(290e) Interaction of the Enzyme with Substrate Is the Most Significant Factor Accounting for Enzyme Deactivation in the Enzymatic Hydrolysis of Cellulose

During enzymatic hydrolysis of biomass, the reaction rate significantly decreases and does not reach 100% conversion even after several days of reaction time,. This may be attributed to enzyme deactivation via a variety of possible mechanisms such as thermal, mechanical, and/or chemical activity. We have sought to determine whether the main source of enzyme deactivation is from enzyme-substrate interactions or other interactions of the enzyme in solution that are unrelated to the substrate (termed here as non-specific deactivation). To accomplish this, a series of tests were performed to compare cellulase's activity loss towards freshly added substrate following initial incubation of the enzyme in a buffer-only solution and in a buffer-substrate solution. For the buffer only solution, the residual activity was 55.3% with a deactivation half-life of 21.5 hours. When enzyme was incubated with 0.1, 0.2 or 0.4 g initial Solka Floc substrate, the residual activity was only 41.5%, 34.2% and 35.7%, respectively, and the deactivation half-life dropped from 21.5 hours to 5.04, 4.56 and 1.37 h, respectively. The rapid decrease of half-life with addition of substrate suggests that enzyme-substrate interactions are the main reason for enzyme deactivation. The extent of the deactivation is also varied for different substrates. We tested deactivation of enzyme toward substrates with various degrees of crystallinity: Solka floc, filter paper, phosphoric acid swollen cellulose (PASC) and cotton fiber. The crystallininity index was measured for each of these cellulose substrates. We observed a positive correlation between the rate of deactivation and crystallininity. A possible explanation is that hydrolysis of crystalline cellulose requires cellobiohydrolases, but the intrinsic activity of cellobiohydrolases decreases due to slow desorption during the incubation with substrate.