Isolation And Purification Of Insecticidal Proteins Produced By Xenorhabdus Nematophila Var.Pekingensis

Xenorhabdus nematophila var. pekingensis strain CB6 is a symbiotic bacterium that was firstly isolated from the gut of insect parasitic nematode Steinernema carpocaposae collected from soil of suburb of Beijing, China. It has been identified and nominated. The previous researches showed that this strain had a high insecticidal activity. To explore this indigenous bacterial resource, in this paper, the insecticidal activity of X. nematophila var. pekingensis was compared with that of other 9 entomopathogenic nematode symbiotic bacteria. The extracellular and intracellular products of 10 strains were isolated, the soluble proteins of each products prepared, and their activity bioassayed. It was indicated that X. nematophila var. pekingensis could produce proteins with a comparatively high insecticidal activity. Then the insecticidal proteins of both extracellular and intracellular sections of this strain were further purified, some technical parameters for purification of this active protein were established, and some of its characteristics were preliminarily analyzed. The results were as the followings:1. The fermentation cultures of 10 strains had oral toxicities against neonatal larvae of Spodoptera exigua and Helicoverpa armigera. The active substances were produced from both extracellular and intracellular sections. The insecticidal activities of different strains and different parts of a fermentation culture from one strain were divergent. And, in general, effect of intracellular products was stronger than that of extracellular ones. The bioassay results showed that the main insecticidal substances of these 10 testing strains were proteins.2. A technical protocol was established to efficiently purify the insecticidal proteins of stain X. nematophila var. pekingensis. Some parameters included:Salting-Out: the optimized saturation of aminium sulphate is 20-50%,Ion Exchange Chromatography: with DEAE Sepharose FF column, the low salt for eluting is25mM Tris-HC1 (pH 7.4), and the high salt is 1mol/L NaCl. The rinsing volume for high salt is atthe range of 15-30%,Hydrophobic Chromatography: with Butyl FF column, the high salt for eluting is 3 mol/L NaCl+25mM Tris-HCl (pH 7.4), and the low salt is 25mM Tris-HCl (pH 7.4), andGel Filtration: with Sephacryl S-200 16/10 column, the eluting buffer is 25mM Tris-HCl (pH7.4).With the protocol above, the concentration of target protein purified was 4.134μg/mL, whosegrowth inhibitory percentage against H. armigera larvae was 97.9%.3. Native-PAGE result revealed that the molecular weight of target proteins from both extracellular and intracellular sections of stain X. nematophila var. pekingensis were higher than 669kD, and they had the same migration location. With SDS-PAGE, only a 220kD band was presented for each of these two target proteins. This indicated the possibility that the target protein from extracellular section was the same as that from intracellular one.4. The target protein was fluctuated on the wavelength between 250-290nm, which meant the complexity of this protein. Staining experiments showed that, this target protein was neither lipoprotein nor glycoprotein.In conclusion, it was shown that the active insecticidal substance of fermentation culture of stain X.