Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Chanell Upshaw - Miami University
Co-Author(s): Rick Page and Thaiesha Wright, Miami University, Oxford, OH
Cellulose is a naturally occurring polymer consisting of 1,4- linked glucose monomers. It is the most abundant organic compound in earth, found in every plant. Cellulases are enzymes that catalyze the breakdown of cellulose into glucose. With biofuel being a growing industry, there has been an increase in the demand of Cellulosic glucose production. With an increase in demand, a more stable and active cellulase enzyme can is needed to increase the production of glucose for biofuel production. Our research seeks to increase the stability, activity, and reusability of the cellulase FnCel5a from the thermophilic bacterium Fervidobacterium nodosum. To improve the stability we plan to site-specifically modify FnCel5a with polymers. Site-directed mutagenesis was done to create the point mutations K94C, K190C, K300C, K94C/K190C, K94C/K300C, and K190C/K300C. The activity assays revealed no change in activity for single mutants, however, the double mutants showed a significant decrease in activity. There was no change in thermal stability for either single or double mutants in comparison to native FnCel5a, as measured by Differential Scanning Fluorimetry (DSF). The effect of point mutations to FnCel5a will be discussed with respect to the goal of producing an enzyme that is more stable under extreme industrial conditions. We will present our initial results with polymer modification of FnCel5a via covalent modification of cysteine residues introduced by site-directed mutagenesis.
Funder Acknowledgement(s): NSF
Faculty Advisor: Rick Page, firstname.lastname@example.org
Role: I performed the activity assays for the native, single mutations and double mutations. I also carried out the protocol for the DSF to determine the thermal stability.