Purification Methods Of Active Material Zh11of Deep-sea Virgibacillus A493

Plant disease is the main harm to the agricultural development of the world, the loss caused by plant disease each year is severe. At present, in the circumstance of chemical pesticide abuse and the worsening of drug resistance, looking for biological pesticide which is broad-spectrum, high-efficiency and low-toxic corresponds with the requirements of eco-agriculture and sustainable agriculture. Researchers hope to discover bioactive compounds from marine microorganisms to control plant pathogen aiming at reducing or replacing chemical pesticides.The research object was Virgibacillus dokdonensis named A493, which had significant inhibition effect on Xanthomonas oryzae. Based on the preliminary separation and purification technologies of antibacterial material ZH11, the purpose of this study was to search new purification methods, analysis its characteristic and preliminarily discuss the antibacterial mechanism.For this study, the original purification methods of ZH11included rough extract of fermentation broth, separation by Cellulose CM-52and DEAE-52ion exchange chromatography and two-step TLC. Because of the low purification efficiency, new purification methods were searched. ZH11crude extract was treated with activated carbon and it had no purification effect, so discarded this method. The crude extract was high-throughput dealed with SP Sepharose F.F. column chromatography and overall purification process was greatly faster than before. Because molecular weight of ZH11was less than700Da, Sephadex G-10gel chromatography was introduced in. And impurity which had smaller molecular weight than ZH11was removed. The next step was CM-52column chromatography. The whole elution process was4h compared with11h previously. The efficiency was greatly improved and much colored impurity was removed, so this method should be maintained. Then the active sample was treated by DEAE-52chromatography, the elution time was2/5of the existing method. And the purification effect was good, so this method should be maintained. The fifth step was separated by TLC. The first TLC was discarded because the difficulty of active material recycling was large of n-BuOH/AcOH/H2O solvent system. And the second TLC was used, the sample was separated to eight components including ZH11and another active component.Comparison was done with the active substances and three known AGs. The results showed that the differences between them on resistance against several common strains and TLC coloration were obvious.Based on purification of the sample, antibacterial mechanism against Xanthomonas oryzae was preliminarily explored. The result of HPLC indicated that inhibition rate of the active ingredients against GlmU enzyme was approximately17.3%, it showed that the active ingredients had some inhibition on GlmU enzyme.