Isolation and Purification of Amylase from Serum of Patients with Pancreatitis and Comparing the Biochemical Properties with Amylase Purified from Healthy People

Abstract

Back Ground: Amylases are a group of enzymes that hydrolyze starch into simple sugars. Amylase is secreted in the human body from saliva and the pancreas. Abnormal levels of the enzyme amylase indicate pancreatitis. Enzyme purification eliminates various proteins and other forms of biomolecules while restoring the majority of enzyme activity. Objectives: Isolation and purification of the alpha-amylase enzyme from the serum of a patient with pancreatitis and a healthy human, and estimation of the values ​​of Michalis constant Km as well as the maximum velocity Vmax to determine the affinity of the enzyme towards the substrate in both cases. Materials and Methods: The enzyme was purified by several steps, including precipitation, by adding ammonium sulfate at a concentration of 30-70%, then dialysis. The extract was transferred through the separation column by gel chromatography containing Sephadex G100 gel. Results: The gel separation chromatography results indicated the appearance of four protein bands, one of which (the third peak) belongs to the amylase enzyme. The specific activity of the enzyme in the last step after concentration of the product was 38 units/gm for the patient and 11.62 U/gm for a healthy human. The yield was 44.67% and 50.53%, while the number of purification times was 9.11 and 9.3 for the patient and the healthy human, respectively. The kinetic constants (Km and Vmax) were estimated using the Leinweaver-Burke plot, Vmax for the patient and the healthy human was 149.3 and 83.3, respectively. the Km for the patient and the healthy human was 1.39 and 2.56, respectively. Conclusion: It is inferred from the results that the affinity of the enzyme to bind to the substrate of patient with pancreatitis is higher than that of the healthy human.