Development and Scale-up of an Efficient and Green Process for HPLC Purification of Antimicrobial Homologues of Commercially Important Microbial Lipopeptides

Lipopeptides are green surfactants with myriad applications in the areas of healthcare, energy, and the environment. However, the commercial potential of these ecofriendly molecules could not be fully realized due to lack of an economically viable and environmentally sustainable purification process. Thus, we endeavored to systematically develop and scale-up a green chromatographic process with zero solvent discharge for rapid and efficient purification of bioactive homologues of three lipopeptide families produced by marine Bacillus megaterium. On optimization of the purification process by analytical HPLC, significant reduction of elution time from 80 to 30 min with simultaneous improvement in peak resolution was achieved. This process was scaled-up to semipreparative HPLC, while maintaining the solvent linear velocity constant to achieve the same purification efficiency and product quality. MALDI-ToF mass spectral analysis of the purified fractions confirmed the presence of the homologues of the iturin, fengycin, and surfactin families of lipopeptides, which were subsequently tested positive for their antimicrobial activities. As evident from minimum inhibitory concentrations, the homologues containing long chain fatty acid exhibited stronger antimicrobial activities than those with shorter chains. Thus, the ecofriendly process can well qualify as a standard operating procedure for efficient purification of a particular lipopeptide homologue with the desired therapeutic properties.