1H NMR Spectroscopy-Based Configurational Analysis of Mono- and Disaccharides and Detection of β-Glucosidase Activity: An Undergraduate Biochemistry Laboratory

A 1 H NMR spectroscopy-based laboratory experiment explores mono- and disaccharide structural chemistry, and the enzyme-substrate specificity of glycosidic bond cleavage by β-glucosidase towards cellobiose (β-linked gluco-disaccharide) and maltose (α-linked gluco-disaccharide). Structural differences between cellobiose, maltose, and the glycolytic product glucose are established in terms of chemical shift changes and J -coupling constants of the anomeric proton peaks. The distinct anomeric proton signals observed in the 4.0–5.5 ppm range of glucose, cellobiose, and maltose spectra serve as a signature NMR feature to monitor the substrate specificity of β-glucosidase towards cellobiose as a substrate. This experiment consolidates carbohydrate structural chemistry and NMR spectroscopy as applied to investigate enzyme specificity in the context of a biochemistry laboratory experiment with hands-on NMR experience for upper-division undergraduates.