昆虫病原线虫共生菌SN52菌株的次生代谢产物和生物活性研究
发布时间：2018-11-22 06:42
【摘要】：昆虫病原线虫共生菌是一类代谢产物丰富的药物资源型细菌,这类细菌主要分为两个属即致病杆菌属和发光杆菌属,其中致病杆菌属的次生代谢产物较多,目前已经发现有10多类40多种化合物。这些结构新颖的化合物拥有着广泛的生物活性,包括抗细菌、抗真菌、抗病毒、抗癌、杀农业害虫等,我国研究工作者也对这类细菌进行了多年的研究并取得了优异的成绩。其发酵液和发酵液有机相粗提物在农业病虫害方面都有较好的开发前景,但国内对这类菌的基础研究以及应用方面的研究不够深入和全面,并且对这类菌次生代谢产物的化学分离纯化研究较少。本文在本实验室的研究基础上,通过利用常规的色谱柱层析技术以及高效液相(HPLC)制备技术对已被鉴定的一株伯氏致病杆菌SN52菌株的次生代谢产物进行了分离纯化。最终鉴定了其化学结构并测试了它们的生物活性。研究结果如下:1.对共生菌SN52的发酵液有机相粗提物进行了初步的抑菌试验评价,发现粗提物对蜡样芽胞杆菌和白色念珠菌有较强的抑制作用。利用色谱柱层析技术和高效液相色谱制备对发酵液有机相粗提物进行分离纯化,最终得到6个黄色粉末单体化合物和2个白色粉末单体化合物。通过1D和2D核磁技术、高分辨质谱等波谱技术和比对文献数据对分离纯化得到的纯化合物进行了结构解析,其结构分别为:4个已知二硫吡咯酮化合物,Xenorhabdin Ⅰ(化合物 3)、Xenorhabdin Ⅱ(化合物 4)、Xenorhabdin Ⅳ(化合物 5)、和Xenorhabdin V(化合物6);2个新的二硫吡咯酮,6-(E-5-甲基己-2-烯酰胺)-5-氧代-1,2-二硫代[4,3-b]吡咯(化合物1),和6-(E-5-甲基己-2-烯酰胺)-4-甲基-5-氧代-1,2-二硫代[4,3-b]吡咯(化合物2)以及4-O-(4-乙酰胺-β-L-鼠李糖苷)-苯甲醛(化合物7),4-0-(4-乙酰胺-β-L-鼠李糖苷)-(Z)-N-苯乙烯基甲酰胺(化合物8)。2.利用微量肉汤稀释法对二硫吡咯酮化合物1-6的抗菌活性进行测试,结果发现化合物3-6对革兰氏阳性细菌枯草芽孢杆菌、蜡样芽孢杆菌、金黄色葡萄球菌具有较强的抑制作用其IC50值为0.8-4.9 μg/mL。而化合物1和2对这三株病原菌表现出中等的抑制活性。此外发现化合物3-6对柞蚕链球菌和白色念珠菌也有一定的抑制活性,分析其结构-活性关系发现这类化合物的N-7位可变酰基侧链对化合物的抑菌活性有强烈的影响。此外化合物1-4对番茄灰霉病菌的抑制活性较差可能与其溶解性有关。3.从昆虫病原线虫共生菌中分离到的含异腈化合物对昆虫免疫系统的关键酶(酚氧化酶)具有纳摩尔级(IC50)的抑制活性,化合物7和8是这类物质的类似物,只是缺少了异腈基。体外测试其对昆虫血腔的酚氧化酶的抑制作用,试验结果支持了异腈基在酶抑制作用中起关键作用的观点。上述结果表明二硫吡咯酮N-7位酰基可变侧链对其抑菌活性有强烈的影响,这为以后合成和衍生这类化合物提供了指向性作用,此外4个新化合物的发现丰富了这类细菌次生代谢产物的多样性为研究和利用这类细菌奠定了一定基础。
[Abstract]:Entomopathogenic nematodes are a kind of drug-based bacteria rich in metabolic products, which are mainly divided into two species, namely, the genus Bacillus and the genus Bacillus, wherein the secondary metabolites of the genus Bacillus are more, and more than 40 compounds have been found at present. These novel compounds possess a wide range of biological activities, including anti-bacterial, antifungal, antiviral, anti-cancer, agricultural pests, and the like, and our researchers have also conducted years of research on this type of bacteria and have achieved excellent results. The crude extract of the fermentation liquor and the fermentation liquor has a good development prospect in the aspects of agricultural diseases and insect pests, but the research on the basic research and application of the bacteria in China is not in-depth and comprehensive, and the research on the chemical separation and purification of the secondary metabolites of the bacteria is less. On the basis of the research of this lab, the secondary metabolites of one of the primary pathogenic bacteria SN52 strains, which have been identified, were isolated and purified by means of conventional column chromatography and high performance liquid chromatography (HPLC). Their chemical structure was finally identified and their biological activity was tested. The results of the study are as follows: 1. The crude extract of the organic phase of the fermentation liquor of the symbiotic bacteria SN52 was evaluated. The results showed that the crude extract has a strong inhibitory effect on the Bacillus cereus and the Candida albicans. and separating and purifying the crude extract of the organic phase of the fermentation liquor by using the chromatographic column chromatography technology and the high-performance liquid chromatography, and finally obtaining 6 yellow powder monomer compounds and 2 white powder monomer compounds. The structure of the pure compound obtained by separating and purifying the literature data by 1D and 2D nuclear magnetic techniques, high-resolution mass spectrometry and the like is carried out, and the structure of the pure compound is as follows: 4 known dithiophenones compounds, Xenophanabdin I (compound 3), Xenophanabdin II (compound 4), Xenorhabdin IV (Compound 5), and Xenorhabdin V (Compound 6); 2 new dithio-pentanone, 6-(E-5-methyl-2-en-amine) -5-oxo-1,2-dithio[4, 3-b][compound 1], and 6-(E-5-methyl-2-en-amine) -4-methyl-5-oxo-1,2-dithio[4, 3-b]-ethanone (compound 2) and 4-O-(4-ethenamine-1-L-rhamnose)-benzaldehyde (compound 7), 4-0-(4-ethenamine-1-L-rhamnose-)-(Z)-N-styryl methylamine (Compound 8). The antibacterial activity of the dithio-1-6 was tested by a micro-broth dilution method. The results showed that the compound 3-6 had a strong inhibitory effect on the Gram-positive bacteria, Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus, and the IC50 value was 0.8-4.9ug/ mL. The compounds 1 and 2 showed moderate inhibitory activity on the three pathogens. In addition, it was found that compound 3-6 had a certain inhibitory activity against Streptococcus mutans and Candida albicans, and its structure-activity relationship was found to have a strong effect on the antibacterial activity of the compound. In addition, the inhibitory activity of compound 1-4 on tomato gray mold is probably related to its solubility. The key enzymes (phenoloxidase), which are isolated from the symbiotic bacteria of the insect pathogenic nematodes, have the inhibitory activity of the nanomolar stage (IC50) of the key enzyme (phenol oxidase) of the insect's immune system, and the compounds 7 and 8 are analogs of such substances, except that the isonitrile group is absent. In vitro, the inhibition of the phenol oxidase in the blood cavity of the insect is tested, and the results of the test support the viewpoint of the key role of the isonitrile group in the inhibition of the enzyme. The results show that the N-7-based variable-side chain of the dithio-bupropion has a strong effect on the antibacterial activity, which provides a directional effect for the later synthesis and the derivatization of the compound. In addition, the discovery of the four new compounds enriched the diversity of the secondary metabolites of this kind of bacteria and laid a foundation for the study and utilization of the bacteria.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
，
本文编号：2348394
[Abstract]:Entomopathogenic nematodes are a kind of drug-based bacteria rich in metabolic products, which are mainly divided into two species, namely, the genus Bacillus and the genus Bacillus, wherein the secondary metabolites of the genus Bacillus are more, and more than 40 compounds have been found at present. These novel compounds possess a wide range of biological activities, including anti-bacterial, antifungal, antiviral, anti-cancer, agricultural pests, and the like, and our researchers have also conducted years of research on this type of bacteria and have achieved excellent results. The crude extract of the fermentation liquor and the fermentation liquor has a good development prospect in the aspects of agricultural diseases and insect pests, but the research on the basic research and application of the bacteria in China is not in-depth and comprehensive, and the research on the chemical separation and purification of the secondary metabolites of the bacteria is less. On the basis of the research of this lab, the secondary metabolites of one of the primary pathogenic bacteria SN52 strains, which have been identified, were isolated and purified by means of conventional column chromatography and high performance liquid chromatography (HPLC). Their chemical structure was finally identified and their biological activity was tested. The results of the study are as follows: 1. The crude extract of the organic phase of the fermentation liquor of the symbiotic bacteria SN52 was evaluated. The results showed that the crude extract has a strong inhibitory effect on the Bacillus cereus and the Candida albicans. and separating and purifying the crude extract of the organic phase of the fermentation liquor by using the chromatographic column chromatography technology and the high-performance liquid chromatography, and finally obtaining 6 yellow powder monomer compounds and 2 white powder monomer compounds. The structure of the pure compound obtained by separating and purifying the literature data by 1D and 2D nuclear magnetic techniques, high-resolution mass spectrometry and the like is carried out, and the structure of the pure compound is as follows: 4 known dithiophenones compounds, Xenophanabdin I (compound 3), Xenophanabdin II (compound 4), Xenorhabdin IV (Compound 5), and Xenorhabdin V (Compound 6); 2 new dithio-pentanone, 6-(E-5-methyl-2-en-amine) -5-oxo-1,2-dithio[4, 3-b][compound 1], and 6-(E-5-methyl-2-en-amine) -4-methyl-5-oxo-1,2-dithio[4, 3-b]-ethanone (compound 2) and 4-O-(4-ethenamine-1-L-rhamnose)-benzaldehyde (compound 7), 4-0-(4-ethenamine-1-L-rhamnose-)-(Z)-N-styryl methylamine (Compound 8). The antibacterial activity of the dithio-1-6 was tested by a micro-broth dilution method. The results showed that the compound 3-6 had a strong inhibitory effect on the Gram-positive bacteria, Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus, and the IC50 value was 0.8-4.9ug/ mL. The compounds 1 and 2 showed moderate inhibitory activity on the three pathogens. In addition, it was found that compound 3-6 had a certain inhibitory activity against Streptococcus mutans and Candida albicans, and its structure-activity relationship was found to have a strong effect on the antibacterial activity of the compound. In addition, the inhibitory activity of compound 1-4 on tomato gray mold is probably related to its solubility. The key enzymes (phenoloxidase), which are isolated from the symbiotic bacteria of the insect pathogenic nematodes, have the inhibitory activity of the nanomolar stage (IC50) of the key enzyme (phenol oxidase) of the insect's immune system, and the compounds 7 and 8 are analogs of such substances, except that the isonitrile group is absent. In vitro, the inhibition of the phenol oxidase in the blood cavity of the insect is tested, and the results of the test support the viewpoint of the key role of the isonitrile group in the inhibition of the enzyme. The results show that the N-7-based variable-side chain of the dithio-bupropion has a strong effect on the antibacterial activity, which provides a directional effect for the later synthesis and the derivatization of the compound. In addition, the discovery of the four new compounds enriched the diversity of the secondary metabolites of this kind of bacteria and laid a foundation for the study and utilization of the bacteria.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
，
本文编号：2348394
本文链接：https://www.wllwen.com/shoufeilunwen/zaizhiyanjiusheng/2348394.html
