Gao Dahai, Shishir Chundawat, Venkatesh Balan, and Bruce Dale. Chemical Engineering and Materials Science, Michigan State University, Great Lakes Bioenergy Research Center (GLBRC), 3900 Collins Rd, Lansing, MI 48910
Cellulase binding to insoluble glucan is the first step towards effective hydrolysis of cell wall polysaccharides. Several studies have shown improvement in substrate digestibility due to synergistic interactions between cellobiohydrolases (CBHs) and endoglucanases (EG). However, due to lack of efficient methods for identification of individual enzymes within hydrolyzate mixtures, the relationship between cellulase adsorption (i.e. productive/un-productive) and synergism is unclear. A novel fast flow liquid chromatography (FPLC) based method was developed to quantify three major cellulases (Trichoderma reesei based CBH I, CBH II and EG I) individually in a heterogeneous reaction environment. The extent of binding for single, binary and ternary component mixtures was studied using various cellulosic substrates. Untreated, Ammonia Fiber Expansion (AFEX) and dilute acid treated corn stover are compared based on their enzyme adsorption capacities. Other substrates such as crystalline cellulose (Avicel or cellulose I), liquid ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and phosphoric acid swollen cellulose (amorphous cellulose) were also studied for determining relative binding affinity for various purified enzyme mixtures. Competitive and cooperative binding between several cellulases was identified for various cellulosic substrates. For AFEX corn stover, presence of EG I significantly enhanced CBH binding unlike untreated corn stover. Cellulase binding to pretreated corn stover was higher than untreated corn stover. However, lesser binding was observed for cellulose III compared to native cellulose I. These results suggest that unproductive/productive enzyme binding is dependent on the type of substrate/pretreatment method and plays a critical role in efficient saccharification of lignocellulosics.
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