PURIFICATION OF ALKALINE PROTEASES FROM A BACILLUS STRAIN AND THEIR POSSIBLE INTERRELATIONSHIP

Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, a s detected by polyacrylamide gel electrophoresis (PAGE). The major pro tease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor pr oteases, designated H protease and N protease, were purified to homoge neity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55 degrees C. N protease consisted of two polypeptide chains with molecul ar masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60 degrees C. The amin o-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic r elationship between the three enzymes was examined after they had been stored at different pH values and at 5 degrees C. M protease was conv erted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 1 1. N protease appeared to be the autolytic product of the M and H prot eases.