Download or read book Characterization of the Binding of Thermobifida Fusca Cellulases and Their Domains to Bacterial Microcrystalline Cellulose written by Hyungil Jung and published by . This book was released on 2002 with total page 292 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Modeling and Analysis of Cellulose Hydrolysis by Thermobifida Fusca CEL5A CEL6B and CEL9A and Binary Mixtures of These Cellulases written by Tina Jeoh and published by . This book was released on 2004 with total page 462 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterization and Genetic Analysis of the Cellulolytic Microorganism Thermobifida Fusca written by Yu Deng and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: 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: 50́9-(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.

Download or read book Cellulose written by Theo G.M. Van De Ven and published by BoD – Books on Demand. This book was released on 2013-08-28 with total page 380 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cellulose is destined to play a major role in the emerging bioeconomy. Awareness of the environment and a depletion of fossil fuels are some of the driving forces for looking at forest biomaterials for an alternative source of energy, chemicals and materials. The importance of cellulose is widely recognized world-wide and as such the field of cellulose science is expanding exponentially. Cellulose, the most abundant biopolymer on earth, has unique properties which makes it an ideal starting point for transforming it into useful materials. To achieve this, a solid knowledge of cellulose is essential. As such this book on cellulose, the first in a series of three, is very timely. It deals with fundamental aspect of cellulose, giving the reader a good appreciation of the richness of cellulose properties. Book Cellulose - Fundamental Aspects is a good introduction to books Cellulose - Medical, Pharmaceutical and Electronic Applications and Cellulose - Biomass Conversion , in which applications of cellulose and its conversion to other materials are treated.

Download or read book Mechanism of Cellulose Hydrolysis and Substrate Binding by Thermobifida Fusca Cellulases written by Yongchao Li and published by . This book was released on 2008 with total page 174 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Mechanistic Studies of Thermobifida Fusca Exocellulase Cel6B written by Thu Van Vuong and published by . This book was released on 2010 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Understanding the mechanism by which cellulases catalyze cellulose hydrolysis can greatly contribute to the development of biofuels. The thermophilic bacterium Thermobifida fusca, a major degrader of plant cell walls in certain environments, secretes seven different cellulases including exocellulase Cel6B. This cellulase acts by an inverting mechanism; however, its catalytic acid and base residues had not been identified. Biochemical approaches confirmed D274 to be the catalytic acid residue. A single catalytic base residue could not be determined, as sodium azide assays showed no activity rescue for any single mutations of candidate residues. However, a double mutation of D226A and S232A knocked out enzymatic activity and its activity was partially rescued by sodium azide. We therefore propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton-transferring network to carry out the catalytic base function. T. fusca exocellulase Cel6B was also engineered to gain knowledge on the relationship between processivity and synergism as these properties are important for hydrolyzing crystalline cellulose. Mutations of several residues in the active site tunnel of Cel6B gave higher processivity. This improvement was confirmed by two assays: the ratio of soluble/insoluble reducing sugars as well as the ratio of oligosaccharide products. Surprisingly, the mutant enzyme, which has the highest processivity, showed the least synergism in mixtures with endocellulases, suggesting that improving exocellulase processivity might not always be an effective strategy for producing improved cellulase mixtures for biomass conversion. The highly processive Cel6B mutant enzymes were successfully fluorescently labeled, so these species can be used to visualize binding and track their movement on cellulose. The catalytic domains of Cel6B was found to bind non-productively to other polysaccharides; therefore, the balance between specific binding and non-specific adsorption should be always considered when engineering cellulases for hydrolyzing complex substrates. Using immuno-precipitation, Cel6B was demonstrated to contribute greatly to the hydrolysis of crystalline cellulose by T. fusca.

Download or read book Dissertation Abstracts International written by and published by . This book was released on 2004 with total page 768 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Investigating the Enzymatic Hydrolysis of Crystalline Cellulose Using Fluorescence Based Assays written by Navaneetha Santhanam and published by . This book was released on 2009 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The hydrolysis kinetics of bacterial microcrystalline cellulose (BMCC) by the cellulases of Thermobifida fusca was studied using fluorescence based assays in three ways. First, the binding of fluorescence-labeled Cel5A, Cel6B and Cel9A in ternary synergistic mixtures was assessed. A rapid high-throughput binding assay using microwell plates was developed to measure the bound fractions of the three cellulases at varying mole ratios of Cel6B and Cel9A, with Cel5A fixed at 10% of the total cellulase loading. This study revealed that the bound fractions of cellulases in ternary mixtures were additive, unlike the hydrolytic activity which was synergistic. Second, an experimental system was developed for the application of high resolution fluorescence microscopy to examine the binding of individual Cel5A, Cel6B and Cel9A to immobilized cellulose with varying morphologies. The immobilization technique allowed deposition of cellulose morphologies ranging from nanoscale cellulose fibers, to microscale cellulose fibril mats to sub-millimeter scale cellulose particles. Fluorescently labeled Cel5A, Cel6B and Cel9A were successfully integrated with fluorescently labeled cellulose to obtain a miniaturized reaction system which retained the intrinsic binding and hydrolytic capabilities of cellulases. Direct visualization of the binding behavior of individual cellulases on cellulose with different morphologies was achieved using this system which showed that the binding behavior depended strongly on the morphology and complexity of cellulose aggregates. Third, the significance of product inhibition by cellobiose as a rateretarding factor in the hydrolysis of BMCC by Cel9A and Cel9A-68, its construct lacking the family 2 cellulose binding module, was investigated. Fluorescently labeled BMCC was used as the substrate for an analysis of initial rates in the presence of exogenous cellobiose. Increasing cellobiose concentrations ranging from 1- 5mM were found to decrease the initial rate by 10 - 30% but increasing cellobiose concentrations from 5 to 60 mM did not cause a further decline in initial rates, clearly ruling out classical competitive inhibition as a possible mechanism. No definitive correlation was observed between binding and cellobiose concentrations for both enzymes indicating that the presence of cellobiose does not lead to significant enhancement or inhibition of binding.

Download or read book Studies of Thermobifida Fusca Lytic Polysaccharide Monooxygenases AA10A and AA10B for Cleavage of Recalcitrant Cellulose written by Nathan Kruer-Zerhusen and published by . This book was released on 2016 with total page 274 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cellulose recalcitrance remains a barrier to producing sustainable fuels and chemicals from renewable plant biomass. The cellulolytic bacterium Thermobifida fusca secretes lytic polysaccharide monooxygenases (LPMOs) to contribute to cellulose breakdown. LPMOs use an oxidative mechanism to synergize with hydrolytic cellulases. LPMOs may play an important role in overcoming cellulose recalcitrance, the physical properties that slow cellulose breakdown. Understanding LPMO structure and function will inform the optimization of industrial cellulase mixtures for use in biomass saccharification. The goal of my work is to explore the relationship between LPMO structure and oxidative cleavage using site-directed mutagenesis, and to determine if the mechanism of LPMO-cellulase synergism relies on changes to cellulose crystallinity. Mutations to residues on the AA10A binding surface change both activity and binding properties, confirming the histidine brace for chelation of copper to be essential for activity. In addition to surface residues that mediate substrate interaction, the conserved cellulose binding domain was shown to be critical for activity of TfAA10B on crystalline cellulose. Kinetics of LPMO mutants were measured by performing secondary hydrolysis to simplify the product distribution to monomeric products, then quantifying using a rapid high performance liquid chromatography (HPLC) approach. The role of LPMOs was initially thought to be amorphogensis, a disruption of crystalline cellulose structure, induced by oxidative cleavages of surface chains. This proposed amorphogenesis model of LPMO action and cellulase synergism was explored using Fourier transform infrared spectroscopy (FTIR). FTIR can penetrate residual substrates to detect changes in multiple measures of crystallinity caused by digestion. While past reports had observed significant changes to cellulose crystallinity imparted by cellulases, these changes were not reproduced after optimization of the measurement approach. Similarly, LPMOs did not induce changes to cellulose crystallinity during oxidative cleavage, suggesting an alternative mechanism of LPMO synergism with cellulases.

Download or read book Index Medicus written by and published by . This book was released on 2004 with total page 2160 pages. Available in PDF, EPUB and Kindle. Book excerpt: Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.

Download or read book High Throughput Data Framework Based Characterization and Evaluations of Thermobifida Fusca for Industrial Applications written by Niti Vanee and published by . This book was released on 2013 with total page 800 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cellulolytic organisms are being heavily studied for the production of biofuels, given that lignocellulosic biomass would be a cheap, abundant, and renewable starting material for chemical production. A challenge with cellulolytic microorganisms is that they are typically poorly characterized and often difficult to genetically manipulate. Our group focuses characterization and engineering of a thermophilic aerobic, cellulolytic actinobacterium, Thermobifida fusca. The wider range of optimal temperature and pH for the growth condition, besides the secretion of several group of cellulases, have made this microbe a potentially efficient host system for industrially application. After the development of first ever successful genetic manipulation protocol by for T. fusca in 2011 in our group the quest continues to better understand and further explore this microbe with such remarkable capabilities. Available genome annotation of the bacteria gives a preliminary clue towards the exploration of its biological system. Genome-scale metabolic reconstruction provides one such framework to populate all the available piece of information to mimic the biological systems to the closest functional state. Further, this skeletal base network can be made more realistic by applying the constraint that controls the flux through various reactions in the pathway network thereby providing the optimal solution space for operation. For the purpose of curation of this in silico model, we aim to integrate the experimental datasets (proteomic and metabolomics) and optimize the agreement between the in silico and in vivo conditions at a steady state condition. Once the model considerably imitates the original biological network, it will be used for the fundamental understanding of the microbial system for the application towards production biofuel and high yields of compound of pharmaceutical interest. The ultimate objective of this project is to design the candidate strain for the cellulolytic production of Natural products. Natural products play an important role in manufacturing of several active pharmaceutical ingredients (APIs). APIs or precursors of APIs can be produced in living organisms with the major challenge of designing and optimizing metabolic pathways to obtain the compounds of interest. In this capacity, living organisms can act as renewable catalysts with high product specificity to produce APIs with potential cost savings over purely synthetic chemistry synthesis routes. This is an effort to understand and design industrially usable microorganism T. fusca to act as a host system for the purpose of production of these compounds. The present project focuses on, in silico characterization and experimental validation of T. fusca, with particular focus on the terpenoids backbone biosynthesis (TBB) pathways using a genome-scale metabolic model, transcriptomics, proteomics and metabolite analysis. The DXP pathway leads to the production of terpenoids precursors that have applications in nutraceutics and pharmaceutics. This study generates the metabolic model, iTFU975 for T. fusca based on the proteomics dataset as the starting point. Further the model and the experimental dataset together helps to characterize the secondary metabolites pathways and compounds in the network associated with the production of terpenoids. In conclusion, this is an effort to characterize the natural products biosynthesis in T. fusca by establishing a bridge between the analytical methodologies and computational efficiencies on "-omics" knowledge to prove the diverse applicability of Systems Biology.

Download or read book IDENTIFICATION AND CHARACTERIZATION OF THERMOBIFIDA FUSCA GENES INVOLVED IN PLANT CELL WALL DEGRADATION written by and published by . This book was released on 2006 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Micro-array experiments identified a number of Thermobifida fusca genes which were upregulated by growth on cellulose or plant biomass. Five of these genes were cloned, overexpressed in E. coli and the expressed proteins were purified and characterized. These were a xyloglucanase, a 1-3,beta glucanase, a family 18 hydrolase and twocellulose binding proteins that contained no catalytic domains. The catalyic domain of the family 74 endoxyloglucanase with a C-terminal, cellulose binding module was crystalized and its 3-dimensional structure was determined by X-ray crystallography.

Download or read book Structure and Functional Characterization of Cellulases and Their Interactions with Cellulose written by Christopher M. Bianchetti and published by . This book was released on 2011 with total page 228 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book American Doctoral Dissertations written by and published by . This book was released on 2001 with total page 776 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels Chemicals and Polymers written by Shang-Tian Yang and published by John Wiley & Sons. This book was released on 2013-05-24 with total page 561 pages. Available in PDF, EPUB and Kindle. Book excerpt: Sets the stage for large-scale production of biofuels and bio-based chemicals In response to diminishing supplies as well as the environmental hazards posed by fossil fuels and petrochemicals, interest and demand for green, sustainable biofuels and bio-based chemicals are soaring. Biomass may be the solution. It is an abundant carbon-neutral renewable feedstock that can be used for the production of fuels and chemicals. Currently, biorefineries use corn, soybeans, and sugarcane for bioethanol and biodiesel production; however, there are many challenges facing biorefineries, preventing biomass from reaching its full potential. This book provides a comprehensive review of bioprocessing technologies that use lignocellulosic biomass for the production of biofuels, biochemicals, and biopolymers. It begins with an overview of integrated biorefineries. Next, it covers: Biomass feedstocks, including sugar, starch, oil, and energy crops as well as microalgae Pretreatment technologies for lignocellulosic biomass Hydrolytic enzymes used in biorefineries for the hydrolysis of starch and lignocelluloses Bioconversion technologies for current and future biofuels such as ethanol, biodiesel, butanol, hydrogen, and biogas Specialty chemicals, building block chemicals, and biopolymers produced via fermentation Phytochemicals and functional food ingredients extracted from plant materials All the chapters have been written and edited by leading experts in bioprocessing and biorefining technologies. Contributions are based on a thorough review of the literature as well as the authors' firsthand experience developing and working with bioprocessing technologies. By setting forth the current state of the technology and pointing to promising new directions in research, Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers will enable readers to move towards large-scale, sustainable, and economical production of biofuels and bio-based chemicals.

Download or read book Sustainable Degradation of Lignocellulosic Biomass written by Anuj Chandel and published by BoD – Books on Demand. This book was released on 2013-05-15 with total page 288 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides important aspects of sustainable degradation of lignocellulosic biomass which has a pivotal role for the economic production of several value-added products and biofuels with safe environment. Different pretreatment techniques and enzymatic hydrolysis process along with the characterization of cell wall components have been discussed broadly. The following features of this book attribute its distinctiveness: This book comprehensively covers the improvement in methodologies for the biomass pretreatment, hemicellulose and cellulose breakdown into fermentable sugars, the analytical methods for biomass characterization, and bioconversion of cellulosics into biofuels. In addition, mechanistic analysis of biomass pretreatment and enzymatic hydrolysis have been discussed in details, highlighting key factors influencing these processes at industrial scale.

Download or read book Biomass Recalcitrance written by Michael Himmel and published by Wiley-Blackwell. This book was released on 2008-06-23 with total page 552 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book examines the connection between biomass structure, ultrastructure, and composition, to resistance to enzymatic deconstruction, with the aim of discovering new cost-effective technologies for biorefineries. It contains chapters on topics extending from the highest levels of biorefinery design and biomass life-cycle analysis, to detailed aspects of plant cell wall structure, chemical treatments, enzymatic hydrolysis, and product fermentation options."--Pub. desc.