Isolation,Purification And Structure Identification Of Active Metabolites From Streptomyces Fungicidicus

Actinomycetes are a kind of important microbial resources that produce abundant active secondary metabolites,which are widely used in biological control of plant disease.A strain of Streptomyces fungicidicus was obtained by preliminary screening in our laboratory.And the fermentation broth of Streptomyces fungicidicus has a good control effect on phytopathogenic fungi.The research on the antibacterial substances in the fermentation broth has been carried out.Streptomyces fungicidicus is large-scale cultured in liquid fermentation,and subsequently the mycelium and the medium components are removed by centrifugation and filtration,after that we collect the fermentation broth,and the active substance in the fermentation broth is adsorbed by XAD-16 macroporous resin,and then we put the macroporous resin in methanol,extracted with dichloromethane,and the dichloromethane phase was collected.After concentration,3.1 g crude extract was obtained.The crude extract was subjected to primary silica gel column chromatography and 15 different components were obtained,numbered 1~15.Using Alternaria alternata as indicator bacteria,the antifungal activity of these 15 components was determined.Among them,six components showed antifungal activity.The diameter of the inhibition zone was measured by the cross method.Sample No.3 and No.6 showed the highest antifungal activity and the inhibitory zone diameter of them were 37.2 mm and 30.8 mm respectively.Sample No.3 was subjected to secondary silica gel column chromatography,Sephadex LH-20 column chromatography to obtain a compound,A3.Sample No.6 was separated by Preparative high performance liquid chromatography,and a total of 4 monomers were obtained,compounds F1,F2,F3 and F8.The structure of five compounds were elucidated based on extensive NMR and MS.Based on the spectral detection data and literature data,it is determined that among the five compounds,A3,F1,F2 and F3 are known compounds,namely: Dibutyl Phthalate,(Cyclo-(Phe-Pro))?(Cyclo-(Pro-Leu))?(Cyclo-(Pro-Ile)),Compound F8 is a new natural product,which is named 6-(5-hydroxy-6-methylheptyl)-5,6-dihydro-2H-pyran-2-one.The optimum carbon and nitrogen sources in the fermentation medium of Streptomyces fungicidicus were identified by single factor test,and results showed that,the best carbon and nitrogen is starch and peanut meal respectively.The Plackett-Burman test was used to screen out the components in the medium which had a great influence on the active substances,namely starch and ammonium sulfate.At the same time,using the response surface method,the Box-Behnken experiment was designed for these three factors and analyzed by multiple regression analysis.The optimized concentration of the three components was 27.15 g/L of starch,27.88 g/L of peanut cake powder and 0.72 g/L of ammonium sulfate.The average diameter of the inhibition zone of the fermentation broth was 33.05 mm,increased by 31.2% than before.To evaluate the possible category of the secondary metabolites,amplification of the functional genes of type I polyketide synthase(PKS-I),type II polyketide synthase(PKS-II)and non-ribosomal peptide synthetase(NRPS)of Streptomyces fungicidicus were amplified by degenerate primers,results showed that the strain contains PKS-? and NRPS genes respectively,and it's possible to produce polyketone and non ribosomal peptide substances.