Production, purification and characterization of antibacterial biomolecules from an alkaliphilic Bacillus

Abstract: Forty alkaliphiles, which were isolated from samples collected in Eastern Africa, have been screened for production of antibacterial compounds. One of these isolates, Bacillus halodurans B20, was able to produce a prominent amount of antibacterial activity against a Gram-positive bacteria on agar plates paper I and selected for further study. Production of the active compound in submerged cultivation by B. halodurans B20 was not successful. However, Solid State Fermentation (SSF) turned out to be the best alternative for production of the antibacterial substance paper I. Optimization of the production conditions for antibacterial compound revealed that the production was stimulated by addition of Mg2SO4 and K2HPO4. The highest production was achieved at a wheat bran/moisture ratio of 1:1.8 and in the presence of 10% (w/w) Na2CO3 paper II. The active substance was purified using hydrophobic interaction chromatography. Mass spectrometer (MS) and tandem MS/MS analysis showed that the purified bioactive compound was composed of two peptides with molecular mass of about 3043 and 2330 Da. The size of the peptides and the strain suggest that the bacteriocin belongs to the two-component lantibiotics of haloduracin type paper I. Its antibacterial effect against clinically important pathogens has been studied alone or in combination with chloramphenicol. A synergistic effect of the haloduracin and chloramphenicol has been observed against strains of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis and different groups of Streoptococcus paper III. In addition, another bacterial metabolite was purified from the solid state culture of B. halodurans 20 using ion exchange chromatography column. The purified compound has shown antibacterial activity only against Gram-positive bacteria. The compound is proposed to be a phenyl fatty acid derivative paper IV. However, further study is necessary to determine the exact structure of this compound.