PURIFICATION AND PARTIAL CHARACTERIZATION OF ANTAGONISTIC SUBSTANCE FROM BACILLUS SUBTILUS B115

Bacillus subtilis B115,an antagonistic bacteria isolatedfrom the mud of aquaculture pond,strongly inhibited the growthof Aeromonas hydrophila which caused the bacterial septicemia disease of freshwater fish. B115 bacteria were cultured in BPYmediumfor 24h at 28 ℃. The supernatant was collected by centrifugation at 6000r/ min for 30min at 4℃,then filtered by a0145 μ mfilter. Crude extract of Antagonistic Substance wasobtained by precipitationof the cell free supernatant of B115 with HCland then extracted by alcohol. Assays for crude extract inhibiting bacteria activity were done on agar plates. The results showedthat crude extract strongly inhibited Aeromonas hydrophila BSK 210 and other pathogens of fish and river crab,and it was thermal2unstable,resistant to trypsin,partial sensitive to proteinase K,sensitive to chloroform and stable in various pH. Its active pHrange was wide,from 4 to more than 9. The activity of Antagonistic Substance would lost when it kept in 4 ℃more than 15d andin 18℃ more than 45d. One antagonistic peak P2 was obtained from the crude extract of B115 by CM iron exchange chro2matography,followed by P260 column chromatography. An antibacterial peptide LP was purified by acetone precipitation and re2versed phase column chromatography. The molecular weight of LP is 80316 D determined by mass spectrometry. 1 mg pure LPcould be obtained from 1L supernatant of B115.