Towards cellulosic ethanol: structure-function studies of glycoside hydrolases

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The experimental plan presented in this proposal represents a continuation of studies on the structure-function relationship of glycoside hydrolases conducted in our laboratory over the past 21 years under the auspices of NSERC. Based on the results of our studies, we hypothesize that the anaerobic rumen bacterium Fibrobacter succinogenes organizes its cellulolytic and xylanolytic enzymes on its cell surface into cellulosome-like complexes and that a soluble factor is released to de-stabilize crystalline cellulose. To test this hypothesis, we propose to clone and express the genes encoding the three previously unidentified xylanases and cellulases, and the hypothetical 180 kDa and 240 kDa scaffoldins. Then, we will investigate the association, if any, of these proteins with one another by affinity chromatographic and surface plasmon resonance techniques. To further investigate the efficiency of cellulose biodegradation by F. succinogenes, we will initiate a search for a factor thought to be released by F. succinogenes for the destabilisation of crystalline cellulose using atomic force and scanning tunnelling microscopy.