Analytical HPLC of the aridicin glycopeptide complex and its application to fermentation development

Summary

A gradient analytical HPLC system was developed to assay titers of the three major components of the aridicin (Ardacin) complex produced byKibdelosporangium aridum (SK&F AAD-216). The separation was performed on a Beckman Ultrasphere column using a gradient of acetonitrile (26–43%) in 0.1 M pH 3.2 phosphate buffer with UV detection at 220 nm. The gradient system was necessary to analyze all three major factors within a reasonable recycle time (14 min) without interference by front eluting impurities. The assay was linear from 12 to 200 μg/ml (multipleR ²=0.998), with a standard deviation for retention time of 1.4%. A SepPAK isolation scheme was developed to assay samples in complex matrices such as fermentation broths. Using this assay as a monitor, fermentation medium optimization increased the total titers of the three factors from approximately 5 μg/ml to over 200 μg/ml. The optimal medium contained glucose, beet molasses and methyl oleate. The latter substrate was particularly effective in enhancingproducation 10-fold, presumably by enhancing the supply of acetly-CoA. This is a biosynthetic precursor of both dihydroxyphenylglycine, present in the nucleus, and the acyl side chains present on the amino-glucuronic acids.