Document Type : Research Paper
Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
Akam Faravardehaye Bahman Arad, Kamalshahr, Karaj, Iran
Razi Vaccine and Serum Research Institute, Karaj, Iran
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
Background: Microbial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity).
Objective: In the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator.
Material and Methods: phytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking.
Results: The results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called “allosteric site”.
Conclusions: The observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.