Purification and Characterization of Novel Delta a-Amylase from Bacillus subtilis KIBGE HAS

• Published in: AAPS PharmSciTech 2011-03, Vol.12 (1), p.255-261
• Main Authors: Bano, Saeeda, Qader, Shah Ali Ul, Aman, Afsheen, Syed, Muhammad Noman, Azhar, Abid
• Format: Article
• Language: English
• Subjects: Indexing in process
• ISSN: 1530-9932; 1530-9932
• DOI: 10.1208/s12249-011-9586-1

Purification of extracellular Delta *a-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified Delta *a-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular Delta *a-amylase showed that the enzyme had a Km and V max value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at 50?C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly (S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of Delta *a-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported Delta *a-amylases from Bacillus strain.