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 Biomass Conversion written by Michael E. Himmel and published by Humana Press. This book was released on 2012-07-28 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biomass conversion research is a combination of basic science, applied science, and engineering testing and analysis. Conversion science includes the initial treatment (called pre-treatment) of the feedstock to render it more amenable to enzyme action, enzymatic saccharification, and finally product formation by microbiological or chemical processes. In Biomass Conversion: Methods and Protocols, expert researchers in the field detail methods which are now commonly used to study biomass conversion. These methods include Biomass Feedstocks and Cellulose, Plant Cell Wall Degrading Enzymes and Microorganisms, and Lignins and Hemicelluloses. Written in the highly successful Methods in Molecular BiologyTM series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting informed, reproducible results in the laboratory.