ISOLATION, PURIFICATION AND CHARACTERIZATION OF Bacillus subtilis ALPHA AMYLASE ISOLATED FROM CASSAVA FERMENTING SITE

Abstract

Pure strain of Bacillus subtilis was isolated from the soil sample obtained from cassava processing site based culture medium. The crude enzyme was purified using a combination of ammonium sulphate precipitation, DEAE Sephadex A-50 and gel filtration. The effect of pH, temperature and metal ions were investigated. Thermal and pH stability as well as Km, and Vmax of the purified enzyme were determined. The molecular weight of the purified enzyme was also determined. Single protein band on SDS-PAGE suggested that the enzyme was homogenous. Two different activity peaks were observed in ion exchange chromatography, the enzyme yield were 7.79 % and 3.95 %, while purification fold of 16.24 and 6.57 were achieved for Pool A and Pool B respectively. The estimated molecular weight of purified amylase from SDS-PAGE was 53 kDa. The two fractions revealed the same optimum pH of 7.0, but different optimum temperature of 60 oC for Pool A and 40 oC for Pool B. The enzyme retained about 70 % of its initial activity after 120 minutes incubation time at 40 oC for pool A and B but less than 20 % residual activity at higher temperature of 60 oC and above for both fractions. The kinetic parameters Km and Vmax of the purified amylase were 5.18 mM, 2.54 mM and 0.29 μmol/min/ml, 0.16 μmol/min/ml for pools A and B respectively. The activity of the enzyme was enhanced in the presence of Na+ and Zn2+, but strongly inhibited by Ca2+ and Hg2. The results revealed a potential for industrial application.