Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONapproach to chemical screening provides an efficient means in elucidatingbiologically active fractions in a chemical heterogeneous mixture.Evaluations determining whether SM were produced intra- or extracellularly, wasshown that the antimicrobial compounds were in most cases present in the brothsupernatant (BSN) and mycelial extracts (ME). Extraction efficiency of SM isdetermined by the polarity of solvent and pH (Schügerl, 1994). The solvent behaviorof the compounds were shown to be highly polar as they were soluble in both waterand organic solvents. Acid-base characteristics of SM extracted in organic solvents,evaluated by small-scale pH extractability studies showed that there was no alterationin the antimicrobial activities or coloration of the extract which may indicate pHdependence of classes of compound such the anthracyclines (Arcamone, 1998). Thepresence of antimicrobial activity in the BSN may indicate that SM were able to beexcreted into the medium, however constituents in the media may have masked someof the antimicrobial activity. Overlooking this masking phenomena can lead to falsenegatives, thus alternative methods of metabolite production detection was used suchas thin-layer chromatography (TLC) which provided a metabolic fingerprint of eachstrain (Zähner et al., 1988). In certain isolates the metabolic banding patterns wereidentical in the BSN and ME represented by fluorescent common bands on on theTLC plate under UV wavelength of 254 nm , however fainter bands were evident in theBSN. The stronger intensities of the metabolic bands in the ME and the presence ofsimilar bands in the BSN, indicated that the compounds were more soluble in organicsolvents, and that partial extractions took place (Figure 33).Zones of inhibition were also detected from the initial points of application of sampleon TLC plates, indicating that the solvent system used may not have been adequatefor efficient separation of the bioactive compounds. A low-polar solvent systemconsisting of butanol-acetic acid-water (4:1:1), was evaluated to separate the bioactivecompounds however this proved to be unsuccessful.4.6 Isolation of Bioactive MetabolitesDevising a isolation scheme for the bioactive metabolites produced by theenvironmental isolates, was devised from the TLC solvent system used where EtAc_____________________________________________________________________128