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 extracellularproteases. 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 andalkaline protease family. The protease activity of this preparation was decreased by 20% at 700C, suggesting thatthere is at least one thermostable protease in it. The protease activities were also partially inhibited byChymostatin (45%), Pefabloc (74%), EDTANa2 (22%); this indicated that there were some of proteases thatbelong to the serine protease family and there was at least one metalloprotease. SDS-PAGE analysis showed thatthere were four proteases in this preparation having molecular weights 67 kD, 48 kD, 30 kD and 20 kD. Thesewere 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 fibrinolyticenzyme. The amino acid sequence at the N-terminal of EPE-20 was about 99% identical to the precursornattokinase and that of EPE-30 and EPE-48 was about 99% identical to mature nattokinase. There were fewdifferences 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 thedatabase. Phylogenetic analysis of this strain showed that it was most closely related to Bacillus mojavensisstrain KL.