title: Purification and characterization of novel α-amylase from Bacillus subtilis KIBGE HAS.
abstract: Purification of extracellular α-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 α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-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 α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain.
authors.affiliation: Pharmaceutical Research Center, PCSIR Laboratories Complex, Karachi, Pakistan.
authors.forename: Saeeda
authors.initials: S
authors.lastname: Bano
authors.forename: Shah Ali
authors.initials: SA
authors.lastname: Ul Qader
authors.forename: Afsheen
authors.initials: A
authors.lastname: Aman
authors.forename: Muhammad Noman
authors.initials: MN
authors.lastname: Syed
authors.forename: Abid
authors.initials: A
authors.lastname: Azhar
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000516:alpha-Amylases