Double Labeling of Binding Sites in Cellulosic Substrates Using Endo- and Exoglucanase-Gold Complexes

Thin sections of cellulose fibers were incubated with an endo- and an exoglucanase labeled with gold particles of differing sizes. The hydrolytic sites were then visualized under transmission electron microscopy (TEM). The potential interaction between the β, 4-glucan substrates and the endo- and the exoglucanases was investigated using cellulosic and lignocellulosic substrates. The simultaneous visualization was very successful in distinguishing preferred substrates for each cellulase in lignocellulosic substrates. When plant lignocellulose was preincubated with endocellulase, density of the gold labeling greatly increased suggesting that preliminary exposure of lignocellulosic material to endocellulase may have enhanced the accessibility of the substrate to endocellulase and exocellulase. This result provided a plausible explanation for the observed endo/exo cellulase co-hydrolysis.