拮抗菌Pseudomonas marginalis CJT23活性物质的分离纯化

Isolation and Purification of the Active Substances from Antagonistic Pseudomonas marginalis CJT23

拮抗菌用于防治病害的本质是其中的活性物质对病原菌具有抑制作用,因此对拮抗菌Pseudomonas marginalis CJT23活性物质进行初步探讨。通过对P.marginalis CJT23活性物质种类的研究,初步判断其活性成分为抗菌蛋白,不同发酵时间和离心速度的离心试验,均表明活性物质属胞内产物;Tris-HCl缓冲为最适缓冲液;缓冲液中不同Na^(+)浓度对活性物质的溶解性无显著性影响(P>0.05);当缓冲液pH为4,6,7时,其细胞破碎液与菌体重悬液有显著性差异(P<0.05);用不同的洗液清洗菌体,结果表明活性物质并不在细胞膜表面;用体积分数为20%的乙醇、丙酮、乙酸乙酯、乙醚等有机溶剂浸泡有活性的菌体,结果显示上述有机溶剂不能提取活性物质,且使活性物质失活;用体积分数为5%的DMSO缓冲液对菌体进行细胞破碎,其上清液有抑菌活性;用超滤管对含有活性物质的上清液进行浓缩,结果表明,其分子质量大于1.0×10^(5) g/mol,且在4℃条件下比在其他温度下储藏抑菌活性强;对含有活性物质的上清液采用硫酸铵分级沉淀法分离,结果显示,当硫酸铵初始饱和度为60%、终饱和度为80%时,其活性物质的抑菌活性最强。该研究可为活性物质分离纯化、拮抗机理研究和开发利用奠定基础。

The essence of antagonistic bacteria used in disease prevention and control is the inhibitory effect of active substances on pathogenic bacteria.Therefore,this study conducted a preliminary discussion on the active substances of antagonistic bacteria Pseudomonas marginalis CJT23.Based on the research on the types of active substances of P.marginalis CJT23,the active ingredients were preliminarily judged to be antibacterial proteins,and centrifugation at different fermentation times and centrifugation speeds all showed that the active substances were intracellular products.Tris-HCl buffer was the optimal buffer solution.Different Na^(+) concentrations in the buffer had no significant effect on the solubility of the active substances(P>0.05).When the buffer pH was 4,6 and 7,there was a significant difference between the cell crushing fluid and the bacterial body weight suspension(P<0.05).Washing the thalli with different lotions showed that the active substances were not on the surface of cell membrane.Soaking the active substance in 20%ethanol,acetone,ethyl acetate and ether,the results showed that the organic solvents could not extract the active substance and made the active substance inactivated.The supernatant of the buffer containing 5%DMSO showed antibacterial activity when the cells were crushed.The supernatant containing active substances was concentrated by ultra filtration tube,the results showed that its molecular weight was greater than 1.0×10^(5) g/mol,and its antibacterial activity was stronger at 4℃than at other temperatures.The supernatant containing active substances was separated by ammonium sulfate fractionated precipitation method,the results showed that when the initial saturation of ammonium sulfate was 60%and the final saturation was 80%,the active substance had the strongest antibacterial activity.This study laid a foundation for separation and purification of active substances,research of antagonistic mechanism and development and utilization.