Jong Hong1, Xinhao Ye1, and Y.-H. Percival Zhang2. (1) Biological Systems Engineering Department, Virginia Tech University, 411 Latham Hall, Blacksburg, VA 24061, (2) Biological Systems Engineering Department, Institute for Critical Technology and Applied Science (ICTAS), Virginia Tech University, 210-A Seitz Hall, Blacksburg, VA 24061
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 the enzymatic cellulose hydrolysis mechanism (Zhang and Lynd, Biotechnol. Bioeng. 2006, 94: 888-898). Quantitative determination of cellulose accessibility to cellulase (m2/g cellulose) was established based on the Langmuir adsorption of the fusion protein containing a cellulose-binding module (CBM) and a green fluorescent protein (GFP) (Hong et al. 2007. Langmuir 23: 12535). One molecule of the recombinant fusion protein occupied 21.2 cellobiose lattices on the 110 face of bacterial cellulose nano-fibers. 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) -- are 41.9, 33.5, 9.76, 4.53, and 2.38 m2/g, respectively. The CAC value of amorphous cellulose made from Avicel is 17.6-fold larger than that of crystalline cellulose - Avicel. Avicel enzymatic hydrolysis proceeds 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.
