Various forms of pure cellulosic substrates were utilized in the study of reaction mechanism in cellulase reaction. The substrates employed were micro-crystalline cellulose (Avicel), α-cellulose, filter paper, cotton, and non-crystalline cellulose (NCC). These substrates were first characterized with respect to Degree of polymerization (DP), crystallinity, surface area and other physical properties. NCC is a product of our laboratory. It is highly amorphous cellulose with crystallinity index less than 10%. When hydrolyzed with cellulase, it produces significant amount of cello-oligosaccharides as reaction intermediates, along with glucose and cellobiose. Cello-oligosaccharides (COS) were categorized into two separate fractions: low DP cello-oligosaccharides (LD-COS) and high DP cello-oligosaccharides (HD-COS). LD-COS, from DP 1-7, are detected by HPLC whereas HD-COS are detected only after secondary hydrolysis. On the basis of the profiles of these sugars during enzymatic hydrolysis, individual actions of Exo-glucanase (Exo-G), Endo-glucanase (Endo-G) and β-glucosidase (β-G) the overall reaction patterns are proposed.

The major findings on the function of individual cellulase components are as follows: (1) Exo-G and Endo-G do not hydrolyze COS. (2) β-G hydrolyzes cellobiose and LD-COS but has negligible reactivity on HD-COS. (3) Low DP is favorable for Endo-G reaction but unfavorable for Exo-G reaction. With regard to the reactivity of cellulase, we find that that crystallinity affects the initial rate of cellulose hydrolysis, whereas the DP of cellulose plays an important role in the determination of terminal glucan digestibility. The BET surface area has a positive effect on the hydrolysis rate but does not show a linear relationship.