title: Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli.
abstract: A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli. During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of the B. subtilis complete alpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with a Km of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50 degrees C, and the purified enzyme was stable at temperatures up to 50 degrees C. While Hg2+, Fe3+ and Al+3 were effective in inhibiting the truncated enzyme, Mn2+ and Co2+ considerably enhanced the activity.
authors.affiliation: Departamento de Biologia Celular, Universidade de Brasilia.
authors.forename: J L
authors.initials: JL
authors.lastname: Marco
authors.forename: L A
authors.initials: LA
authors.lastname: Bataus
authors.forename: F F
authors.initials: FF
authors.lastname: Valência
authors.forename: C J
authors.initials: CJ
authors.lastname: Ulhoa
authors.forename: S
authors.initials: S
authors.lastname: Astolfi-Filho
authors.forename: C R
authors.initials: CR
authors.lastname: Felix
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D000516:alpha-Amylases