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High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium

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A marine Bacillus strain produced biosurfactant during its growth in a defined glucose-containing medium. An efficient method for separation and purification of biosurfactant isoforms was developed and optimized in high-performance liquid chromatography (HPLC) by manipulating solvent gradient program and flow rates. Starting with an initial run time of 60 min, the final optimized method had a significantly reduced run time of 20 min. By using this method, all the surface-active isoforms (fractions A–D) were eluted within 12 min of elution with much shortened retention time of each component. The purity levels of the isoforms were enhanced using the optimized method as evident from their lower CMC values. Among the four surface-active fractions, antimicrobial action was solely displayed by HPLC fraction A. FTIR analysis revealed all the HPLC fractions to be lipopeptide in nature and MALDI-ToF mass spectral analysis showed that these belonged to the fengycin family containing C15, C16, and C17 fengycins.

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RS and CS acknowledge the Department of Biotechnology (DBT), the Government of India for the project grant (BT/PR-6827/AAQ/03/263/2005) in marine biotechnology. SM acknowledges CSIR, New Delhi for the Senior Research Fellowship. RS acknowledges Department of Biotechnology, IIT Kharagpur. Authors acknowledge Palashpriya Das for taking pains in strain isolation and for her all-round support throughout this work.

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Correspondence to Ramkrishna Sen.

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Sivapathasekaran, C., Mukherjee, S., Samanta, R. et al. High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium. Anal Bioanal Chem 395, 845–854 (2009).

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