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UBC Theses and Dissertations

Can we enhance cellulose hydrolysis by minimizing enzyme inhibition resulting from pretreatment-derived inhibitors? Zhai, Rui

Abstract

Any pretreatment process used to enhance the enzymatic deconstruction of lignocellulosic substrates, although opening up and enhancing access to the cellulose, will typically generate inhibitory compounds (i.e. soluble mono/oligomeric sugars, phenolics, furans, extractives, etc.) that will limit or restrict the efficiency of cellulose hydrolysis. To develop more effective inhibition mitigation strategies, it would be beneficial if we had a better understanding of the inhibitory mechanisms of these soluble compounds on the enzyme components of cellulase cocktails. Most of the previous studies which have tried to assess the effects of inhibitors on cellulase enzymes, have used “synthetic mixtures of inhibitors” and “traditional” cellulase preparations such as Celluclast. The work presented in this thesis assessed the major inhibitory compounds derived from a range of “real-life” lignocellulosic biomass substrates that were steam pretreated at various severities. The major inhibitory mechanisms, such as reversible/irreversible inhibition of the major enzyme activities (e.g. exo/endo-glucanase, β-glucosidase, xylanase activities, etc.), were investigated and potential inhibitor mitigation strategies were evaluated. Initial work showed that, although the more recent cellulase mixture CTec3 was more inhibitor tolerant than the older Celluclast enzyme preparation, they were still strongly inhibited by pretreatment derived inhibitors. Of the various inhibitors, sugars and phenolics were shown to be the major groups that significantly contributed to the observed decrease in cellulose hydrolysis. This was mostly because of the strong inhibition and deactivation of β-glucosidase and cellobiohydrolase activities present within the cellulase mixture. Surprisingly, although hemicellulose derived sugars did not appear to inhibit individual enzyme activities, they did inhibit overall cellulose hydrolysis. Subsequent work suggested that hemicellulose-derived sugars inhibited the processive movement of cellobiohydrolase Cel7A and thus restricted cellulose hydrolysis. In addition to sugars, pretreatment derived phenolics were shown to be more influential, with the molecular size and carbonyl content of the phenols playing major roles in influencing the extent of phenolic inhibition. However, the phenolics could be modified to minimize enzyme inhibition and allow the use of lower enzyme loadings.

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