Purification and characterization of pepsin enzyme from Scomberomorus commerson mackerel’s stomach by aluminum hydroxide, and improved its thermal stability by graphene oxide nanosheet

Abstract In the present study, pepsinogen enzyme was purified from the S. commerson viscera in 7 steps, including; (1) using a buffer containing NaCl and CaCl 2 , (2) Acidification, (3) precipitation by dried sulfate ammonium, (4) Aluminum hydroxide gel, (5) Saturated ammonium sulfate, (6) Gel filtration Sephadex G-50, and (7) Anion-exchange DEAE-cellulose. Purified pepsinogen converted into pepsin quickly at pH 2.0, and its optimum pH and temperature were 2, and 37 °C. Hence, ammonium sulfate with 67/5 % saturation showed the highest activity and protein precipitation. Besides, results showed that 18% Alum gel had the highest enzyme activity in the precipitate formed during dialysis. Furthermore, pepsin activity was stopped above 50 °C, but immobilized pepsin on GO-PEG maintained it up to a temperature of 65 °C. Purified pepsin was completely inactive in the presence of 0.1 M pepstatin A. Catalytic constants K M and k cat for proteolysis of acid-denatured hemoglobin were 35/39 ± 0.03 M, and 5.3 ± 0.002 × 10 -5 S -1 , respectively. Finally, based on the obtained results, it can be suggested that the use of aluminum hydroxide gel and graphene oxide can be a suitable approach for purifying pepsin enzyme from fish viscera and improving their thermal stability.