Purification and Structure Elucidation of the Polyether Antibiotic Alborixin from Streptomyces pulcher CRF17

Aims: To screen Streptomyces sp. from saline soil in Pakistan, for its antimicrobial activity and to purify and identify the active metabolites produced by mass spectrometry and NMR spectroscopy. Strain identification by morphological, biochemical, physiological characterization and by 16S rRNA gene sequencing. Study Design: Cultivation in lab fermenter, solvent extraction and purification of the compounds by column chromatography, identification of the compounds by mass spectrometry and NMR spectroscopy, determination of the antimicrobial activity and cytotoxicity. Place and Duration of Study: Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan and Institute of Organic and Bio molecular Chemistry, University of Göttingen, Germany, between February, 2007 and April, 2009. Methodology: The strain was cultivated in a 20 liter fermenter (working volume 10 liters) and the culture broth was extracted with ethyl acetate, acetone and methanol. The resultant crude extract was fractionated on silica gel and the components were purified by column chromatography (silica gel, sephadex column and preparative TLC). The pure component was identified by mass spectrometry (ESI and HRESI-MS), NMR analysis (1H and 13C NMR) and by comparison with reference data, the antimicrobial activity and cytotoxicity was determined by disc diffusion assay and by micro well cytotoxicity assay against Artimia salina. Results: The morphological, biochemical and physiological characterization suggested that isolate CRF17 belongs to the genus Streptomyces. Further NCBI BLAST of the partial 16S rDNA gene sequence 1420 bp (gene bank accession number: EU294134) from the isolate CRF17 showed 99% identity and 98% query coverage towards Streptomyces pulcher. The scale up fermentation of the isolate CRF17 yielded active compound and was identified as alborixin (1). Conclusion: The isolate Streptomyces pulcher CRF17 is a potent producer of the antibiotic alborexin and can be exploited for its commercial production.