Purification and Characterization of α-Amylase from Bacillus subtilis Isolated from Cassava Processing Sites

Abstract

This study was designed to purify and characterize of α-amylase from pure strain of Bacillus subtilis. The crude α-amylase was purified by ammonium sulphate precipitation, then loaded on DEAE Sephadex A-50 ion exchange chromatography and gel filtration. The effect of pH, temperature and metal ions were investigated on the purified enzyme. The single protein band on SDS-PAGE suggested that the enzyme was homogenous. Two different activity peaks were observed in ion exchange chromatography designated pool A and pool B with the 8% and 4% yield, 15.93 and 6.44 purification fold and specific activity 2.55 μmol/min/mg and 1.03 μmol/min/mg respectively. The two fractions revealed the same optimum pH 7.0 for the α-amylase activity while the enzyme was relatively stable at pH 4.0 and 7.0 between 20 to 40 minutes and 60 to 80 minutes for pool A and pH 8.0 between 40 and 100 minutes for pool B. At 40°C, optimum temperature was reached, and amylase activity was maintained at 75% and 70% temperature stability between 60 to 80 minutes for pool A and B, less than 20%, the residual activity at 60°C and 70°C was recorded. The incubation of α-amylase with Na + and Zn 2+ ions enhanced/activate the enzyme activity correspondingly, Al 3+ and K + ions exhibited varied degree of inhibition while Ca 2+ and Hg 2+ ions caused total inhibition on α-amylase activity. The ability of purified α-amylase from Bacillus subtilis under wide range of temperatures and pH suggests its applications in industries and bioremediation of effluent discharge on food processing sites.

Production of thermostable multiple enzymes from Bacillus amyloliquefaciens KUB29

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