Browsing by Author "Mhlongo, Sizwe Innocent"

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    Impact of inhibitors associated with lignocellulosic hydrolysates on recombinant cellulolytic enzymes
    (Stellenbosch : Stellenbosch University, 2017-03) Mhlongo, Sizwe Innocent; Van Zyl, Willem Heber; Viljoen-Bloom, Marinda; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Enzymatic hydrolysis contributes a significant cost towards the production of bioethanol and is estimated to comprise 15% of the minimum ethanol selling price. One of the areas of concern during the enzymatic hydrolysis is the non-productive adsorption of enzymes by pretreatment by-products that may lead to the inhibition/deactivation of cellulases. Nonproductive adsorption of cellulases onto lignin is mainly driven by hydrophobic interactions and the extent of adsorption varies depending on the hydrophobicity of the lignin. Most fungal cellulases are bimodular with a catalytic domain and a carbohydrate binding domain (CBM) connected by a flexible linker. To achieve high yields of fermentable sugars for subsequent conversion to ethanol, it is desirable to include sugars from both cellulosic- and hemicelluloserich fractions, which implies the presence of inhibitory degradation compounds during enzymatic hydrolysis. To reduce the enzyme loading for hydrolysis, the inhibitor compounds in lignocellulosic biomass should be reduced to below toxic levels or be removed from hydrolysates. The first aim of the study was to investigate the role of individual lignocellulose-associated compounds in the inhibition and/or deactivation of the Talaromyces emersonii cellobiohydrolase (TeCel7A) fused to the Trichoderma reesei carbohydrate binding domain (TrCBM), Trichoderma reesei endoglucanase TrCel5A and Saccharomycopsis fibuligera β- glucosidase (SfCel3A) cellulases. The second aim was to explore detoxification strategies in the alleviation of the cellulose inhibition. The final aim was to investigate the mechanism(s) involved in the inhibition of cellulases. The impact of selected inhibitor compounds on the hydrolysis of Avicel was also investigated using a combination of TeCel7A-TrCBM and TrCel5A in the presence of Novozyme 188 Cel3A to prevent feedback inhibition by cellobiose. The study revealed that polymeric phenols, such as tannic acid, are strong inhibitors of cellulases, whereas monomeric phenols with aldehyde groups showed a strong inhibition of cellulose with increased contact time. This further confirmed that compounds with increased surface hydrophobicity have a strong inhibition effect. TrCel7A was shown to be quite resistant to inhibition and only hydroxymethyl furfural (HMF) strongly inhibited this cellobiohydrolase. This selective inhibition of retaining cellulases (TrCel7A), but not inverting cellulases (TrCel5A), was also observed with acetic and formic acid. This suggests that the non-processive nature and groove-shaped active site of TrCel5A allows it to escape non-productive binding to inhibitor compounds through the same mechanism it employs during cellulose hydrolysis. Further investigation revealed that increasing inhibition was not linked to contact time, but rather ascribed to increased concentration of inhibitor compounds. Detoxification strategies were explored as enhancers of enzymatic hydrolysis and tools to alleviate inhibition in biomass conversion processes. The results indicated that reducing agents (sodium dithionite and sodium sulfite) strongly reacted with coniferyl aldehyde and syringaldehyde, but not tannic acid. The addition of reducing agents substantially increased the hydrolysis of Avicel containing 10% bagasse pretreatment liquid. Application of the differential scanning fluorimeter (DSF) technique showed that increased concentrations of furans and acetic acid sharply increased unfolding of TeCel7A. This study showed that DSF could be developed as a tool to study cellulase binding, but this will depend on the development of dyes not based on hydrophobic interactions.