Isolation, Purification And Identification Of Secondary Metabolites Produced By Soil Actinomycete â…¢-172

A batch of mutant strains producing ascomycin provided by Shanghai Pesticide Research South Center, were fermented and fostered separately, then a series of procedures were dealt with them. During the process, a lot of biological activity tests were done to them, their antimicrobial activities and the growth condition of strain on the slant were confirmed as two screening indicators, finally the soil actinomycete Ⅲ-172 was obtained, which can produce metabolites with strong inhibitory activity against plant pathogens. In this paper, the antimicrobial ingredients of soil actinomycete Ⅲ-172 were isolated and purified.Before fermentation, some culture conditions were simply optimized such as the fermentation time and organic solvent extraction. By comparing the inhibition activity, the fermentation time was determined as 7 days, and n-butyl alcohol was chosen as the best organic solvent. Through fermentation with the optimized conditions, highspeed centrifuge, solvent extract, silica gel chromatography, thin layer chromatography, and high performance liquid chromatography and other ways were used, combined with the tracking test of the biologic activity of plant pathogen, ascomycin chosen as a comparison standard to isolate and extract the active substances from its metabolites. When the ratio of expansion agent was CH2Cl2:CH3OH=10:1, and the Rf value was about 0.43,one active strip signed strip 5 was detected on the TLC plate. High performance liquid chromatography(HPLC) analysis was carried out on the strip 5. Finally one seperate active peak came when the mobile phase was 10% methanol, and the retention time was between 24-26 mins. Then preparative high performance liquid chromatography was done, at last one antimicrobial compound was isolated from the culture extract of strain Ⅲ-172, named as Ⅲ-172-Ⅰ-a. By the purity test of high performance liquid chromatography, the purity of the compound was 92.9%.Followed by its mass and NMR analysis. Then the spectra data of UV, ESI-MS, 1H-NMR, 13C-NMR etc. and other methods were used to identify the structure of the compound, and analyze its molecular formula. Through these spectra, its molecular weight was 138.1, and its molecular formula was C8H10O2. By looking for some relavent information, some properities of the compound were found out, such as its melting point was 85℃, and its boiling point was 310℃, it can dissolve in water, ethanol, ethyl ether, acetone, etc. And biological activitity test was used to determine its minimal inhibitory concentration(MIC) against plant pathogens, and finally its MIC was determinded as 31.25 ppm.Extracted compound Ⅲ-172-Ⅰ-a had strong inhibitory activity against the common plant pathogenic fungi Po. The MIC was tested against pathogenic fungi, which made it more economical to be applied to agricultural production.