Extraction, Purification and Characterization of Lipoxygenase from Aspergillus niger

Lipoxygenase was extracted from a local isolate of Aspergillus niger that was obtained from maize. The isolate was grown and biomass was harvested after 5days of incubation at 30°C for detecting enzymatic activity. Three steps were used to purify lipoxygenase: concentration by using ammonium sulfate with (30-90%) saturation , then ion exchange chromatography by using DEAE Sephadex A-50 with NaCl (0-1)N. Two peaks appeared at the washing step and four peaks appeared at the elution step, Only one peak had specific activity ,which was 592.14 u/mg, That peak was chosen for the third step of purification, using gel filtration chromatography with Sephadex G-100. Only one peak showed specific activity (1069.7u/mg). The purity of enzyme was tested by gel electrophoresis using polyacrylamide, The enzyme was show to be purified by appearance as band. The characteristics of purified lipoxygenase were then studied: The molecular weight was104 KDa as shown by SDS- polyacrylamide gel electrophoresis, The optimum temperature and pH of enzyme activity were 35 ̊Cand 6.5 respectively. Heat stability and optimum pH of stability were (0-45) ̊C and (7-6) respectively. The effect of some mineral ions on enzymatic activity, had been noticed that CaCl2, KCl, NiCl2 , MgCl2 and MnCl2 increased the activity while the enzymatic activity was decreased when using ZnCl2,FeCl3 , and CuCl2. EDTA inhibited the enzyme activity. Studying of kinetic constants showed that Michaelis constant (Km) and maximum velocity (Vmax) of the enzyme were 0.2 mg/mLand 45 µm/mL/min respectively. The activation energy was 8 kcal/mole, and the denaturation energy was 54 kcal/mole.

کلیدواژه ها:

Lipoxygenase ، Extraction ، Purification ، Characterization ، Activity ، Stability