Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 3: RESULTS3.2.4 Thin Layer Chromatography (TLC) of AntimicrobialMetabolites from Fermented ActinobacterialCultures3.2.4.1 Liquid FermentationsAs a result of preliminary antimicrobial screening optimal fermentation medium wasselected for each actinobacterial culture based on the strongest antibacterial activity.Extracted fractions from these actinobacterial cultures were collected and furtheranalysed using thin layer chromatography to obtain a chemical metabolic bandingprofile. Three fractions were obtained these consisted of the broth supernatant,methanolic and EtAc extracts of the mycelium As shown in figure 33, metaboliteswere present in the MeOH or EtAc extracts. The EtAc extracts from isolates A0350,A1113 and A2707 in lanes 4, 7 and 12 respectively, displayed a banding pattern thatwas stronger in intensity than those extracted from the broth supernatant (lanes 1, 5,and 8) and methanol (lanes 3 & 6), indicating that EtAc was better suited forextraction of these metabolites. Similar chemical banding profiles were present in theMeOH extracted mycelium from isolates A0350 and A1113, indicating that the samecompounds were present in the mycelium (lanes 3 & 6), however the bandingintensity was not as strong as that seen in the EtAc extracts (lanes 4 & 7). In certainextractions, metabolites were only extractable using EtAc for isolate A2381 (lane 10),indicating a selective isolation step which can be incorporated into the purificationprocess for this class of compound.1 2 3 4 5 6 7 8 9 10 11 12 13 14Lane 1: Dextrin fermentation medium with A0350 isolate (Broth Supernatant)Lane 2: Dextrin fermentation medium with A0350 isolate extracted twice with EthylAcetate_____________________________________________________________________103