Purification of surfactin compounds produced by a Bacillus subtilis strain

Surfactins are lipopeptide-type biosurfactants produced mainly by Bacillus species containing a peptide loop of seven amino acids and a hydrophobic fatty acid chain. These molecules exhibit various biological activities; therefore, their therapeutic and environmental applications are in the focus worldwide. In our work, a multi-step purifi cation and separation process was developed to isolate surfactins from the ferment broth of B. subtilis SZMC 6179J strain. The process incorporates normal phase fl ash chromatography for pre-purifying the crude extract and two consecutive reverse phase HPLC separations for the isolation of the various surfactin molecules. The determination of the relative amounts of lipopeptides both in the crude extract and in each fraction of every separation step were carried out by HPLC-HESI-MS examinations. The ratio of surfactins in the crude extract was 21.35%, but after the preparative fl ash chromatographic separation the relative amount of surfactins was observed to be 30.44%. The preparative HPLC purifi cation step resulted 85.39% purity of the surfactins. Nine diff erent surfactin variants were isolated and identifi ed from the fractions of this fi nal semi-preparative HPLC purifi cation, out of which three compounds were completely purifi ed, and three others were detected in relative amounts of more than 95% in some fractions.