Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONwas the major organic solvent. The highly polar solvent system, ethylacetate:methanol (9:1) was successfully used in the separation of compounds by TLCfrom both fermentation broth and organic fractions (Figure 33). This also indicatedthat the compounds were soluble in the solvents used. Small scale extractions usingboth methanol and ethyl acetate showed that compounds were able to be separated. Apurification scheme was devised incorporating ethyl acetate as the solvent to be usedin solvent extraction. In order to isolate sufficient amounts of the antimicrobialmetabolites, a liquid fermentation was scaled-up from 50 mls upto 1 litre to obtain adry yellow-orange powder from isolates A0350, A1113 and a oily brown residue fromisolate A2381. The average recoveries were 4.0 mg of semi-purified extract.4.7 UV-Vis Spectroscopy scanning of organic extractsMany microbial metabolites have characteristic absorption spectra in the ultraviolet –visible region, such spectra; (1) can be used for initial identification by comparisonwith spectral libraries to determine if the compound belongs to a particular chemicalclass, (2) for identifying components related to a known structure and (3) formonitoring the production of secondary metabolites (Stead., 1998; Bystrykh et al.,1996). Properties of classes of compounds such as polyketides, which haveabsorbance maxima between 205 –280 nm or polyenes which absorb between 300 –380 nm , can be used to partially identify constituents in microbial fermentation extracts(Williams & Fleming, 1973). UV spectra of extracts from strains A1113, A0350 andA3675 showed the presence of polyene type compounds, and an anthraquinonechromophore (Rohr personal commun.). The presence of this four peak profile hasbeen detected in microbial extracts possessing antifungal activity (Gagoś et al., 2001).Interestingly, extract A3675 containing this profile contained only antifungal activity,however extracts A0350 and A1113 containing the extended chromophore showedboth antibacterial and antifungal activities (Table 56 and Figure 35). From thedetected antimicrobial activities indications are that the chemical species representedby the four peak UV spectrum may be responsible for the antifungal activity. Theextended chromophores detected in UV-Vis spectral profiles of A1113 and A0350(Table 56) are characteristic of aromatic compounds (Doyle et al., 1979). UV-Visspectroscopy of extract A2381 showed a number of highly absorbing chemicalspecies indicated by the bell shaped UV spectra profile in the 250 – 400 nm range._____________________________________________________________________129