Enzyme-substrate interaction significantly affects activity loss during enzymatic hydrolyis of cellulose.
Date on Master's Thesis/Doctoral Dissertation
Berson, Robert E. (Eric)
Rising concerns about dependency on fossil fuels in the 21st century has sparked a growth in research for renewable energy sources. One renewable energy production process of interest is the reduction of cellulose into fermentable sugars by means of enzymatic hydrolysis. The reaction requires a residence time on the order of seven or more days and usually does not achieve complete conversion. The slow reaction rate and incomplete conversion is generally attributed to loss of enzymatic activity during the reaction. Deactivation of the enzyme is classified here as either substrate related deactivation or nonspecific deactivation. The general term of nonspecific deactivation refers to any activity loss of the enzyme not attributed to interaction with substrate. Reasons for deactivation due to enzyme-substrate interaction are still uncertain and deactivation may possibly be attributed to factors such as poor desorption of enzyme from the substrate and product inhibition. In this research, the nonspecific deactivation was quantified by activity measured following enzyme incubation in a substrate-free buffer for 2, 4, 8, 16, 24, 48, or 72 hours, followed by a second incubation of one hour with 2.0 grams of substrate. Testing for enzyme-substrate interaction was performed by adding an initial substrate load to the first incubation in the amount of 0.1, 0.2, or 0.4 grams, and then substrate was added during the second incubation to bring the total in all cases up to 2.0 grams. The amount of enzyme in the solution was held constant at 0.6 mL, for all cases. Two substrates of different crystallinity, filter paper (CrI = 45%) and dewaxed cotton (CrI = 90%) were studied here. The cellulase enzyme showed slight deactivation after incubating for varying times during the initial incubation in a substrate-free buffer. Enzyme-substrate interactions also resulted in deactivation and generally contributed to more of the overall deactivation than did nonspecific deactivation. Deactivation was seen to depend on the initial incubation time, substrate load, and substrate type (crystallinity). There did not appear to be a consistent trend in relative percent deactivation for nonspecific deactivation and deactivation due to enzyme-substrate interaction for initial incubations less than 24 hours for either substrate, but the relative amount of nonspecific deactivation appeared to increase between 24 and 72 hours. However, the enzyme-substrate interaction still contributed to more than fifty percent of deactivation for all but one case. The lack of a trend prior to 24 hours is likely attributed to glucose concentrations that are within the range of error of the YSI analyzer.
Hatfield, Kristen M., "Enzyme-substrate interaction significantly affects activity loss during enzymatic hydrolyis of cellulose." (2010). Electronic Theses and Dissertations. Paper 583.