Purification and characterization of a truncated Bacillus subtilis α-amylase produced by Escherichia coli

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 (48kDa), 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 α-amylase (57.7 kDa) and the truncated protein (54.1kDa). This truncated enzyme form hydrolysed starch with a K m of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purifed enzyme was stable at temperatures up to 50°C. While Hg 2+ , Fe 3+ and Al +3 were effective in inhibiting the truncated enzyme, Mn 2+ and Co 2+ considerably enhanced the activity.