PRODUCTION OF NODULATION FACTORS BY RHIZOBIUM-MELILOTI - FERMENTATION, PURIFICATION AND CHARACTERIZATION OF GLYCOLIPIDS

Lipooligosaccharides, synthesized by soil bacteria of the genera Rhizo bium, are known to have multifunctional effects on a wide variety of p lants as signal substances in symbiosis initiation, cell response elic itation and growth regulation. These so called nodulation (Nod-) facto rs represent interesting biotechnological products with respect to fun damental studies of symbiotic interactions as well as for potential ap plications. Therefore, a batch fermentation process on a scale of 30 I has the Rhizobium meliloti strain R. m. 1021 (pEK327) strongly overex pressing the genes for the been developed by means of synthesis of Nod factors. Induction by the flavone luteolin led to growth associated p roduction of the lipooligosaccharides. Ultrafiltration was used for se parating the biomass from the filtrate containing the extracellular No d factors. Simultaneously, ultrafiltration reduced the amount of lipop hilic substances, which would otherwise interfere with processes downs tream. The second separation step consisted in adsorption on XAD-2, a nonspecific hydrophobic adsorptive resin. Adsorption of Nod factors wa s carried out by batch operation of a stirred tank. Desorption was per formed by elution with methanol in a fixed bed column. A semi-preparat ive reversed phase HPLC (Polygoprep 100-30 C18) was chosen as the fina l purification step. The Nod factors were obtained after evaporation a nd lyophilization. Thus, about 600 mg of Nod factors were produced fro m 20 I of fermentation broth. The Nod factors produced by Rhizobium me liloti R. m. 1021 (pEK327) were identified by liquid secondary ion mas s spectrometry and by reversed-phase HPLC as fluorescent derivatives o f 2-aminobenzamide. The biological activity of the products was demons trated by means of the root hair deformation (HAD-) assay.