Studies on Bacterial Proteases

An alkaline protease (pep tidy I peptide hydrolase, EC class 3.4.4) of Bacillus natto strain Ns was purified by column chromatographies on Duolite A‐2 anion exchange resin, CM‐cellulose, and DEAE Sephadex A‐50, and crystallized from aqueous solution. The enzyme was most active at pH values between 10.3 and 10.8 toward casein substrate and completely inactivated by incubation with DFP. Ultracentrifugal analyses indicated that the molecular weight and S 20,w were 27,000 and 2.8, respectively. The enzyme consisted of a single polypeptide chain with alanine as the amino terminus and was composed of 271. residues of amino acids, the composition of which was quite similar to that of subtilisin Carlsberg (EC class 3.4.4. 16). The alkaline protease of B. natto Ns strain was clearly distinguished electrophoretically from subtilisin BPN’and subtilisin Amylosacchariticus but not from subtilisin Carlsberg. The fact that most strains of Bacillus natto so far investigated by us have also produced this type of protease suggests that Bacillus natto mainly secretes the alkaline protease belonging to the group of subtilisin type Carlsberg.