06 16 2006 09 55 21 198

Isolation of new actinomycetes strains and taxonomic characterization

■ Various selective and non-selevtive methods are applied to isolate actinomycetes from soils and sediments from various habitats. Marinę sources are of special interest, such as sediments from deep sea trenches from the Pacific and Atlantic Ocean. Besides the well known families Streptomycetaceae and Micromonosporaceae, members of rare actinomycete genera are isolated for screening purposes. The new isolates are determined by morphological and chemotaxonomical methods and 16S rDNA sequencing to the genus level. The actinomycete collection of the group contains 1100 terrestrial and 800 marinę actinomycete strains.

Single colonies

HPLC-DAD screening for novel secondary metabolites

• Extracts from new isolated actinomycete strains are cultivated in the shake fiask scalę and extracts are prepared from biomasses and culture filtrates to determine the Chemical diversity by means of our HPLC-UV-Vis-Database. The equipment of the group consists of three fully automated HP 1090 liquid chromatographs with diodę array monitoring and rapid ChemStations.

Scale-up fermentation and metabolite isolation

• The group is equipped with eightfermenters in the scalę 1-8 litres, eleven fermenters in the scalę 10-20 litres, one airlift fermenter with 3.5 litre working volume, and one dialysis fermenter. Ali reactors can be used in the batch or fed-batch modę. One 100 litre stirred tank fermenter (P-100, BioEngineering) and one 200 litre intensor fermenter (b200, Giovanola) are availabel for the production scalę.

Isolation

• Metabolite isolation from the biomass is done by solvent extraction and from the culture filtrate by polystyrene resin chromatography (Amberlite XAD, Diaion Sepabeads). A specific purification protocol is developed in case of each metabolite dependent on its hydrophilic or lipophilic properties. Polar metabolites are purified by subsequent ion-exchange chromatographic steps (anion/cation exchange) followed by aqueous size exclusion chromatography. Lipophilic metabolites are purified by adsorption chromatography (silica gel, diol modified silica gel), size exclusion chromatography (Sephadex, Toyopearl) and preparative reversed-phase HPLC. The isolation steps are evaluated by HPLC-DAD analysis and biological assays.