Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODS2.6.1.1 Small scale actinobacterial fermentation metabolite extractionand processingAliquots (1 ml) of actinobacterial fermented culture grown in various productionmedia were centrifuged at 4000 rpm for 20 min to pellet the mycelium. Aftercentrifugation the broth supernatant was separated out by siphoning it off with amicropipette and it was kept for testing. 200 μls of MeOH or EtAc was added to thepelleted mycelium to extract metabolites. Following addition of MeOH or EtAc tothe mycelium a vortex mixer was used for thorough mixing and then left standing for30 min. Following this incubation time the mycelium extract was centrifuged asabove, and the MeOH or EtAc mycelial extract was siphoned off with a micropipette.Both the broth supernatant and MeOH or EtAc mycelial extract were tested forbioactivity (section 2.7).2.6.2 Large scale production and recovery of antimicrobial metabolitesGenetic and bioassay directed screening identified a number of the environmentalactinobacterial cultures as producers of antimicrobial metabolites (Table 55). Three ofthese cultures were selected for large-scale production and purification studies. Thesecultures were A0350, A1113 and A2381. Using optimal fermentation conditions,identified in section 3.7 for each of the cultures, the fermentation was scaled up to 1liter. In order to partially identify and characterise the active constituents from thesemetabolite producing cultures, a semi-purified organic extract was recovered usingthe conditions outlined below. A general purification regime was applied to each ofthe cultures to produce a suitable quantity (>1mg) of semi-purified organic extractsfor chemical characterisation studies. The purification regime involved using 1 litre ofactinobacterial fermented culture, dispensed into 200 ml centrifuge bottles andcentrifuged at 4000 rpm for 30 min at 4 o C to pellet the mycelium. The brothsupernatant was placed into a separating funnel and extracted twice with an equivalentvolume of EtAc to obtain the non-polar extractable metabolites. The mixture wasallowed to stand for 1 hr until two layers were visible, the top organic layer and anextracted broth supernatant bottom layer. An intermediate layer formed whichconsisted of globules was also apparent. This globule layer was passed through nonabsorbentcotton wool (Smith+Nephew, Aus., Cat. No.131578A) and sodium sulphate_____________________________________________________________________63