Separation And Purification Of Bioactive Compounds From Endophytic Bacteria EBS05 And Its Antibiotic Activity On Tobacco Mosaic Virus

Abstract: EBS05, an endophytic bacteria strain from Cinamonum camphora periderm organization, had strongly antagonist activity to many kinds of the plant pathogenic fungus and the tobacco mosaic virus, and had many excellent properties as biological controls factor, such as tolerance of acid and alkali, salt, heat, etc. Taxonomy of EBS05 was studied. Separation and purification of metabolic bioactive compounds produced by EBS05 and its antibiotic activity on tabacco mosaic virus were conducted too. Results were as follows:1. EBS05 was identified as Bacillus subtilis by means of 16S rDNA and 16S-23S rDNA intergenic spacer regions sequence analysis based on the morphological, physiological and biochemical characteristics.2. Properties of antimicrobial compounds produced by EBS05 were assayed. The active compound had the maximum absorbance peak atλ213.5 nm. The antimicrobial activity was stable in solution with pH value from 5 to 8, and decreased significantly in solution with pH value less than 2 or more than 10. The antimicrobial compound had thermodynamics stability. Its activity changed a little after treated at 60℃-80℃for two hours, and compared with 65% original activity after treated at 1×105 Pa for 30 minutes. The active substance had high resistance to ultraviolet radiation and protease K. Antimicrobial compound was soluble in alcohol solution, which was easily dissolved in methanol and ethanol, but not dissolved in ethyl acetate, acetonitrile and petroleum,et al.3. Separation and purification process of active compound was investigated. Crude extract obtained after fermentation broth was treated by hydrochloric acid with pH2.0 and extracted three times by the methanol. It was purified by Sephadex LH-20 column chromatography and silica gel column with mixture of chloroformrmethanol: water(65:25:4,V/V/V) and methanol as the eluent respectively. It was purified by reverse phase C18 HPLC, and pure compound, one of active peaks in HPLC pattern, was obtained from collection.4. Molecular structure of bioactive compound was assayed by the amino acid component and time-of-flight mass spectrometry and electrospray ionization mass spectrometry/mass spectrometry (TOF-MS and ESI-MS/MS) analysis. Bioactive compound is a series of homologous lipopeptides, a surfactin with different molecular weights 1035.65D,1021.64D,1007.63D and 993.65D,respectively. The peptide chain of lipopeptides is [Glu·Leu·Leu·Val·Asp·Leu·Leu], and the molecular structural formula is5.Anti-TMV activities of active compounds from endophytic bacteria EBS05 were determined using TMV/Tobacco as test system. Active compound strongly inhibited TMV infection in vitro, and also effectively inhibited TMV replication in vivo. The bioactive substance can restrain polymerization of TMV coat protein and interfered from antigen site of TMV too. There is no influence on the TMV particle structure.