Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis.

A strong fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4, subtilisin DFE, was isolated from douchi, a traditional Chinese soybean-fermented food. Based on the high homology between the N-terminal sequence of subtilisin DFE and that of subtilisin BPN, PCR primers were designed that allowed for the amplification and cloning of the intact subtilisin DFE gene. Sequence analysis indicated the presence of a 1149-bp open reading frame encoding 382 amino acid residues. The enzyme was actively expressed by the Escherichia coli-Bacillus subtilis shuttle expression vector pSUGV4 in the protease-deficient strain B. subtilis WB600, and its biochemical characteristics were the same as those of the original subtilisin DFE isolated from the donor strain, i.e., its molecular weight is approximately 28 kDa and it is a serine protease.