films colorés. La structure interne de ces films a été déterminée par réflectivité de neutrons à l’état sec et en solution pour évaluer les différences de structures. Les cinétiques de dégradation des films par les cellulases ont également été comparées par QCM-D. Il s’avère que la détection de l’activité de cellulases avec les films nanométriques est plus rapide et plus sensible qu’une méthode colorimétrique utilisée usuellement. L’utilisation des films nanométriques pour la détection d’activité est également applicable à d’autres systèmes enzyme/substratCellulose and xyloglucan represent a major network from plant cell wall. Theses molecules are linked by Van der Waals interactions and hydrogen bonds and we have used these interactions in order to elaborate nanometric multilayered films. According to their thickness and refractive index, nanometric films can present bright colours due to interference phenomena. Such films composed of cellulose nanocrystals and xyloglucan can be used as cellulases enzymatic activities detector. Hydrolytic enzyme action can actually be detected by film thickness decrease and results in a colour change. Growth mode has been studied for two kinds of film and the better conditions were chosen to obtain coloured films. Films internal structure has been investigated by neutron reflectometry on dry films and films in solution in order to evaluate the structures differences. Films hydrolysis kinetics has also been compared by QCM-D. Cellulases activities detection using nanometric films is faster and more sensitive than a colorimetric method usually used. We have also proved that this technique can be used for other enzyme/substrate systems
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Langsford, Maureen Lynn. "The purification and characterization of two cellulose-binding, glycosylated cellulases from the bacterium Cellulomonas fimi." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28852.
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Cellulomonas fimi secretes several cellulase activities as well as protease activity into the culture medium. In contrast, few activities are bound to the cellulose in the culture. To characterize the cellulase system and to Identify cloned gene products, it was necessary to purify native, intact cellulases. We hypothesized that the cellulose-bound cellulases would be protected from proteolysis, and therefore represent the intact enzymes.
Two cellulases were purified from Cellulomonas fimi. Avicel was recovered from cultures and the proteins were eluted from it with guanidine-HCl (Gdn-HCl). The Gdn-HCl extract was fractionated by Concanavalin A-Sepharose affinity column chromatography and by Mono Q anion exchange column chromatography. The cellulases purified by this procedure were an endoglucanase, EngA, and an exoglucanase, Exg. The purified enzymes were characterized. EngA has Mr 57,000, pi 8.2, and is 10 % mannose by weight. Exg has Mv 56,000, pi 5.8, and is 8 % mannose by weight.
Two recombinant DNA plasmids were identified as encoding EngA and Exg. The recombinant gene products were not glycosylated. The role of glycosylation was studied by comparing some properties of the recombinant EngA and Exg with the native EngA and Exg. Both glycosylated and unglycosylated forms bound to Avicel. Sensitivity to the C. fimi protease was also compared. The glycosylated enzymes were protected from proteolysis when bound to cellulose. In contrast, the unglycosylated forms were processed to yield active, truncated products with greatly reduced affinity for cellulose. The cleavage site was predicted based on size of the products and reactivity with anti-PT serum. The N-terminal region of EngA and the C-terminal of Exg show 50 % conservation of sequence (Warren et al., 1986). This region appears to be the cellulose-binding domain and is not required for the hydrolysis of soluble substrates. The C. fimi protease can partially degrade glycosylated EngA when it is not bound to cellulose. Some of the multiple CMCase activities in culture supernatants are derived from EngA by partial proteolysis.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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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 organismesThe 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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Imai, Makiko. "Analysis of interaction between cellulosic biomass and saccharification enzymes." Kyoto University, 2020. http://hdl.handle.net/2433/252998.
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Deng, Yu. "Characterization and genetic analysis of the cellulolytic microorganism Thermobifida fusca." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2440.
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Currently, one of the hurdles hindering efficient production of cellulosic biofuel is the recalcitrant nature of cellulose to hydrolysis. A wide variety of cellulase enzymes are found natively in microorganisms that can potentially be used to effectively hydrolyze cellulose to fermentable sugars. Thermobifida fusca is a high G-C content, thermophilic, gram-positive soil actinobacterium with high cellulolytic activity. The phenomenological and mechanistic parameters affecting cellulase activity were studied in T. fusca and two mechanisms have been found: 1) transcriptions of cellulase-related genes were not closely associated with measured differences in cellulase activity and 2) cellular energetics (intracellular ATP) correlated more closely to changes in specific cellulase activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat: 5’-(T)GGGAGCGCTCCC(A) that is upstream of many known cellulase genes. An efficient procedure for creating precise chromosomal gene replacements has been developed and this procedure was demonstrated by generating a celR deletion strain. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR potentially acts as a repressor for some cellulase genes and as an activator for other cellulase genes. Based on the protocol of disrupting celR gene, the direct conversion of untreated cellulosic biomass to 1-propanol in aerobic growth conditions using an engineered strain of T. fusca was demonstrated. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase (encoded by adhE2) was added to T. fusca leading to production of 1-propanol during growth on glucose, cellobiose, cellulose (Avicel), switchgrass, and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. The adaptive evolution of T. fusca was conducted to find a high cellulase-yield strain. The evolved strains of T. fusca were generated for two different scenarios: continuous exposure to cellobiose (strain muC with specialist phenotype) or alternating exposure to cellobiose and glucose (strain muS with generalist phenotype). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains and 18 and 14 point mutations in the muC and muS strains, respectively were verified. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. The experiment results were used to test genome-scale metabolic model of T. fusca built in this study.
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Abadie, Alicia Renée. "QUANTIFYING CELLULASE IN HIGH-SOLIDS ENVIRONMENTS." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_theses/548.
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In recent years, fungal and bacterial cellulases have gained popularity for the conversion of lignocellulosic material to biofuels and biochemicals. This study investigated properties of fungal (Trichoderma. reesei) and bacterial (Clostridium thermocellum) cellulases. Enzymatic hydrolysis was carried out with T. reesei using nine enzyme concentration and substrate combinations. Initial rates and extents of hydrolysis were determined from the progress curve of each combination. Inhibition occurred at the higher enzyme concentrations and higher solids concentrations. Mechanisms to explain the observed inhibition are discussed. Samples of C. thermocellum purified free cellulase after 98% hydrolysis were assayed to determine the total protein content (0.15 ± 0.08 mg/mL), the enzymatic activity (0.306 ± 0.173 IU/mL) and the cellulosome mass using the Peterson method for protein determination, the cellulase activity assay with phenol-sulfuric acid assay, and the indirect ELISA adapted for C. thermocellum cellulosomes, respectively. Issues regarding reproducibility and validity of these assays are discussed.
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Porter, Suzanne L. "Evidence of multiple cellulase forms in Trichoderma harzianum E58 and their significance in cellulose hydrolysis." Thesis, University of Ottawa (Canada), 1990. http://hdl.handle.net/10393/5829.
Full textAbstract:
The occurrence of multiple cellulase components of Trichoderma harzianum E58 and the implications of their existence on the hydrolysis of cellulose were examined. A single commercial enzyme preparation, Novo-Celluclast, showed different extents of hydrolysis of several cellulosic substrates over time. The filter paper activities of six batches of T. harzianum E58 showed poor correlation with the ability of these enzymes to hydrolyze other cellulosic substrates over extended periods of time. Hydrolysis of a single substrate by a single enzyme preparation resulted in similar slopes in reducing sugar production with enzyme concentration, between one-hour and twenty-four-hour hydrolyses. The multiplicity of the cellulase components of T. harzianum E58 was examined, and the number of endoglucanase components and their specificities towards $\beta$-1,4-linkages were studied. Several types of endoglucanases were produced by the fungus. The role of the exoglucanase was examined using sub-saturation concentrations of T. harzianum E58 cellulase. No significant increase in hydrolysis was observed when purified exoglucanase was added to the cellulase mixture. The high proportion of non-specific endoglucanases and the need for an efficient endoglucanase-to-exoglucanase ratio are discussed in terms of a modified model for cellulose hydrolysis. (Abstract shortened by UMI.)
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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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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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Barnard, Damian Kelly. "Design and construction of modular genetic devices and the enzymatic hydrolysis of lignocellulosic biomass." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/7688.
Full textAbstract:
The enzymatic deconstruction of lignocellulosic plant biomass is performed by specialist microbial species. It is a ubiquitous process within nature and central to the global recycling of carbon and energy. Lignocellulose is a complex heteropolymer, highly recalcitrant and resistant to hydrolysis due to the major polysaccharide cellulose existing as a crystalline lattice, intimately associated with a disordered sheath of hemicellulosic polysaccharides and lignin. In this thesis I aim to transfer the highly efficient cellulolytic mechanism of the bacterium Cellulomonas fimi, to that of a suitably amenable and genetically tractable expression host, in the hopes of better understanding the enzymatic hydrolysis of lignocellulose. Using tools and concepts from molecular biology and synthetic biology, I constructed a library of standardised genetic parts derived from C. fimi, each encoding a known enzymatic activity involved in the hydrolysis of cellulose, mannan or xylan; three of the major polysaccharides present in lignocellulose. Characterization assays were performed on individual parts to confirm enzymatic activity and compare efficiencies against a range of substrates. Results then informed the rational design and construction of parts into modular devices. The resultant genetic devices were introduced into the expression hosts Escherichia coli and Citrobacter freundii, and transformed strains were assayed for the ability to utilize various forms of xylan, mannan and cellulose as a sole carbon source. Results identified devices which when expressed by either host showed growth on the respective carbon sources. Notably, devices with improved activity against amorphous cellulose, crystalline cellulose, mannan and xylan were determined. Recombinant cellulase expressing strains of E. coli and C. freundii were shown capable of both deconstruction and utilization of pure cellulose paper as a sole carbon source. Moreover, this capacity was shown to be entirely unhindered when C. freundii strains were cultured in saline media. These findings show promise in developing C. freundii for bioprocessing of biomass in sea water, so as to reduce the use of fresh water resources and improve sustainability as well as process economics. Work presented in this thesis contributes towards understanding the complementarities and synergies of the enzymes responsible for lignocellulose hydrolysis. Moreover, the research emphasizes the merits of standardizing genetic parts used within metabolic engineering projects and how adopting such design principles can expedite the research process.
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Takahashi, Schmidt Junko. "Functional studies of selected extracellular carbohydrate-active hydrolases in wood formation /." Umeå : Dept. of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 2008. http://epsilon.slu.se/200876.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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Ravachol, Julie. "Rôle des glycosides hydrolases de famille 9 dans la dégradation de la cellulose et exploration du catabolisme de xyloglucane chez Ruminiclostridium cellulolyticum." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4054.
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R. cellulolyticum est une bactérie mésophile, anaérobie stricte et cellulolytique, qui sécrète des macro-complexes multienzymatiques (cellulosomes) très performants dans la dégradation des polysaccharides de la paroi végétale. Les Glycoside Hydrolases de famille 9 (GH9) sont toujours surreprésentées chez les bactéries à cellulosomes. Le génome de R. cellulolyticum code 13 GH9 dont 12 participent aux cellulosomes. Mon travail de thèse a consisté à étudier l’ensemble des GH9 de R. cellulolyticum, en déterminant leurs activités à l’état libre et en complexes, afin d’élucider leurs rôles dans la dégradation de la cellulose. Les GH9 ont chacune des activités et des spécificités de substrats différentes. Deux GH9 présentent des activités atypiques, puisque l’une d’elles est inactive et l’autre est une xyloglucanase. Les caractérisations en complexes ont souligné l’importance de la diversité des GH9 et ont montré qu’elles agissent en synergie dans la dégradation de la cellulose. De plus, l’élargissement du panel des GH9 de R. cellulolyticum par l’introduction d’une cellulase exogène de Lachnoclostridium phytofermentans a permis d’améliorer les capacités cellulolytiques de la clostridie. L’activité xyloglucanase d’une des GH9 m’a poussé à étudier le catabolisme du xyloglucane chez R. cellulolyticum. Ce travail a mis en exergue la présence d’un équipement spécialisé dans l’utilisation de ce sucre. Ainsi, après une dégradation du xyloglucane par les enzymes cellulosomales en xyloglucane dextrines, ces dernières sont importées dans le cytoplasme par un transporteur ABC spécifique puis hydrolysées séquentiellement par les enzymes cytoplasmiques en mono et disaccharides assimilablesRuminiclostridium cellulolyticum is a mesophilic and strictly anaerobic bacterium. It produces multienzymatic complexes called cellulosomes which efficiently degrade the plant cell wall polysaccharides. Family-9 Glycoside Hydrolases (GH9) are plethoric in cellulosome-producing bacteria. The genome of R. cellulolyticum thus encodes for 13 GH9 enzymes, 12 of them participate to the cellulosomes.My Ph. D. aimed at characterizing all GH9 enzymes from R. cellulolyticum, by determining their activities in a free and complexed states, in order to elucidate their role in cellulose degradation. All GH9 enzymes exhibit various activities and substrate specificities. Two of them have atypical activities, since one is inactive and one is a xyloglucanase. Results obtained when all GH9 are in complex highlighted the importance of GH9 diversity and revealed they act synergistically in cellulose depolymerization. Moreover, expanding the panel of GH9 enzymes by introducing an exogenous cellulase from Lachnoclostridium phytofermentans improved the cellulolytic capacities of R. cellulolyticum. The xyloglucanase activity of one GH9 enzyme prompted me to investigate the xyloglucan catabolism in R. cellulolyticum. This work uncovered the presence of a specialized equipment for xyloglucan utilization. After extracellular digestion of xyloglucan by cellulosomal enzymes, xyloglucan dextrins are imported into the cytoplasm via a specific ABC-transporter and sequentially hydrolyzed by cytoplasmic enzymes into fermentable mono and disaccharides
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Klose, Holger [Verfasser]. "Recombinant cellulase production in plants / Holger Klose." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2014. http://d-nb.info/105943136X/34.
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Zhang, Qin. "COLLECTION OF TRICHODERMA REESEI CELLULASE BY FOAMING." University of Akron / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=akron1195069754.
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Tominaga, Rumi. "Cellulase-induced Cell Growth in Higher Plants." Kyoto University, 2001. http://hdl.handle.net/2433/150771.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第9000号
農博第1182号
新制||農||821(附属図書館)
学位論文||H13||N3519(農学部図書室)
UT51-2001-F330
京都大学大学院農学研究科森林科学専攻
(主査)教授 伊東 隆夫, 教授 酒井 富久美, 教授 島田 幹夫
学位規則第4条第1項該当
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Chahal, Parminder Singh. "Cellulase production from lignocellulosic materials by Trichoderma reesei." Thesis, University of Ottawa (Canada), 1987. http://hdl.handle.net/10393/5536.
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Gough, Clare Linda. "Molecular genetics of cellulase production by Xanthomonas campestris." Thesis, University of East Anglia, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327816.
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Maamar, Hédia. "Etude in vivo du système cellulolytique de Clostridium cellulolyticum : caractérisation du premier mutant d'insertion cipC." Aix-Marseille 1, 2003. http://www.theses.fr/2003AIX11034.
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La bactérie anaérobie Clostridium cellulolyticum, produit des complexes multiprotéiques (cellulosomes) qui dégradent la cellulose. Ces complexes sont composés de plusieurs enzymes ancrées sur une protéine d'assemblage (CipC). La plupart des gènes codant pour les sous unités des cellulosomes sont groupés dans un cluster de 26 kb. Deux mutants spontanés d'insertion du gène cipC (1er gène du cluster) ont été obtenus. CipC est interrompu par une seule séquence d'insertion (IS) (ISCce1) dans un cas ou par deux ISs (ISCce1 interrompue par ISCce2) dans l'autre cas. ISCce2 a été classée dans la famille des IS256, alors que ISCce1 pourrait être le premier membre d'une nouvelle famille d'IS. L'insertion dans cipC affecte la synthèse des cellulosomes et la capacité de la souche à dégrader la cellulose cristalline. Le mutant produit une protéine cipC tronquée et assemble des complexes qui n'incluent aucune des enzymes codées par les gènes du cluster. Ces complexes contiennent au moins 12 protéines à dockérine dont la majorité sont inconnues. Le mutant produit également 3 protéines majoritaires inconnues ne faisant pas partie des cellulosomes. La complémentation d'un mutant par clonage du gène cipC n'a pas permis de restaurer le phénotype sauvage. Les perturbations observées chez le mutant et la souche complémentée sont dues à l'effet polaire puissant de l'interruption de cipC sur l'expression des gènes en aval. L'intégration du plasmide dans le chromosome par recombinaison homologue a seule permis de restaurer le phénotype sauvage en rétablissant la succession des gènes dans le cluster. L'étude du mutant suggère un lien transcriptionnel entre les gènes du cluster.
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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)0048
新制・課程博士
博士(農学)
甲第19758号
農博第2154号
新制||農||1039(附属図書館)
学位論文||H28||N4974(農学部図書室)
32794
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 佐藤 健司, 教授 山下 洋, 准教授 豊原 治彦
学位規則第4条第1項該当
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Mattinen, Maija-Liisa. "Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy." Espoo : VTT, 1998. http://www.vtt.fi/inf/pdf/publications/1998/P342.pdf.
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Academic dissertation : Chemistry : Helsinki : 1998.Textes et résumés en anglais. ISBN de la version électronique 951-38-5226-1. Pagination multiple pour les articles reproduits en annexe. ISSN de la version électronique : 1455-0849. Bibliogr. p. 56-72. Bibliogr. à la suite des articles.
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O'Dwyer, Jonathan Patrick. "Developing a fundamental understanding of biomass structural features responsible for enzymatic digestibility." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4137.
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Lignocellulosic biomass is one of the most valuable alternative energy sources
because it is renewable, widely available, and environmentally friendly. Unfortunately,
enzymatic hydrolysis of biomass has been shown to be a limiting factor in the
conversion of biomass to chemicals and fuels. This limitation is due to inherent
structural features (i.e., acetyl content, lignin content, crystallinity, surface area, particle
size, and pore volume) of biomass. These structural features are barriers that prevent
complete hydrolysis; therefore, pretreatment techniques are necessary to render biomass
highly digestible.
The ability to predict the biomass reactivity based solely on its structural features
would be of monumental importance. Unfortunately, no study to date can predict with
certainty the digestibility of pretreated biomass. A concerted effort with Auburn
University and Michigan State University has been undertaken to study hydrolysis
mechanisms on a fundamental level. Predicting enzymatic hydrolysis based solely on
structural features (lignin content, acetyl content, and crystallinity index) would be a
major breakthrough in understanding enzymatic digestibility.
It was proposed to develop a fundamental understanding of the structural features
that affect the enzymatic reactivity of biomass. The effects of acetyl content,
crystallinity index (CrI), and lignin content on the digestibility of biomass (i.e., poplar
wood, bagasse, corn stover, and rice straw) were explored.
In this fundamental study, 147 poplar wood model samples with a broad
spectrum of acetyl content, CrI, and lignin were subjected to enzymatic hydrolysis to
determine digestibility. Correlations between acetyl, lignin, and CrI and linear hydrolysis profiles were developed with a neural network model in Matlabî. The
average difference between experimentally measured and network-predicted data were
ñ12%, ñ18%, and ñ27% for 1-, 6-, and 72-h total sugar conversions, respectively. The
neural network models that included cellulose crystallinity as an independent variable
performed better compared to networks with biomass crystallinity, thereby indicating
that cellulose crystallinity is more effective at predicting enzymatic hydrolysis than
biomass crystallinity. Additionally, including glucan slope in the 6-h and 72-h xylan
slope networks and glucan intercept in the 6-h and 72-h xylan intercept networks
improved their predictive ability, thereby suggesting glucan removal affects later-stage
xylan digestibility.
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Shen, Hua. "The construction and characterization of a Pro-Thr box deletion of a Cellulomonas fimi endoglucanase (Cen A)." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/28744.
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The catalytic domain is separated from the cellulose-binding domain in Cellulomonas fimi endoglucanase CenA by a proline-threonine rich sequence called the Pro-Thr box. To study the function of the Pro-Thr box region, a deletion mutant, cenAAPT, was made from cenA by an oligonucleotide directed in vitro mutagenesis. The truncated enzyme, CenAAPT, was purified to homogeneity by affinity chromatography on cellulose and characterized. Comparing CenAAPT to CenA, the following characteristics were observed: 1) the Pro-Thr box affected the migration of CenA on SDS-PAGE; 2) the deletion of the Pro-Thr box altered the high affinity interaction with cellulose; 3) the truncated enzyme showed 40-50% reduction in catalytic activity towards both microcrystalline and amorphous cellulose; 4) the truncated enzyme was as sensitive as CenA to a C.fimi protease, and both enzymes were cleaved at the same site adjacent to the binding domain. The Pro-Thr box is not essential for the catalytic activity of CenA or its binding to cellulose, but it does contribute to both functions.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Reverbel-Leroy, Corinne. "La cellulase CelF : un composant majoritaire du cellulosome de Clostridium cellulolyticum." Aix-Marseille 1, 1996. http://www.theses.fr/1996AIX11073.
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Clostridium cellulolyticum, bacterie anaerobie stricte mesophile a gram positif, secrete des complexes multiproteiques, appeles cellulosomes, qui lui permettent de se multiplier sur cellulose comme seule source de carbone et d'energie. Les cellulases, composants majoritaires des cellulosomes, ont ete divisees en deux categories: les endocellulases (qui coupent au hasard a l'interieur des chaines) et les exocellulases (qui attaquent a partir d'extremites et poursuivent leur degradation en glissant le long des chaines, selon un mode processif). Un fragment d'adn, contenant un ensemble de genes ('celf, celc, celg, cele') codant pour des elements du cellulosome de c. Cellulolyticum, a ete isole et sequence en 1992. Le travail de cette these a debute par le clonage de l'integralite du gene celf par la technique de pcr inverse. Ce travail a egalement permis d'identifier en amont de celf, le gene cipc codant pour la proteine d'assemblage du cellulosome. Le gene celf a ete place dans le vecteur d'expression pet22b(+) afin de produire dans e. Coli une proteine fusion recombinante portant a son extremite c-terminale une queue de 6 histidines, permettant une purification simplifiee de la proteine heterologue. L'activite enzymatique de la proteine purifiee a ete mise en evidence sur divers substrats cellulosiques et son mode de fonctionnement a ete caracterise comme etant de type processif. Neanmoins, plusieurs experiences ont montre que l'attaque de celf sur les chaines de cellulose se faisait selon un mode endo. Celf a alors ete appelee une endocellulase processive. Une nouvelle classification des cellulases en endocellulases d'une part et cellulases processives d'autre part, a ete proposee. La fabrication d'anticorps polyclonaux contre la proteine recombinante a permis d'identifier la proteine celf au niveau des structures cellulosomales parentales de la clostridie. Celf constitue l'un des trois composants majeurs du cellulosome. De plus, l'interaction de celf avec le facteur d'assemblage cipc, ou une forme recombinante tronquee minicipcl, a ete etudie. Des etudes preliminaires de cooperation entre celf et les differentes cellulases recombinantes purifiees au laboratoire ont revele des synergies d'action sur cellulose. Enfin, la cristallisation de la proteine recombinante celf a ete realisee
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Rojas-Cuellar, Tania Raquel. "Utilisation of cellulose waste for the production of a chemical intermediate of economic interest." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/utilisation-of-cellulose-waste-for-the-production-a-chemical-intermediate-of-economic-interest(81b2984b-adf0-48a9-8e06-f3e9da548c19).html.
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Currently biomass is considered to be one of the main options to substitute the fossil fuels. Nevertheless, bioenergy is not the only alternative use for organic waste materials. In recent years, the utilisation of cellulolytic waste from industries, such as the paper industry and agriculture (in fields and in processing plants), is considered to be a good source of renewable carbon to produce chemical intermediates, such as glucose, lactic acid, ethanol and acetic acid, which can be returned to the productive chain. However, the principal obstacle in the use of this material for enzymatic degradation lies in the nature of the cellulose polymer. There are still many engineering, technological and chemistry related issues which remain to be resolved. The main objective of this study is to enable the production of glucose from the enzymatic hydrolysis of cellulose waste, arising from the waste of a recycle paper plant (paper crumb) by using Trichoderma reesei strain directly, instead of the commonly used mixture enzymes. This procedure, known as the single-step glucose production process, aims to reduce the costs associated in the use of pure enzymes and pre-treatments that are usually necessary to carry out the enzymatic degradation. The paper crumb is high in cellulose fibres (32%) with an alkaline characteristic, which carries a wide variety of impurities. This study recommends using existing knowledge with regards the enzymatic activity of the fungus and demonstrates its ability to degrade this substrate; regardless of the complex matrix linked to the cellulose polymer. Due to the nature of paper crumb a number of issues had to be solved during the development of the single-step production process. Firstly, the identification of an analytical method to monitor the enzymatic degradation of the paper crumb without interference of the inorganic compounds present in the substrate. The glucose analyser GL6 proved to be most suitable in this study. Secondly; the verification of the fungus’ ability to grow in this substrate by using PDA/Paper crumb plate, which allowed its adaptation gradually and reduced the time to produce enzymes. Finally, the evaluation of the enzymatic activity under acid and alkaline conditions was undertaken. It is demonstrated that the single-step process is feasible under acid conditions. The study also found that the fermentation time was the key parameter (up to 9 h.) to avoid the consumption of the glucose. The results show that the single-step process produces the same amount of glucose as the multi-step process (0.4 g/l), however the lower glucose production making it less economically attractive and less feasible to be expanded into an industrial scale. Nevertheless, the findings of this research contribute to establishing the basics for the optimisation of the glucose production process as an alternative for cellulose waste management. This adds economic value to the organic waste minimisation, which will lead to reduce cost in production processes.
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Jonsson, Rudsander Ulla. "Functional studies of a membrane-anchored cellulase from poplar." Doctoral thesis, KTH, Träbioteknik, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4520.
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Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana. Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario. The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus.QC 20100802
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Greenberg, Norman Michael. "Cellulase gene transcription in Cellulomonas fimi and an Agrobacterium." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28836.
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Transcriptional analysis was used to investigate the molecular mechanisms which effect cellulase gene expression in the gram-positive bacterium Cellulomonas fimi strain ATCC 484 and the gram-negative bacterium Agrobacterium sp. strain ATCC 21400. The cenA, cex and cenB genes of C. fimi encoding the extracellular β-1,4-endoglucanase, EngA (EC 3.2.1.4; Mr 48,700), the extracellular β-1, 4-exoglucanase, Exg (EC 3.2.1.91; Mr 47,300) and the extracellular β-1,4-endoglucanase EngB (EC 3.2.1.4; Mr 110,000) respectively, were characterised. By northern blot analysis, cenA mRNA was detected in C. fimi RNA prepared from glycerol- and carboxymethylcellulose (CMC)-grown cells but not in RNA from glucose-grown cells. The cex mRNA was found only in RNA from CMC-grown cells. The cenB mRNA was found in all three preparations of RNA. Therefore, the expression of these genes is subject to regulation by the carbon source provided to C. fimi. High resolution nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, were used to map the 5' termini of cenA and cex mRNAs. Two cenA mRNA 5' ends, 11 bases apart, mapped 51 and 62 bases upstream of the cenA start codon, suggesting that in vivo, cenA transcription was directed from two promoters in tandem. The cex mRNA 5' end was found to map 28 bases upstream of the cex start codon. Using SI mapping with unlabeled DNA probes and C. fimi RNA which had been isolatedin vivo but which had been 5'-labeled in vitro with vaccinia virus capping enzyme confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. The SI mapping revealed mRNA 3' termini 1,438, 1,449, and 1, 464 bases from the major cenA start site, and one 3' terminus 1,564 bases from the major cex mRNA start site, in good agreement with the northern blot data. High resolution SI studies were also used to show that abundant mRNA 5' ends mapped upstream of the cenB start codon in RNA prepared from CMC-grown cells, while less-abundant species mapped 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results seem to indicate a tandem promoter arrangement with an ATG-proximal promoter directing low-level constitutive cenB transcription and a more distal promoter directing higher levels of cenB transcription as a result of C. fimi growth on cellulosic substrate. Steady- state levels were determined for cenA, cex and cenB mRNAs with RNA prepared from glycerol-, glucose-, and CMC-grown cultures of C. fimi in slot-blot hybridisations with radiolabeled oligodeoxyribonucleotide probes. A cex-linked gene (clg) was identified by sequence inspection and SI mapping.
Transcripts of the abg gene encoding the β-glucosidase (Abg, EC 3.2.2.21/ Mr 50,000) of Agrobacterium sp. strain ATCC 21400 were also characterised. Northern blot analysis of Agrobacterium RNA revealed the size of the in vivo abgmRNA was approximately 1,500 bases in length. High resolution SI mapping determined abg mRNA 5' ends 22 bases upstream of the abg ATG codon and 3' ends 71 bases downstream of the abg stop codon.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Tu, Maobing. "Enzymatic hydrolysis of lignocellulose : cellulase enzyme adsorption and recycle." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/31175.
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Producing ethanol from the bioconversion of lignocellulosic substrates is one of the most promising technologies to decrease fossil fuel utilization. However, the current economics of the bioconversion process prohibit its commercialization due to the high cost of cellulase enzymes. One potential means to decrease enzyme costs is to recycle enzymes during the bioconversion process. The initial work focused on comparing the distribution of cellulases among the solid and liquid phases after a typical enzymatic hydrolysis of Avicel and an organosolv pretreated Douglas fir substrate. It was shown that 50% of the applied cellulases desorbed into the liquid phase after the hydrolysis of an ethanol pretreated D. fir substrate compared to 76% in the case of Avicel. By exploiting the natural affinity of cellulases for cellulosic substrates, the free enzymes were recovered via readsorption onto fresh substrates. Using this approach, 85% of the free enzymes could be recovered, compared to an 82% recovery predicted by the Langmuir isotherm model. A novel recycling strategy for recovering both the free and bound enzymes was developed where Tween 80 was added at the beginning of the hydrolysis, followed by the addition of fresh substrate to recover free enzymes after hydrolysis. The cellulases from T. reesei ( Hypocrea jecorina ) could be recycled for four consecutive rounds of hydrolysis of an ethanol pretreated (EPLP) substrate with the addition of 0.2% Tween 80, compared to one round with a steam exploded (SELP) substrate, presumably due to the higher lignin content of the SELP substrate. Comparing isolated lignin preparations from SELP and EPLP, it was shown that CEL-SELP lignin exhibited a greater capacity to bind cellulases than CEL-EPLP lignin. A reduction in the adsorption of cellulases to lignin was achieved by the addition of Tween 80. The recycling of β-glucosidase was achieved by immobilization on an inert carrier, Eupergit C. The immobilized β-glucosidase exhibited improved operational stability and an increase in the apparent K[sub m] and V[sub max]. Overall, the results demonstrated that enzyme recycling using a combination of surfactants, readsorption onto substrates and enzyme immobilization could potentially decrease enzyme costs in the hydrolysis of softwoods during the bioconversion process.Forestry, Faculty of
Graduate
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Bannari, Rachid. "Mathematical modeling of cellulase production in an airlift bioreactor." Thèse, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/1930.
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Fossil fuel is an important energy source, but is unavoidabiy running out. Since the cellulosic material is the most abundant source of organic matter, the ethanol, which is produced from cellulosic waste materials, is gaining more and more attention. These materials are cheap, renewable and their availability makes them superior compared to other raw materials. The cellulose must be hydrolyzed to glucose before it can be fermented to ethanol. The enzymatic hydrolysis of cellulose using cellulase enzymes is the most widely used method. The production cost of cellulase enzymes is the major cost in ethanol manufacture. To optimize the cost of ethanol production, enzyme stability needs to be improved through maintaining the activity of the enzymes and by optimizing the production of the cellulase. The aim of researchers, engineers and industrials is to get more biomass for the same cost. The filamentous fungus Trichoderma reesei has a long history in the production of the cellulase enzymes. This production can be influenced strongly by varying the growth media and culture conditions (pH, temperature, DO, agitation,... ). At present, it is my opinion that no modelling study has included both the hydrodynamic and kinetic aspects to investigate the effect of shear and mass transfer on the morphology of microorganisms that influence the rheology of the broth and production of cellulase. This thesis presents the development of a mathematical model for cellulase production and the growth of biomass in an airlift bioreactor. The kinetic model is coupled with the methodology of two-phase flow using mathematical models based on the bubble break-up and coalescence to predict mass transfer rate, which is one of the critical factor in the fermentation. A comparison between the results obtained by the developed model and the experimental data is given and discussed. The design proposed for the airlift geometry by Ahamed and Vermette enables us to get a high mass transfer and production rate. The results are very promising with respect to the potential of such a model for industrial use as a prediction tool, and even for design.
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Lo, Chi-Ming. "Cellulase Production by Trichoderma Reesei Rut-C30." University of Akron / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=akron1205776927.
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Kimura, Giselle Kobata 1985. "Investigação do potencial celulolítico de bactérias oriundas de processo de compostagem." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316714.
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Orientadores: Fabiana Fantinatti Garboggini, Suzan Pantaroto de VasconcellosDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Bactérias e fungos têm sido largamente explorados devido às suas habilidades em produzir uma grande variedade de enzimas, entre elas, as celulases que se destacam devido ao seu potencial em degradar materiais lignocelulósicos em açúcares fermentáveis, que podem então ser convertidos, por exemplo, em biocombustíveis. O presente trabalho visou a bioprospecção de bactérias isoladas a partir do processo de compostagem realizado pela Fundação Parque Zoológico de São Paulo (FPZSP), quanto à produção de enzimas celulolíticas, além da caracterização taxonômica das linhagens de interesse. Para tanto, os micro-organismos oriundos do processo de compostagem da FPZSP foram isolados, preservados e caracterizados macroscopicamente. Dentre as linhagens isoladas, 168 foram testadas numa triagem qualitativa para a produção de celulases, obtendo-se 135 micro-organismos com potencial celulolítico evidenciado pela formação de halos de hidrólise em meio de cultura contendo carboximetilcelulose. Destes, 10 linhagens apresentaram halos translúcidos com diâmetros entre 1,3 cm e 1,9 cm, as quais foram avaliadas quanto a atividade celulotíca em ensaios quantitativos monitorados durante 7 dias, em duas condições de pH distintas: 4,8 e 7,4. Os melhores tempos de incubação verificados foram de sete e cinco dias para os valores de pH 4,8 e 7,4, respectivamente. Em seguida, foram selecionados linhagens para os ensaios de delineamento experimental e otimização das atividades enzimáticas. No planejamento P&B, a melhor atividade celulolítica verificada foi de 3,6392 FPU/mL obtida a partir da linhagem FPZSP 143, no pH 4,8. Esta linhagem foi então selecionada e o planejamento do tipo Delineamento Composto Central Rotacional ¿ DCCR aplicado, promovendo dessa maneira, um aumento 0,4574 FPU/mL em relação ao experimento do planejamento anterior. Posteriormente, o experimento foi validado e o resultado máximo alcançado para atividade celulolítica da linhagem FPZSP 143 foi de 4,6435 FPU/mL. Cinco linhagens selecionadas com atividade celulolítica foram identificadas por análise de sequências do gene RNA ribossomal 16S como membros do gênero Bacillus, bactérias frequentemente encontradas em ambientes da compostagem e que tem sido largamente reportada como produtoras de enzimas celulolíticas. O processo de compostagem demonstrou ser um ambiente em potencial para a produção de celulases de interesse para diversos ramos da indústria, sendo os representantes do gênero Bacillus os melhores produtores de enzimas celulolíticas. Embora as bactérias tenham sido isoladas de um ambiente com pH em torno de 7,4, há um potencial para a produção de celulases em pH mais ácidos, evidenciando sua aplicabilidade em diferentes condições. Essa característica torna-se relevante quando se leva em consideração os processos industriais, onde uma condição diferente e específica é exigida em cada processo, tornando as enzimas celulolíticas oriundas de processo de compostagem grandes aliadas no desenvolvimento e otimização de processos industriais
Abstract: Bacteria and fungi have been extensively explored due to their ability to produce a variety of enzymes, including the cellulases that stand out because of their potential to degrade lignocellulosic materials to fermentable sugars, which can then be converted, for example, in biofuels. The present work aimed bioprospecting of bacteria isolated from the composting process conducted by Fundação Parque Zoológico de São Paulo (FPZSP), for the production of cellulolytic enzymes and the taxonomic characterization of strains of interest. For both, the microorganisms derived from the composting process of FPZSP were isolated, preserved and characterized macroscopically. Among the isolates, 168 were tested for production in a qualitative screening of cellulases, obtaining 135 cellulolytic microorganisms with potential evidenced by the formation of halos of hydrolysis in culture medium containing carboxymethylcellulose. These, 10 strains showed translucent halos with diameters between 1.3 cm and 1.9 cm, which were evaluated for activity in cellulolytic quantitative assays monitored for 7 days under two different pH conditions: 4.8 and 7.4. The best times of incubation recorded were seven and five days to pH 4.8 and 7.4, respectively. Then, strains for testing experimental design and optimization of enzymatic activities were selected. In Planning P&B, the best cellulolytic activity verified was 3.6392 FPU/mL obtained from FPZSP 143, at pH 4.8. This strain was selected and the DCCR applied, thus promoting an increase of 0.4574 FPU/mL compared to the previous experiment planning. Subsequently, the experiment was validated and the maximum score achieved for cellulolytic activity FPZSP 143 strain was 4.6435 FPU/mL. Five strains with cellulolytic activity were identified by sequence analysis of the 16S ribosomal RNA gene as members of the genus Bacillus, bacteria frequently encountered in composting environments and has been widely reported as producing cellulolytic enzymes. The composting process proved to be a potential environment for the production of cellulases of interest for various branches of industry, being the representatives of the genus Bacillus the best producers of cellulolytic enzymes. Although bacteria have been isolated from an environment with a pH around 7.4, there is a potential for the production of cellulases in more acidic pH, indicating their applicability in different conditions. This feature becomes important when one takes into account the industrial processes, where a different and specific condition is required in each case, making the cellulolytic enzymes derived from composting process a good allied in developing and industrial process optimization
Mestrado
Microbiologia
Mestra em Genética e Biologia Molecular
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Liu, Zelin. "Studies of Biomacromolecule Adsorption and Activity at Solid Surfaces by Surface Plasmon Resonance and Quartz Crystal Microbalance with Dissipation Monitoring." Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/39455.
Full textAbstract:
Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 Nâ ¢sâ ¢m-2 and elastic shear moduli of ~ 105 Nâ ¢m-2.
Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli.
Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn â ¥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn â ¥ 0.7).
These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces.Ph. D.
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Xu, Hui. "Genetic Modification of Thermotoga to Degrade Cellulose." Bowling Green State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1430913637.
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Veivers, Pamela Christine. "Biochemical aspects of symbiosis in carbon and nitrogen metabolism in higher termites." Thesis, The University of Sydney, 1995. https://hdl.handle.net/2123/26814.
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A comprehensive study of carbon metabolism has been carried out on representatives of two genera of higher termites, Nasutitermes walkeri and the fungus gardeners, Macrotermes spp. Substantial activity of cellulase and its components endo-B-l,4—glucanase and B—glucosidase, was found in workers of N. walkeri, Macrotermes michaelseni M. subhyalinus, M. bellicosus and M. mulleri. Less activity was found in the soldiers and none in the larvae of the Macrotermes spp. In fungal material associated with Macrotermes spp. only the fungal nodules had significant enzyme activity. In N. walkeri and M. michaelseni at least 90% of all enzyme activities was found in the midgut, with the exception of endo-B-l,4-xy1anase activity which was not measured in M. michaelseni. Low levels of endo-fi-l,4—xylanase activity were evenly distributed throughout the gut of N. walkeri. Midgut enzyme activity was restricted to the anterior region. The salivary glands contained variable amounts of enzyme activities. Less than 5% of the endo-B-l,4— glucanase activity was located in the hindgut. The endogenous cellulase from N. walkeri consisted of multiple B-glucosidase and endo-B-1,4- glucanase components. Elution profiles of fungal endo-B—l,4—glucanase activities on Bio-Gel® P-150 indicate that each Macrotermes spp. has a different Termitomyces sp. associated with it, consisting of one to three endo-B-l,4-glucanase activities. Endo-B-1,4-glucanase components from termite workers also consisted of one to three enzymes. A comparison of the elution profiles on Bio-Gel® P-60 of endo-B-lA—glucanase and Bglucosidase activities from fungal and termite material was used to show the absence of fungal enzymes in the termite gut. Similar results were found with fungal and termite material from M. bellicosus and M. mulleri using Bio-Gel® P—150 chromatography.
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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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Awafo, Victor Ankang. "Biosynthesis of cellulase-system from Trichoderma reesei and its characteristics." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0002/NQ29881.pdf.
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Duff, Sheldon Joseph Blaine 1956. "Studies on cellulase production with pure and mixed fungal fermentations." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=72840.
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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.
Full textAbstract:
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.The adsorption behavior of the four enzyme fractions was examined, with respect to pH, temperature and ionic strength. This was accomplished by using ($ sp3$H) radiolabeled cellulase fractions as tracers. The adsorption of the cellulases occurred within 60 minutes, and was described by a Langmuir type correlation. Increasing the adsorption temperature increased the saturation uptake of the endoglucanases but not of the cellobiohydrolases. Changes in pH and ionic strength affected both the degree and strength of adsorption of all the fractions, likely due to protein structure conformational changes.
Direct evidence of exchange between adsorbed and free enzymes was obtained for each component using ($ 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.
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.