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摘要巨大芽胞杆菌（Bacillus megaterium）B196菌株产生的拮抗物质对水稻纹枯病菌（Rhizoctonia solani）的生长具有较强的抑制作用，明确该菌产生拮抗物质的种类是进一步研究该菌的抑菌机制及其应用的基础。本文采用盐酸沉淀B196菌株的去菌体培养液，再用甲醇抽提获得拮抗物质的粗提物。利用反相HPLC系统，将粗提物过C18柱，收集有抑制水稻纹枯病菌生长作用的活性化合物。运用质谱测得其分子量分别为1 042.592 7 u，再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片，结果表明其一级结构为ProAsnSerβAAAsnTyrAsnGln (βAA为14个碳原子的氨基脂肪酸)。综合以上信息将该化合物鉴定为Iturin A2。
关键词巨大芽胞杆菌；iturin A2；分离纯化
中图分类号:S482.292文献标识码：ADOI：10.3969/j.issn.05291542.2014.02.004Isolation and purification of an antimicrobial substance produced
by Bacillus megaterium strain B196 Liao Ting,Qin Jian,Yuan Gaoqing,Li Qiqin,Lin Wei,Peng Haowen(College of Agriculture,Guangxi University, Nanning530005, China)AbstractAn antimicrobial substance produced by Bacillus megaterium strain B196 can strongly inhibit the growth of Rhizoctonia solani．Identification of the antimicrobial substance is very important for further research on the inhibition mechanism and its application．Crude antimicrobial substances were extracted with methanol from the precipitate which was obtained by adding HCl to the cellfree culture broth and then stored at 4 ℃ overnight．The crude extract was further purified by reversedphase HPLC with a C18 column（PRCODS，20×250 nm）to separate the substances．An antifungal compound with strong inhibitory activity against R.solani was purified．The molecular weight of the compound was 1 042.592 7 u by Matrixassisted laser desorption ionization timeofflight mass spectrometer(MALDITOFMS)．TESI collision induced dissociation mass spectrometry analysis demonstrated that the compound had a primary structure of ProAsnSerβAAAsnTyrAsnGln. Therefore, the compound was identified as Iturin A2.
Key words Bacillus megaterium；iturin A2 ；isolation and purification 巨大芽胞杆菌（Bacillus megaterium de Bary）是一类好氧、革兰氏阳性、可生长在土壤和植物体内的芽胞杆菌细菌，它可以被用来吸附金属Au3 ［1］、净化水体［2］、降解农药［3］、解磷解钾［4］等，被广泛地应用在环境保护方面。此外，巨大芽胞杆菌还可以产生抑制植物病原菌的抗菌物质［5］，因而在植物病害防治方面也受到人们的关注。目前已有报道分离出一些对植物病原菌有较强拮抗作用的菌株［6］，一些菌株对一些植物病害有较好的防治作用［7］，巨大芽胞杆菌可产生的抑菌物质有蛋白类［5］、挥发性物质［8］、细菌素［9］和脂肽类物质等［10］。
巨大芽胞杆菌B196菌株是广西大学农学院植物病理研究室从水稻上分离出来的拮抗细菌，在离体、盆栽和大田试验中，该菌株对水稻纹枯病均表现出较好的防效［11］。前期工作发现，该菌株的发酵滤液对玉米小斑病菌［Bipolaris maydis （Nisik
关键词巨大芽胞杆菌；iturin A2；分离纯化
中图分类号:S482.292文献标识码：ADOI：10.3969/j.issn.05291542.2014.02.004Isolation and purification of an antimicrobial substance produced
by Bacillus megaterium strain B196 Liao Ting,Qin Jian,Yuan Gaoqing,Li Qiqin,Lin Wei,Peng Haowen(College of Agriculture,Guangxi University, Nanning530005, China)AbstractAn antimicrobial substance produced by Bacillus megaterium strain B196 can strongly inhibit the growth of Rhizoctonia solani．Identification of the antimicrobial substance is very important for further research on the inhibition mechanism and its application．Crude antimicrobial substances were extracted with methanol from the precipitate which was obtained by adding HCl to the cellfree culture broth and then stored at 4 ℃ overnight．The crude extract was further purified by reversedphase HPLC with a C18 column（PRCODS，20×250 nm）to separate the substances．An antifungal compound with strong inhibitory activity against R.solani was purified．The molecular weight of the compound was 1 042.592 7 u by Matrixassisted laser desorption ionization timeofflight mass spectrometer(MALDITOFMS)．TESI collision induced dissociation mass spectrometry analysis demonstrated that the compound had a primary structure of ProAsnSerβAAAsnTyrAsnGln. Therefore, the compound was identified as Iturin A2.
Key words Bacillus megaterium；iturin A2 ；isolation and purification 巨大芽胞杆菌（Bacillus megaterium de Bary）是一类好氧、革兰氏阳性、可生长在土壤和植物体内的芽胞杆菌细菌，它可以被用来吸附金属Au3 ［1］、净化水体［2］、降解农药［3］、解磷解钾［4］等，被广泛地应用在环境保护方面。此外，巨大芽胞杆菌还可以产生抑制植物病原菌的抗菌物质［5］，因而在植物病害防治方面也受到人们的关注。目前已有报道分离出一些对植物病原菌有较强拮抗作用的菌株［6］，一些菌株对一些植物病害有较好的防治作用［7］，巨大芽胞杆菌可产生的抑菌物质有蛋白类［5］、挥发性物质［8］、细菌素［9］和脂肽类物质等［10］。
巨大芽胞杆菌B196菌株是广西大学农学院植物病理研究室从水稻上分离出来的拮抗细菌，在离体、盆栽和大田试验中，该菌株对水稻纹枯病均表现出较好的防效［11］。前期工作发现，该菌株的发酵滤液对玉米小斑病菌［Bipolaris maydis （Nisik