Investigating the Relationship between Characteristics of Heterogeneous Cellulose and Cellulase Activities Y-H Percival Zhang, Biological Systems Engineering, Virginia Tech Blacksburg, VA 24061. Tel: 540-231-7414 Email: biofuels@vt.edu Heterogeneous cellulose accessibility is an important substrate characteristic, but all methods for determining cellulose accessibility to the large-size cellulase molecule have some limitations. Characterization of cellulose accessibility to cellulase (CAC) is vital for better understanding of the enzymatic cellulose hydrolysis mechanism. We have developed a nano-scale tool (recombinant protein) containing a green fluorescent protein and cellulose-binding module. The adsorption of this protein will be used to more accurately represent cellulose accessibility to cellulase than other technologies. Quantitative determination of cellulose accessibility to cellulase (m2/g of cellulose) was established based on the Langmuir adsorption of the fusion protein containing a cellulose-binding module (CBM) and a green fluorescent protein (GFP) (right figures for the protein and adsorption) 1 2 3 Quantitative determinations of the total substrate accessibility to cellulase (TSAC) and cellulose accessibility to cellulase (CAC) based on adsorption of a non-hydrolytic recombinant protein TGC were for the first time established without and with blocking of bovine serum album, respectively. The COSLIF-pretreated corn stover had a CAC of 11.57 m2/g, nearly twice of that of the dilute acid (DA)-pretreated biomass (5.89 m2/g). All results suggest that CSLF can break lignocellulose structure more efficiently than can DA, resulting in more CAC accompanied by faster hydrolysis rates and higher sugar digestibility. Langmuir , 23 (25), 12535 -12540, 2007. Biotechnol. Bioeng. In prep. Thrombin Thrombin