Jornal Europeu de Biologia Experimental Acesso livre

Abstrato

Purification and comparison properties of crude enzyme with purified α-amylase from Bacillus licheniformis ATCC 6346

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 85^oC. The optimum pH was 7.0 for the crude and purified at 85^oC. When the crude enzyme was pre-incubated at 85^oC 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 85^oC 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.