Purification and characterization of amylase enzyme produced by Aspergillus sp.

The extracellular amylase produced by Aspergillus fumigatus HBF50 was purified 3.0 folds using ultrafiltration, DEAE sepharose CL-6B, phenyl sepharose CL-4B and gel filtration chromatography. The molecular weight of amylase was estimated to be about 55 kDa by SDS-PAGE. The temperature and pH for optimum activity of the enzyme were 55°C and 5.0, respectively. It was completely thermostable between 27°C and 40°C. It was found to be stable pH range between 4.0 and 8.0. Km and Vmax values for soluble starch of the enzyme were calculated to be 1.55 mg/ml and 9091 U/mL, respectively. The amylase exhibited broad substrate specificity. The enzyme was stimulated by Mn2+ and Co2+ and strongly inhibited by Cu2+, Fe2+, Al3+ ve Hg2+.The enzyme activity was inhibited in the presence of NBS, PMSF, IAA and CMC, suggesting that tryptophan, serine, histidine residues and carboxyl groups play an important role in the catalytic process. The activity of amylase was found to be tolerant up to 5M NaCl concentration. Raw corn starch adsorption of amylase was found to be 48%. Thin layer chromatography of the starch hydrolysis products showed glucose, maltose and maltotriose as the main end products, indicating that the purified enzyme was ?-amylase.