Purification And Structure Identification Of Water-Soluble Bioactive Substance From Fermentation Broth By Streptomyces Lavendulae X33

Streptomyces lavendulae X33,isolated from the rhizospheric soil of citrus by our laboratory,synthesizes a secondary metabolite with a strong inhibitory activity against Penicillium digitatum,Penicillium italicum and many other pathogenic fungus.The previous researches indicated that the component was a small nontoxic molecule compound with strong polarity and easy to dissolve in water,so it plays a vital role in the field of citrus preservation.On the basis of establishing the detection method of the component,the purification and structure identification of the component was researched systematically.Simultaneously,the whole genome sequencing results of Streptomyces lavendulae X33 were systematically analyzed via bioinformatics.The details of this paper's result were described as follows:(1)The detection method of the component was established.The active substance detection was through thin layer chromatography and the composition of developing solvent been ethanol-isopropanol-water-stronger ammonia water(3:3:1:3,V/V).The HPLC used the chromatographic column(XBridge(?)BEH Amide 5?m,4.6*250mm,Waters).The detection conditions:The mobile phase was acetonitrile aqueous solution(65%),the flow rate was 1 m L/min,the sample amount was 15?L,the temperature was 30?,and the detection wavelength was 201 nm.The retention time of active substances was approximately 15-16 min.And the trace of inhibitory activity was displayed by cup-plate method and bioautography.(2)The separation and purification process of fermentation product was discussed.This paper covered five column chromatography methods embracing D151 ion exchange column chromatography,D101 macroporous adsorption column chromatography,silica gel column chromatography,ODS C18 column chromatography and sephadex LH-20 column chromatography.The separation extraction process is determined as:the concentration of fermentation broth,silica gel column chromatography and 0.01M HCl solution elution system,ODS C18 column chromatography and water elution system.(3)The active material was predicted as ? -three polylysine,based on the nuclear magnetic spectrum analysis(¹³C spectrum,¹H spectrum,DEPT-90 spectrum,DEPT-135spectrum,¹H-¹H COSY spectrum,HSQC spectrum and HMBC spectrum),acid degradation experiment,thin layer chromatography detection and specific rotation.The stereostructure needs further analysis.(4)The genetic prediction analysis,GO analysis,KEGG analysis and secondary metabolite synthesis gene cluster analysis were used to analyze the whole genome sequencing results of Streptomyces lavendulae X33.The genome with a 72.32%GC content contained 7 871 365 nucleotides,comprised 7 216 coding sequences involved in168 metabolic pathways.Among of them,there were 860 coding sequences belonging to26 secondary metabolites synthesize gene clusters,and one,named PEG 6966,was highly similar to polylysine synthase.(5)The bacteriostatic test of the active substance revealed that it could inhibit mycelial growth and spore formation and germination of Penicillium digitatum.When the sample concentration was 0.5 mg/m L,the growth inhibition rate of mycelia was 100%,and the inhibition rate of spore germination was 98%.Furthermore,the active substance could cause the abnormal development of mycelia and sporophore,even influence the morphology of the conidia significantly.