Production, purification and characterization of extracellular α-amylase from Aspergillus fumigatus HBF125

In our study, screening of industrial enzymes and temperature limits of the 91 fungi was done. Aspergillus fumigatus HBF 125 strain was chosen as the best one showing amylase activity from thermotolerant and thermophilic fungi. Culture condition was optimized for this fungus.. The best enzyme production from A. fumigatus HBF125 strain were determined in medium including 7 days sporulation medium, 5% inoculum ratio, temperature 35 °C, starting pH 5.0 and 1.5% bran as carbon source. Extracellular amylase produce by A. fumigatus HBF125 in optimal growth medium purified 54.4 fold with a recovery of 4.7% using starch affinity chromatography. Molecular weight of enzyme was found to be about 160 kDa by SDS-PAGE method. Those two subunits of molecule weight of the enzyme was found to be 86.2 and 73.8 kDa. The temperature and pH for maximum activity of the enzyme were 60 oC and 5.5, respectively. In a wide range of pH and temperature of the enzyme was found to be stable. It was found that the Km and Vmax of amylase were 1.43 mg/mL and Vmax 909 U/mL. In addition, the amylase exhibited broad substrate specificity. The enzyme activity was markedly inhibited in the presence of NBS suggesting that tryptophan residues play an important role in the catalytic process. The enzyme activity was not affected from detergents as Tween 20, but it was found inhibited by 1.4-dioxan and n-propanol. The enzyme by Hg2+ was strongly inhibited while Mn2+, Co2+, Ca2+ and Ba2+ were activated. The salt tolerance of the enzyme was found to be very good. The amylase was found to be metalloenzyme.