Gene Expression of the Micrococcus luteus Fibrinolytic Enzyme (MLFE) in Bacillus subtilis WB600

Abstract:

MLFE (Micrococcus luteus fibrinolytic enzyme) is a fibrinolytic enzyme produced by Micrococcus luteus ML909 strain. The promoter and signal peptide-coding sequence of α-amylase gene from Bacillus amyloliquefaciens DC-4 was cloned and fused to the sequence coding for mature peptide of MLFE (Gen-Bank: EU232121), forming the fusion gene called amymlfe. This hybrid gene was inserted into the Es-cherichia coli-Bacillus subtilis shuttle plasmid vector pSUGV4 and expression plasmid pSU-AmyMLFE was constructed. After transformation with B. subtilis WB600, transformant WB600/pSU-AmyMLFE was obtained and produced clear hydrolyzed zones on fibrin plates. The fibrinolytic activity in supernatants of WB600/pSU-AmyMLFE fermented for 24 hours was tested and found to be 238 UKU/mL. The results of SDS-PAGE analysis showed that there was indeed recombinant protein in supernatants. The Western blotting showed that the molecular weight of the expressed protein was the same as expected. These results indicate that the gene, amymlfe, is successfully expressed in B. subtilis WB600.

Key words:Micrococcus luteus, Fibrinolytic enzyme, α-amylase gene promoter, Fusion gene, Bacillus subtilis