Abstract 1802 A Colorimetric Assay to Determine Substrate Specificity of Beta-Amylases

Starch is the primary form of energy storage for plants. During the day, starch is synthesized, stored, and then broken down in the chloroplast during the night to form usable sugar in the form of maltose. Among the enzymes that catalyze this degradation are β-amylases, which cleave two glucose units from the reducing end of a polyglucan chain, forming β-maltose. The Arabidopsis β-amylase (BAM) gene family is commonly used as a model system to study amylases. β-amylase (BAM) proteins are diverse in both structure and function, and collectively contribute to a number of cellular processes including gene expression and starch metabolism in plants. There are five catalytically active BAMs in Arabidopsis that are expressed under different conditions and are localized either in the chloroplast or the cytoplasm, although their specific functions are unclear. The goal of this project is to examine the substrate specificity and selectivity of β-amylases found in Arabidopsis as a means to understand their broader roles in the physiology of the plant. In order to quantify the generation of new reducing sugars, the bichinchoninic acid (BCA) assay was used, which is a colorimetric assay that operates by the reduction of copper ions and the formation of a colored copper-bicinchoninate complex. Data was collected for the activity of several β-amylases from Arabidopsis and compared them to a well characterized ortholog found in sweet potatoes (Ipomoea batatas BAM5). Kinetic data was collected for various substrates, including soluble starch, maltodextrin, and the individual components of soluble starch: amylopectin and amylose. Future directions include expanding the tested substrates, and collecting kinetic data for other catalytically active BAMs, ultimately leading to a comprehensive understanding of the way different Arabidopsis β-amylases interact with substrates and function within the plant This work was supported by the JMU Department of Chemistry and Biochemistry, NSF REU CHE-2150091, NSF MCB RUI-193275, and NSF MCB RUI-2322867.