ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS
This research is on Isolation, purification and characterization of free and immobilized alpha-amylase from bacillus licheniformis. The bacteria Bacillus licheniformis was cultured in nutrient agar and then incubated for 15h at 35oC. The bacteria cells were harvested by centrifugation after incubation. The cell free supernatant was used to estimate alpha-amylase activity. The alpha-amylase obtained was isolated and purified using ammonium sulfate precipitation, gel filtration and ion exchange chromatography. It was purified up to 15.5 fold and a yield of 20.2% on DEAE- Sephadex column with a final specific activity of 12.14 u/mg. The alpha-amylase was immobilized by entrapment in calcium alginate beads. The free and immobilized enzyme had broad temperature ranges from 20oC to 70oC with optima of 60oC and 70oC respectively and optimum pH of 7.0 and 8.0 respectively. Initial velocity studies for the determination of kinetic constants with maltose as substrate revealed a KM value of 2.5 mg/ml and 1.0 mg/ml for the free and immobilized enzyme respectively and a Vmax value of 0.4unit/mg/min and 0.95unit/mg/min for the free and immobilized enzyme respectively. Both the free and immobilized enzyme activity were enhanced by Ca2+ , Mn2+ , and Na+ while Hg2+ and Zn2+ were found to be strong inhibitors of both the free and immobilized enzyme.
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