The Purification And Identification Of The Antimicrobial Peptide EdeineB Secreted By Brevibacillus Brevis X23

The strains X23isolated from the rhizosphere soil of solanaceae crops in Yongzhou, Hunan Province, and it was identified as Brevibacillus brevis, the strain possesses a stable antibacterial effect and a broad antimicrobial spectrum, which has a great potential for a wide application in biological control. In order to test the antimicrobial active component generated by strain X23, this work has conducted the relevant researches including antibacterial activity test of fermentation broth and culture medium optimization for strainX23, purification and identification of AMPs, and antimicrobial spectrum and minimal inhibitory concentration test for edeine B. The main results are as follows:1The fermentation broth of strain X23has found to possess a strong inhibiting effect for Ralstonia solannacearum, through confrontation cultivation on plates. The NB medium has been improved with single factor analysis method; through the comparison of bacteriostatic ring, the best inhibiting effect was obtained when beef extract is of1g/L, peptone of5g/L and glucose of10g/L2AMPs were separated and purified with inverse high performance liquid chromatography (HPLC). Under the optimized elution conditions, the multi-step separation was conducted for fermentation broth; and two activity peaks were found with the gradient elution set as Omin0%,15min4%,35min4%, and using XAqua C18column. Two active compounds X23-3-1-land X23-3-1-9were finally isolated, with the purity of97.51%and96.67%, respectively.3The structural analysis was conducted for two separated compounds with nuclear magnetic resonance (NMR) spectra and mass spectrometry. Compound X23-3-1-1was preliminarily determined as edeine A through high-resolution mass spectrometry, and further NMR analysis is just in progress. The molecular formula of compound X23-3-1-9was speculated as C34H60Ni2O10through high-resolution mass spectrometry and then, X23-3-1-9was identified as edeine B through NMR analysis.4The antimicrobial spectrum and minimal inhibitory concentration test for edeine B shows that this compound has antimicrobial effect for multi bacterium including Ralstonia solanacearum GMI1000, Escherichia Coli DH5a, Phytophthora parasitica, Staphylococcus aureus, Alternaria alternate and Magnaporthe oryzae; the minimal inhibitory concentration of edeine B was tested as3μg/ml on Ralstonia solanacearum, and6μg/ml on Escherichia Coli and staphylococcus aureus.