Maturation of an NH2-terminally extended thermostable alpha-amylase in Bacillus subtilis: A possible mechanism examined by in vitro experiments

An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilis α-amylase signal peptide and the mature thermostable α-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH 2 -terminally extended thermostable α-amylase were analyzed and the results were compared with those of the mature form of the α-amylase. It is suggesteded that the cleavage of the NH 2 -terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH 2 -terminally extended peptides by the alkaline protease was also found in other hybrid thermostable α-amylases obtained.