Miniscale identification and characterization of subtilisins from Bacillus sp. strains

Abstract

Subtilisin (EC 3.4.21.14) is the major extracellular alkaline serine protease of Bacillus species. Previously, we found that subtilisins did not migrate in the electrophoretic field in the Laemmili buffer system due to their high pI values (over 8.8); however, it formed a "binding mode" at the top of the separating gel. Utilizing this characteristic, four subtilisins from Bacillus sp. strains (e.g., B. subtilis 168, B. subtilis KCTC 1021, B. amyloliquefaciens KCTC 3002, and Bacillus sp. DJ-1 and DJ-4) were easily and quickly identified by an over-running electrophoretic technique with a miniscale culture supernatant (less than 20 ml) without any column chromatographic steps. Two subtilisins (DJ-1 and a recombinant version) from Bacillus sp. DJ-1 were characterized, and the enzymatic properties were determined by SDS-fibrin zymography and densitometric analysis. Based on this observation, the recombinant pro-subtilisin DJ-1 showed the same "binding mode," similar to native subtilisin DJ-1. On the other hand, mature subtilisin DJ-1 without pro-peptide showed no enzymatic activity.