Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 3: RESULTS3.2.8 Determination of antibacterial activity of HPLC fractionsfrom Organic ExtractsFractions corresponding to the HPLC peaks were collected and assayed using wholecell antibacterial activity screens by the industry partner. Extracts from A0350 andA1113 showed similar chromatographic profiles exhibiting two main peaks 1 and 2with retention times 11.6 and 11.9 minutes respectively (Figure 36), both exhibitingthe same chromaphore (UV-Vis Spectra). Fractions recovered from 11.0 to 12.5minutes from extract A0350 showed strong antibacterial activity againstStaphylococcus aureus achieving 98% inhibition in the whole cell antibacterialscreen. In extract A1113 two antibacterial activities were recovered from fractionscollected from 11.0 to 12.5 minutes. Strong inhibition was detected against S.aureusachieving 100 % inhibition and at 11.5 minutes strong inhibition againstStreptococcus pneumoniae achieving 100% inhibition. A number of peaks weredetected in both chromatograms in the UV-Vis region from extracts A0350 andA1113, exhibiting distinct chromophores however no antibacterial activity wasdetected (Figure 36). A number of peaks were detected in the chromatogram ofextract A2381 exhibiting different chromophores. Fractions recovered from between14 and 18 minutes in the non-polar region of the chromatogram showed strongantibacterial activities against S.aureus achieving 100 % inhibition and S.pneumoniae100% inhibition. No antibacterial activity was recorded in the course of screening theGram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumoniae for allthe extracts tested.3.2.9 Electrospray Ionisation High Performance Liquid-Chromatography Mass-Spectrometry (ES HPLC-MS) ofOrganic ExtractsLC-MS analysis in the negative mode revealed that both extracts A0350 and A1113contained two major peaks at 26.2 and 26.5 minutes, these dominant peaks yieldedmolecular ions at m/z 1253 to 1255 in the negative ion mode. The compounds did notionize well in the positive ion mode. LC-MS analysis in the negative ion mode ofextract A2381 showed the presence of multiple peaks. The two major compoundsshowed peaks at 25.53 and 26.3 with respective molecular ions in the negative ionmode at m/z 425.5 and m/z 385.3 (Table 57)._____________________________________________________________________110