Purification and characterization of α-amylase fromAspergillus flavus

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheKm andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively.