Determining Invertase Enzyme Activity Using Isothermal Titration Calorimetry

Abstract

Invertase catalyzes the hydrolysis of sucrose producing fructose and glucose. Invertase is widely used in the food industry because it possesses a lower crystallinity than sucrose at higher concentrations and is sweeter than sucrose. Invertase enzyme activity is typically measured using an endpoint coupled‐secondary reaction. The glucose produced by the invertase‐catalyzed reaction reacts with 4‐hydroxybenzoic acid hydrazide to produce yellow anions that can then be measured spectrophotometrically. Contrary to the standard method for measuring invertase activity, single injection isothermal titration calorimetry (ITC) directly measures heat generated by invertase continuously. ITC provides rapid and efficient measurement for enzyme activity. However, recent studies have shown that the single injection ITC measurements cannot separate km and kcat because the enzyme does not reach saturation. We recently demonstrated that single injection ITC can be used to measure the stability of invertase activity. Here we further characterize the relationship between the peak invertase activity and the peak heat‐rate. Additionally, previous ITC measurements of invertase activity have demonstrated that there is a delay in peak invertase activity. We elucidate the dependence of this delay in activity on the sucrose concentration. Single injection ITC is uniquely positioned to provide insight into the mechanistic action of invertase that cannot be determined using coupled reactions.Support or Funding InformationBuild DairyBYU MEG GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.