PURIFICATION AND CHARACTERIZATION OF A TRUNCATED BACILLUS-SUBTILIS ALPHA-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 carboxyterminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous pro tein. 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 calculat ed from the derived amino acid sequences of the B. subtilis complete a lpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This tru ncated enzyme form hydrolysed starch with a K-m of 3.845 mg/ml. Activi ty was optimal at pH 6.5 and 50 degrees C, and the purifed enzyme was stable at temperatures up to 50 degrees C. While Hg2+, Fe3+ and Al+3 w ere effective in inhibiting the truncated enzyme, Mn2+ and Co2+ consid erably enhanced the activity.