Effect of Different Durations of Assay and Substrate Concentrations on Kinetic Parameters of Amylase with Criteria for Validity in Focus

Abstract

Introduction: Researchers adopt different duration of the assay for a given concentration of the enzyme using different substrate concentration ranges. The kinetic parameters (KP) may not be the same for the different duration of the assay. Therefore, it is imperative to determine the KP arising from different durations of the assay and different substrate concentrations. It is also necessary to investigate the validity of the KP given the substrate concentration regime used for assay. Method: The experiment entails Bernfeld method of enzyme assay for the generation of data. (Modified or alternative) direct linear plots and Lineweaver – Burk plots were carried out using Microsoft Excel. The validity of kinetic parameter was examined using the validating equations such as standard quasi-steady-state approximation (sQSSA), total QSSA (tQSSA), reverse QSSA (rQSSA), and reactant stationary assumption (RSA). Results and Discussion: Kinetic parameters, KP, Michaelis – Menten constant (Km) and maximum velocity of hydrolysis (vmax) generated from different linear plots generally differed in magnitude. Larger magnitude of KP was generally observed at the shorter duration of the assay. The pseudo first-order rate constant, k was higher at lower substrate concentration. The KP values satisfy the condition for the validity of rQSSA regardless of the duration of the assay.  Conclusion: Increasing duration of assay of Aspergillus oryzea alpha amylase leads to a decrease in the magnitude of kinetic parameters, enzyme-substrate complex dissociation constant (ks) and maximum velocity of catalysis (vmax) and increasing concentration of the substrate leads to decreasing magnitude of the pseudo first-order rate constant, k for the utilization of substrate. The duration of the assay does not influence or alter the criterion for the validity of kinetic parameters if the potato starch concentration range is « enzyme concentration.