【作者】 杨少波；

【导师】 刘训理；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2009， 硕士

【摘要】 多粘类芽孢杆菌(Paenibacillus polymyxa) Cp-S316 (GenBank注册号为AY292989)由本实验室从泰山林地土壤中分离获得,该菌株可产生对多种动、植物病原细菌和病原真菌具有较强拮抗作用的环肽类抗细菌活性物质和抗真菌活性物质。并且,在不同的发酵培养基中,抗细菌活性物质和抗真菌活性物质的产量有很大差异。本研究以Cp-S316菌株为出发菌株,通过紫外诱变对抗细菌活性物质高产菌株进行了选育,分离纯化了Cp-S316菌株产生的抗细菌活性物质,研究了活性物质对大肠杆菌的抑制作用。主要研究结果如下。1、运用紫外线诱变以及对自身产生的抗细菌活性物质的抗性筛选法,通过预筛、初筛和复筛,获得多粘类芽孢杆菌Cp-S316突变株A17、E13、E18、E33和F28,其发酵效价分别为出发菌株的191%、167%、163%、160%和162%。A17、E18菌株的高产性能遗传特性稳定。2、Cp-S316菌株发酵液经DK110大孔弱酸性阳离子交换树脂吸附、0.5mol/L的硫酸动态解析,抗细菌活性物质解吸液经旋转蒸发、冷冻干燥以后,进一步经过甲醇抽提,Sephadex G25分子筛层析,得到6个洗脱峰,收集抗细菌活性最高的部分,经CM Sepharose FF离子交换柱层析,0-0.3mol/L的NaCl梯度洗脱得到3个抗细菌活性物质洗脱峰,旋转蒸发浓缩后,进行Sephadex G25分子筛层析脱盐处理,得到抗细菌活性物质的中间纯化物。Cp-S316抗细菌活性物质A组分的最佳制备色谱条件为：流动相为乙腈：水(0.1%TFA)= 23:77 (v/v); Venusil XBP C18柱(250mm×20mm,5μm),流速5 mL/min,柱温30℃,进样量5mL,检测波长为210nm。在此色谱条件下,分离得到保留时间(Rt)为36min(A-1)和51min(A-2)的两个抗细菌活性物质组分；活性物质E组分的最佳制备色谱条件为：流动相为乙腈：水(0.1%TFA)=17:83 (v/v); Venusil XBP C18柱(250mm×20mm,5μm),流速5 mL/min,柱温30℃,进样量5mL,检测波长为210nm。在此色谱条件下,分离得到保留时间(Rt)为17min(E-1)和20min (E-2)的两个抗细菌活性物质组分。E-2、A-2两组分经waters超高效液相色谱工作站检测均显示为单峰,峰形呈正态分布,无杂质峰,初步判断制备成功。3、经试管肉汤二倍稀释法测定,Cp-S316抗细菌活性物对大肠杆菌K12的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为12.5μg/mL和25μg/mL。通过测定大肠杆菌K12菌悬液的OD260的变化,研究了多粘类芽孢杆菌Cp-S316抗细菌活性物质对大肠杆菌K12细胞膜完整性的影响,结果表明Cp-S316抗细菌活性物质可损伤大肠杆菌K12的细胞膜,从而引起胞内RNA、DNA等大分子物质的泄漏。更多还原

【Abstract】 A new antagonistic bacterium Paenibacillus polymyxa Cp-S316 isolated from soil of Mountain Tai and further identified with registration number AY292989 on Genbank. It showed broad inhibitory spectrum and great inhibition against animal and plant pathogenic bacteria and fungi. The shake-flask fermentation properties of Cp-S316 were excellent, and It can produced antibacterial and antifungal active substances, but the production of both active substances fluctuated greatly with different medium. Paenibacillus polymyxa Cp-S316 could produce polymyxin E and polymyxin E1 and at least three novel antibiotics against pathogenic bacteria. Meanwhile it could produce at lease one kind of antifungal active substances. As an original strain, Paenibacillus polymyxa Cp-S316 was treated by ultraviolet mutagenesis. The mutated strains were obtained, and several antibacterial active substances were isolated, purified, and identified, and the effect of antibacterial active substances from Paenibacillus polymyxa Cp-S316 on the integrity of Escherichia coli K12 membrane was studied by determining the variation of OD26o values of bacterial suspensions. The main results were as follows:1 Paenibacillus polymyxa Cp-S316 was treated by ultraviolet mutagenesis. The mutated strains of A17, E13, E18, E33 and F28 were obtained by prescreening of antibacterial active substances, screening and re-screening of shake flask culture. Compared with Cp-S316, the yield of antibacterial substances obtained from mutant A17 was increased by 91%. The subculture experiments indicated that the hereditary characteristic of high productivity of Paenibacillus polymyxa A17 and E18 were stable.2 Isolation and purification of antimicrobial active substances were operated. antimicrobial active substances were absorpted with cation exchange resin and desorbed with the 0.5 mol/L H2SO4 in dynamic. The desorption liquid was concentrated by vaporization and vacuum freezy-desiccation. antimicrobial active substances were further isolated gradually by methanol extraction, Sephadex G25 column chromatography, CM-Sepharose Fast Flow ion exchange column chromatography with 0-0.3mol/L NaCl as grads desorption solution. The antimicrobial active substances contained less impurity were got by Sephadex G25 column chromatography.Finally antimicrobial active substances were refined by reversed-phase high performance liquid chromatography (RP-HPLC). The method of RP-HPLC was set up for preparation of the antimicrobial active substances A. The conditions of RP-HPLC were suggested as Venusil XBP C18 column(250mm×20.0mm,5μm), detection wavelength 210nm, the mobile phase gradient elution of acetonitrile-buffer(23:77, v/v) at 30℃, with the buffer containing 0.1% TFA, flow rate 5.0mL/min, the volume of injection 5.0mL. Two antimicrobial active substances component was separated under the condition. The conditions of RP-HPLC for preparation of the antimicrobial active substances E were suggested as Venusil XBP C18 column (250 mm×20.0mm,5μm), detection wavelength 210nm, the mobile phase gradient elution of acetonitrile-buffer(17:83, v/v) at 30℃, with the buffer containing 0.1% TFA, flow rate 5.0mL/min, the volume of injection 5.0mL. Two antimicrobial active substances components were separated under the condition. At last antimicrobial active substances components E-2 and A-2 were showed single peak in UPLC chromatography.3 Inhibition of antibacterial active substances from Paenibacillus polymixa Cp-S316 on Escherichia coli were explored. The effect of antibacterial active substances from Paenibacillus polymyxa Cp-S316 on the integrity of Escherichia coli K12 membrane was studied by determining the variation of OD260 values of bacterial suspensions. The results showed that the antibacterial substances could damage the Escherichia coli K12 membrane which leaded to the leakage of RNA, DNA and other cytoplasmic macromolecules.更多还原