Characterization of Multiple Extracellular Proteases Produced by a Bacillus subtilis Strain and Identification of the Strain

A Bacillus subtilis strain isolated from shrimp by-product in Vietnam produced a mixture of extracellular
proteases. The enzyme preparation precipitated by ethanol (EPE) obtained from this strain was characterized.
The effect of pH on the protease activities showed that the proteases in this preparation belong to neutral and
alkaline protease family. The protease activity of this preparation was decreased by 20% at 700C, suggesting that
there is at least one thermostable protease in it. The protease activities were also partially inhibited by
Chymostatin (45%), Pefabloc (74%), EDTANa2 (22%); this indicated that there were some of proteases that
belong to the serine protease family and there was at least one metalloprotease. SDS-PAGE analysis showed that
there were four proteases in this preparation having molecular weights 67 kD, 48 kD, 30 kD and 20 kD. These
were named EPE-67, EPE-48, EPE-30 and EPE-20 respectively. EPE-48, EPE-30 and EPE-20, but not EPE-67,
were found to match with nattokinase by MS analysis. Among these EPE-20 was a thermostable fibrinolytic
enzyme. The amino acid sequence at the N-terminal of EPE-20 was about 99% identical to the precursor
nattokinase and that of EPE-30 and EPE-48 was about 99% identical to mature nattokinase. There were few
differences in the amino acids between the identified sequences and the known sequences in the database.
Sequencing analysis of 16S rDNA gene showed 99% identity of the tested strain with the Bacillus subtilis in the
database. Phylogenetic analysis of this strain showed that it was most closely related to Bacillus mojavensis
strain KL.