UBC Theses and Dissertations
The role of amorphogenesis in the enzymatic deconstruction of lignocellulosic biomass Gourlay, Keith Ian
Agricultural and forestry-derived fibres can be converted into fuels and chemicals via a biorefinery. However, the densely-packed fibrillar architecture of lignocellulosic biomass makes the cellulose inherently inaccessible to the enzymes involved in this bioconversion process. This limits the efficiency of enzymatic deconstruction and necessitates relatively high enzyme/protein loadings, which decreases the economic viability of the overall process. It has previously been suggested that the rate-limiting step of cellulose hydrolysis is not the depolymerisation of the carbohydrate chains, but rather the rate at which the enzymes can gain access to the cellulose buried within the biomass. Recently, several proteins such as the Expansins, Swollenin and Loosenin have been shown to disrupt the cellulosic structure without directly depolymerizing the carbohydrates. This protein-induced “amorphogenesis” is thought to occur as a delamination, splitting, peeling, swelling, or decrystallizing of the biomass, thereby enhancing accessibility of the entrenched carbohydrates to the depolymerizing enzymes. However, a key challenge when studying these amorphogenesis-inducing proteins involves quantifying their disruptive effects. While depolymerizing enzymes can be readily quantified by measuring the amount of liberated soluble sugars, amorphogenesis-inducing proteins are thought to promote a variety of disruptive effects without releasing soluble products. As the undefined nature of the amorphogenesis end product makes quantification challenging, one of the initial goals of the work was to refine/develop techniques to better quantify amorphogenesis. Two distinct carbohydrate binding modules (CBMs), one of which preferentially binds to crystalline cellulose and the other to amorphous cellulose were used to track changes in cellulose accessibility and surface morphology. When various substrates were treated with the amorphogenesis-inducing protein, Swollenin, CBM adsorption revealed that Swollenin promoted the dispersal and disruption of the more amorphous regions of biomass, increasing the access of the depolymerizing enzymes to the cellulose component. Subsequent work involving the fluorescent tagging of these CBMs and confocal microscopy further suggested that Swollenin was targeting the less-ordered regions of the cellulosic substrate. When Swollenin was assessed for its ability to disrupt an industrially-relevant substrate, steam pretreated corn stover, it primarily targeted amorphous regions where it synergised strongly with xylanases (~300%), promoting the release of hemicellulosic oligomers.
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