Vengadaramana, A., Balakumar, S. and Vasanthy Arasaratnam
The α-amylase from Bacillus licheniformis ATCC 6346 was purified by ion-exchange chromatography (DEAE-Sepharose). The spent medium contained 37.5 UmL-1 a-amylase activity and 1.77 mgL-1 protein. Highest specific activity (65.54Umg-1) was obtained at 50% (NH4)2SO4 saturation and 66.6% recovered. The precipitated and dialyzed enzyme was purified using DEAE-Sephorose at pH 8.0, and eluted with the 0.01M Tris buffer containing 0-0.8 M NaCl. The recovery of a-amylase by ion-exchange chromatography was 7.5%, with 8.2 fold purification, showing the specific activity of 173.8Umg-1 protein. The purified a-amylase was tested for purity by SDS-PAGE. The purified enzyme showed a single band with an apparent molecular weight of 55.54 kDa. Crude a-amylases showed zero order kinetics for 10min while purified a-amylase showed zero order kinetics for 8min. The optimum temperature for the activities of crude and purified enzymes was 85oC. The optimum pH was 7.0 for the crude and purified at 85oC. When the crude enzyme was pre-incubated at 85oC and at pH 7.0, it lost 40% of its initial activity at 10min while the purified enzyme lost 75% of its initial activity at 10min. Crude and purified enzymes showed 119, 77.7 & 20.3 and 107, 60, & 20% of relative activities respectively with amylose, amylopectin, and maltose when compared to soluble starch at 85oC and pH 7.0. Both crude and purified enzymes showed no activity with cellulose, sucrose and pullulan. Therefore substrate specificity indicated, that both purified and crude a-amylases were able to hydrolyse mainly starch, amylose and amylopectin.
