Purification and characterization of a truncatedBacillus subtilis α-amylase produced byEscherichia coli

ABacillus subtilis amylase gene was inserted into a plasmid which transferred toEscherichia coli. During cloning, a 3′ region encoding 171 carboxyterminal 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 theB. subtilis complete α-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with aKm of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purified enzyme was stable at temperatures up to 50°C. While Hg2+, Fe3+ and Al3+ were effective in inhibiting the truncated enzyme Mn2+ and Co2+ considerably enhanced the activity.