7-28: The potential application of substructure-specific CBMs to track changes in cellulose surface morphology during the initial stages of hydrolysis

Despite significant progress in reducing the cost of the hydrolysis step of biomass-to-ethanol processes, commercialisation has been limited, at least in part, by the large amount of enzymes (protein) that is still required to achieve fast, complete hydrolysis of the cellulosic component. We and other groups have shown that it is not the actual slow rate of hydrolysis of the β-1,4 linkages within the cellulose that limits the rate of the reaction but rather the degree of amorphogenesis (which has been described as the non-hydrolytic ‘opening up’ or disruption of a cellulosic substrate), which is one of the key steps in the enzymatic hydrolysis that needs to be improved. It is apparent that our current understanding of the possible mechanisms involved in amorphogenesis is limited by a lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during this process.  We will describe a quantitative technique for measuring amorphogenesis where carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes (i.e. the accessibility of the amorphous and crystalline regions) in the surface morphology of cotton fibres. This technique was used to try and quantify the relative contribution that amorphogenesis makes in determining the actual rate and effectiveness of cellulose hydrolysis over a range of cellulosic substrates.