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45348-G4
Investigating the Relationship between Characteristics of Heterogeneous Cellulose and Cellulase Activities

Y.-H. Percival Zhang, Virginia Polytechnic Institute and State University

Cellulose is the most abundant renewable resource (ca. 1x10 E11 tons/year). Heterogeneous cellulose hydrolysis expedited by cellulases -- endoglucanase, exoglucanase, and beta-glucosidase -- plays an important role in the global carbon cycle, is important in agricultural processes and waste pretreatment, and will be an important part of emerging biobased products and biofuels industries. The production of chemicals and energy from renewable cellulosic materials is vital to sustainable development of human beings because it reduces reliance on non-renewable fossil fuels, decreases emission of greenhouse gases, and offers benefits to the national interests.

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 (Zhang and Lynd, Biotechnol. Bioeng. 2004, 88, 797-824; 2006, 94, 888-898). With the help of this award, 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). One molecule of the recombinant fusion protein occupied 21.2 cellobiose lattices on the 110 face of bacterial cellulose nanofibers. The CAC values of several cellulosic materials-regenerated amorphous cellulose (RAC), bacterial microcrystalline cellulose (BMCC), Whatman No. 1 filter paper, fibrous cellulose powder (CF1), and microcrystalline cellulose (Avicel) - were 41.9, 33.5, 9.76, 4.53, and 2.38 m2/g, respectively. The CAC value of amorphous cellulose made from Avicel was 17.6-fold larger than that of crystalline cellulose-Avicel. Avicel enzymatic hydrolysis proceeded with a transition from substrate excess to substrate limited. The declining hydrolysis rates over conversion are mainly attributed to a combination of substrate consumption and a decrease in substrate reactivity. Declining heterogeneous cellulose reactivity is significantly attributed to a loss of CAC where the easily hydrolyzed cellulose fraction is digested first (Langmuir , 23 (25), 12535 -12540, 2007)

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