Quantification Of Synergy In Mixtures Of Glycosyl Hydrolases On Cellulosic Biomass

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Abstract
Cellulases are an important class of cell-wall degrading enzymes that are being deployed for biochemical conversion of cellulose to fermentable sugars. Individual cellulases hydrolyze cellulose relatively slowly; however, mixtures of cellulases and other plant cell wall degrading enzymes act synergistically to amplify hydrolysis. A major question affecting cellulase binding and synergistic behavior is to what extent does cellulose morphological structure influence cellulase accessibility to cellulose polymers and how does this accessibility change with different cellulases? In nature, cellulose is contained within a complex matrix of polymers that comprise the plant cell wall. Organisms that rely on cellulose use a cocktail of enzymes to access the cellulose within the plant cell wall matrix quickly and efficiently. Identifying novel enzymes from the organisms that can be incorporated into synergistic mixtures will allow for greater diversity in biomass feedstocks and greater efficiency in conversion. To this end, a high through-put method was developed to screen plant pathogenic fungi for cellulolytic ability. Within a small sample, some isolates had crude activities comparable to that of Trichoderma reesei, particularly at lower temperatures and on more complicated biomass substrates. Cellulase binding and synergistic behaviors on simple and complex cellulose structures can be studied using advanced imaging techniques with high temporal and spatial resolution. A method for observing Thermobifida fusca cellulases Cel5A and Cel6B binding on immobilized cellulose using fluorescence microscopy techniques was established. This method allows for simultaneous imaging of iii binding on cellulose fiber and mat structures at a range of temperatures to encourage or discourage hydrolysis. Having established this method, equimolar mixtures of Cel5A and Cel6B were applied to the cellulose substrates and binding was observed. The binding data's temporal resolution obtained by this method allows for the calculation of degree of synergistic binding throughout the time course on both cellulose fibers and mats. Hydrolytic activity enhances the DSB early in the time course for fibers and throughout the time course for mats, indicating a significant effect of cellulose structure and accessibility on cellulase binding in a synergistic mixture. iv
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2013-08-19
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cellulase; fluorescence microscopy; cellulose
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Walker, Larry P
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Wilson, David B
Craighead, Harold G
Gibson, Donna Marie
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Agricultural and Biological Engineering
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Ph. D., Agricultural and Biological Engineering
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Doctor of Philosophy
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dissertation or thesis
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