Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp strain DJ-4 screened from Doen-Jang

Bacillus sp. strain DJ-4, which produces extracellular proteases, was scree ned from Doen-Jang, a traditional Korean fermented food. A fibrinolytic enz yme (subtilisin DJ-4) was purified using commercial chromatographic techniq ues. The relative molecular mass of the isolated protein was 29 kDa by SDS- PAGE and fibrin zymography assay. The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amido lytic assay using a chromogenic substrate. The enzyme was inhibited by PMSF , but not by EDTA or leupeptin. The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN', but the activity of sub tilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively.