Reduced cellulose accessibility slows down enzyme-mediated hydrolysis of cellulose.

Enzyme-mediated hydrolysis of cellulose always starts with an initial rapid phase, which gradually slows down, sometimes resulting in incomplete cellulose hydrolysis even after prolonged incubation. Although mechanisms such as end-product inhibition are known to play a role, the predominant mechanism appears to be reduced cellulose accessibility to the enzymes. When using Simon's stain to quantify accessibility, the accessibility of mechanically disintegrated and phosphoric acid-swollen cellulose substrates decreased as hydrolysis proceeded. In contrast, the poor initial accessibility of Avicel remained low throughout hydrolysis. However, washing the residual cellulose increased cellulose accessibility, likely due to the removal of tightly bound but non-productive enzymes which blocked access to more active enzymes in solution. Atomic force microscopy (AFM) analysis of the initial and residual cellulose collected when the hydrolysis plateaued, showed an increase in the roughness of the cellulose surface, possibly resulting in the tighter binding of less active cellulases.