Production, Encoding Genes Variability and N-Terminal Sequences of Fibrinolytic Enzymes Produced by Bacillus Strains Isolated from Fermented Cassava Leaves Ntoba Mbodi at Brazzaville, Republic of Congo

Nine (9) Bacillus strains isolated from Ntoba Mbodi, already characterized by their 16S rDNA and listed as: Bacillus safensis NM1, Bacillus safensis NM5, Bacillus pumilusNM28 Bacillus pumilus NM29, Bacillus subtilis NM39, Bacillus subtilis NM41, Bacillus megaterium NM56, Bacillus licheniformis NM74 and Bacillus amyloliquefaciens NM75, have been explored for Production, encoding genes variability and molecular characterization of their fibrinolytic enzymes. All of them produce proteolytic enzymes using casein as a substrate. Using fibrin, two of them as Bacillus safensis NM1, Bacillus safensis NM5 could not produce fibrinolytic enzymes. For the same conditions fibrinolytic enzymes production was different as was the growth. To explore encoding fibrinolytic enzymes genes variability, DNA was extracted and using three selected sets of primers in the literature, PCR amplification of corresponding genes was done with the optimization of annealing temperature (58C, 60C and 62 C). Only three strains including. Bacillus subtilis NM39, Bacillus subtilis NM41and Bacillus licheniformis NM74 displayed specific bands in the 1 % Agarose Gel electrophoresis for one to another set for 58C. Sequencing of specific bands in two strains Bacillus subtilis NM41 and Bacillus licheniformis NM74 was performed, followed by a bioinformatic analysis. The gene encoding fibrinolitic enzyme in B. subtilis NM41 exibits 99 % of similarity with subtilisin gene of B. subtilis G1, accession number EF061457.1 in GenBank. While that encoding fibrinolitic enzyme in B. licheniformis NM74 exibits 98 % of similarity with Alkaline protease (apr) gene of B. licheniformis MP1, accession number HM147766 in GenBank. All the two fibrinolytic enzymes have some modifications in the pre, the pro and the mature peptide. The N-terminal sequence has shown in the two mature peptides, for the twelve first amino acids two kinds: AQTVPYGIPLIK for NFE-1 (Ntoba Mbodi fibrinolytic enzyme-1) of Bacillus licheniformis NM 74 and AQSVPYGISQIK for NFE-2 (Ntoba Mbodi fibrinolytic enzyme-2) of Bacillus subtilis NM41. The sequences of fibrinolytic enzymes in Bacillus strains of Ntoba Mbodi comprise the two kinds of organizations in N terminal sequences which are already described.