Isolation And Structure Elucidation Of A Novel Biopesticide--Mildiomycin

Mildiomycin(MIL) is a new kind of nucleoside agro-antibiotic which showed strong activity against powdery mildews and is remarkably low toxicity in mankind and animals, so it was a high promising product. The objective of the thesis is to establish a comprehensive process to extract MIL from fermentation broth by ion exchange and prepare the pure MIL. The chemical structure of MIL will be identified also.At fiest, the analytic methods for MIL were established. Both Sakagnchi-Reaction method and HPLC method for quantitative analysis of MIL were respectively established. Sakagnchi-Reaction method was fast and simple, but was easily interfered by metal ions and arginine. It was only suitable for the analysis of relative pure MIL. HPLC method was fast, accurate and seldom interfered, which was very suitable for quantitative analysis of MIL in fermentation broth and during extracting process. The chromatographic conditions were as followings. Column: Agilent Zorbax SB-C18(4.6x250mm, Sum); mobile phase: 5%methanol-0.005M citrate buffer(pH5.8); detect wave length: 254nm; flow rate: 0.7mL/min.; injection volume: 15 uL; column temperature: room temperature.A separation and purification process of MIL from fermentation broth was developed. The process included four steps: l)Solid-liquid separation: the fermentation broth was regulated by adding oxalic acid to pH3.0. After being kept for 30min, it was centriruged or filtrated. The supernatant containing MIL was obtained for further separation. In this step, the recovery of MIL was almost 100%. 2) Ion exchange separation. After the pH value of the supernatant was adjusted to pH 7.5, it passed through an ion-exchange column filled with macroreticular cation ion-exchange resin, D115, at a flow rate of IBV/h. The MIL was adsorbed on the resin. Then the column was eluted with 2% ammonia at a flow rate of 0.5BV/h to desorb MIL. Experimental data showed that the average adsorption capacity of D115 resin for MIL was 40~50mg/g dry resin and the average recovery in ion-exchange step was about 80%. 3) Decolor. The elution from ion-exchange column was adjusted to pH 6.5; then passed through a column containing D750 resin at a flow rate of 0.5BV/h for decolorization. The experimental results indicated that the average recovery of MIL was about 90% and the treatment capacity of D750 was 150mL elute/ lOmL wet resin. 4) Precipitation and drying. After decolorization, the elution was concentrated and precipitated with ethanol to yield crude mildiomycin. The crude MIL was freezing dried. The recovery of MIL in the whole procedure was above 70%,ABSTRACTand the purity of the crude product was about 80%.The crude product could be further purified by ion exchange chromatograph method. After adsorbed on HD-2 resin, the MIL was eluted with 5% methanol-0.3M lithium borate buffer(pH8.8) and the effluent was collected. The resultant active fractions were decolorized, desalted and precipitated with ethanol to yield MIL product with purity above 95%. Mass spectrums, IR spectnuns, H-NMR spectrums and C-NMR spectrums were applied to identify the product. The results showed that the product was the same as standard MIL sample.