Masters Thesis

The influence of Sucralose on the enzymatic conversion of sugars using real-time NMR spectroscopy

The Michaelis and Menten initial rate approach to enzyme kinetics using sucrose hydrolysis by invertase was first performed over 100 years ago. Their approach allows for the measurement of an enzyme’s binding affinity (KM), and the maximum rate of substrate turn over (Vmax). This approach was so significant that it is still used as the main method of measuring enzyme kinetics in today’s time, however many things have changed since the 1900s. There are now non- caloric artificial sweeteners, such as sucralose, used as a sugar substitute. The artificial sugar sucralose is a sucrose derivative which is perceived to be biologically inert as it enters and exits the body fully intact without being metabolically consumed. This, however, does not necessarily mean that sucralose is not affecting the body as it passes through. To investigate the effects of sucralose, this research performs the Michaelis Menten enzyme kinetics approach on using real-time nuclear magnetic resonance (NMR) spectroscopy. NMR not only provides real-time measurement of enzyme-catalyzed substrate conversions but also permits for the complete analysis of the full substrate conversion. Investigation of the full time-course of substrate conversion allows for both initial rate approach and progress curve analysis from the same set of experiments. These results tend to suggest that both invertase and β-galactosidase in the presence of sucralose shows that sucralose acts as a competitive inhibitor.

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