Isolation, Purification And Identification Of Antibacterial Components, Not Spectinomycin From Streptomyces Spectabilis

Streptomyces spectabilis LXR1H-8was obtained by genome shuffling combing traditional mutagenesis, the antibacterial ability of its cultural broth was dramatically improved and its antibacterial spectrum was changed by repeatedly confirming fermentation.Methods of resins isolation and ethyl acetate extraction were applied to extract the antibacterial components from cultural broth. The effects of pH value on resins adsorption and extraction were studied, and the kinds of eluents and its concentration were also investigated. The eluents was extracted by third times with4liters of ethyl acetate. Then the organic part was evaporated in vacuo to dryness to obtain2.00g of a brown residue. This residue was redissolved in methanol and applied to a column of MCI gel.The column was eluted with a mixture of methanol-water (from20:80to80:20)to obtain, successively, fraction A, fraction B, fraction C,and fraction D. Fraction A and B had no biological activity, and fraction C and D showed high antibacterial activity. Fraction C, containing the components Ci, C2, C3, C4,was evaporated under reduced pressure to yield860mg of a crude, which was applied to a column of silica gel. Elution was carried out with a mixture of chloroform-methanol (from16:1to10:1).On the basis of TLC and dipped disk assay vs Sarcina lutea appropriate fraction Ci containing antibacterial components was tracked. Fraction Ci was applied to a column of HW-40C gel, Lichrosorb RP-18and purified by HPLC on Sepax Biol C-18(5μm,250×30mm φ)with methanol-water(48:52) as eluent. The solvent was vacuumly condensed to yiled:1.3mg Ⅱa-1and3.3mg Ⅱa-2. Fraction C2was applied to a column of silica gel with petroleum ether-acetone(1:1)as eluent, and purified by HW-40C gel eluted with methanol. On the basis of TLC analysis and bioautography appropriate fraction C2containing antibacterial components was tracked.3.0mg Cv2was obtained under reduced pressure.1D NMR and2D NMR of Ⅱa-2were taken while1D NMR of Cv2was taken, Liquid chromatograph-mass spectrum of both were obtained.On the basis of spectra data analysis, Ⅱa-2was classified as aromatic ester compound with mass weight754, it was presumably close analogues of streptovaricins. mass weight of Cv2was1448.