The effect of hemicellulose on the binding and activity of cellobiohydrolase I, Cel7A, from Trichoderma reesei to cellulose

Hydrothermal pre-treatments decrease lignocellulose recalcitrance against enzymatic hydrolysis by removing the majority of the hemicellulose, thus increasing cellulase accessibility. However, a small amount of the hemicellulose may remain and become adsorbed to the cellulose, leading to cellulase inhibition. Here, we produced hemicellulose bound cellulose, using glucuronoxylan and galactomannan, to simulate hydrothermally pre-treated hardwoods and softwoods, respectively, and evaluated how this can affect cellulose hydrolysis by Trichoderma reesei derived cellobiohydrolase I (Cel7A). Based on X-ray powder diffraction (XRD), histochemistry, scanning electron microscopy and Simon's staining, hemicellulose binding onto cellulose affected the physical properties of the biomass, which subsequently affected its hydrolysis rate. As a result of hemicellulose binding onto cellulose, the adsorption of Cel7A was significantly impacted (up to 45%), leading to lowered activities (a 40% reduction), especially for glucuronoxylan. The bound hemicellulose may be released from the cellulose during agitation and hydrolysis. We therefore evaluated the effect of free hemicellulose on Cel7A. Free xylan was more inhibitory to Cel7A than free mannan, demonstrating non-competitive inhibition, while mannan exhibited uncompetitive inhibition. The recalcitrant effect of both bound and free hemicellulose could be relieved by the addition of hemicellulolytic enzymes (i.e. XT6 and Man26A) during cellulose hydrolysis. During the degradation of cellulose in "realistic" woody biomasses by Cel7A, the addition of hemicellulases led to a significant improvement in cellulose hydrolysis. This study showed that hemicellulose remains a critical factor regarding biomass recalcitrance and that the addition of hemicellulolytic activities in commercial enzyme cocktails is required (especially the mannanolytic activities lacking from most commercial enzyme cocktails), in order to realise high sugar yields at low enzyme protein loadings for low-cost biofuel production. Graphic abstract