Quantification of bound and free enzymes during enzymatic hydrolysis and their reactivities on cellulose and lignocellulose

Enzymatic hydrolysis of insoluble biomass is a surface reaction. Part of the enzyme adsorb on the surface of biomass, whereas the others stay in the liquid phase. In this study, three substrates (Avicel cellulose, bleached hardwood pulp, and green-liquor pretreated hardwood pulp) were used to study the reactivity of bound and free enzyme. In a continuous enzymatic hydrolysis, 35–65% initially added enzymes became bound enzymes, which were primarily responsible for enzymatic hydrolysis. The contribution from free enzymes became insignificant after a certain period of reaction time. SDS–PAGE analysis showed that CBH I was significantly decreased in the free enzyme, which might be the reason for the low digestibility of free enzymes due to the loss of synergistic effect. When Tween 80 was added during enzymatic hydrolysis, the digestibility of free enzyme on Avicel was greatly enhanced. However, the benefit of surfactant was not noticeable for lignocellulosic pulps, comparing to Avicel.