Isolation And Purification & Preliminary Identification Of Antibiotic Substance Produced By Endophytic Bacteria A11

An endophytic bacterium A11 was isolated from the foliage of Aloe Chinenesis(Haw) Baker,whose secondary metabolite has been found to have a high inhibitory on gram-postive (bacteria) such as staphylococcus aureus and bacillus subtilis.In order to obtain its molecular formula information,the fundamental work is to isolate simple active component from the fermentation broth..In the paper,it was staphylococcus aureus as indicator that the active substance was isolated and purified with different rourte.Study one:fementation broth was foretreated by breaking intopieces of supersonic wave,105℃steriliration,10000r/min centrifugalization for five minutes,then supermatant solution was remained and dyliazed(trapping molecular weight 3500), dyliasate was collected and condensed to some volume by evaporating under low temperature and reduced pressure,then extracted with n-butanol(1:1),organic phase of extraction was evaporated to dryness under low temperature and reduced pressure and dissolved in the methanol to get crude sample.It was isolated and purified by the silica gel column chromatagraphy and mass spectrographic analysis.resulted as followed:By the silica gel column chromatagraphy,showed the active substance eluted with MeOH: EtOAt 4:6 to afford A;Eluted with MeOH:EtOAt 5:5 to afford B.By mass spectrographic analysis,A and B both include two identical components,molecular weight was 475.2,499.5.The active substance could not be isolated using the silica gel column.Study two:fermentation broth was fore treated:6M/L HC1 was used for changing PH to 3,standing for hours,then centrifuge for 5 minutes at 10000r/min,hybridprotein was removed, the PH of supemalant liquid was changed from 3 to 7 with NaOH.Dehydrated alcohol of 3 times volume was used for removing hybridprotein and polysaccharide.then fiiltered in the vacuum condition,the filtration vacuum liquid was evaporated to dryness under the low temperarure and reduced pressure.The light yellow powder was dissolved in the minimum MeOH,and the crude sample was obtained.Active substance was isolated and purified by the silica gel column chromatagraphy and sephadex-LH20 molecular sieve,crystal and so on,By the mass spectrographic and H-NMR analysis,resulted as followed:By the silica gel column chromatagraphy,eluted with MeOH:EtOAt 2:8 to afford A-1;with MeOH:EtOAt 3:7 to give A-2;A-1 was isolated and purified by silica gel column again,the crystal was educed under condensing and standing.A-2 was isolated and purified by silica gel column again and again and sephadex-LH20 molecular sieve,By the mass spectrographic analysis,A-1 molecular weight was460.17;A-2 molecular weight was 460.2.By the H-NMR analysis,presumed that A-2 possiblly exclude bezene ring and aldehyde group and include amide linkage and alkyl group and so on..