昆蟲(chóng)病原線蟲(chóng)共生菌SN52菌株的次生代謝產(chǎn)物和生物活性研究
發(fā)布時(shí)間：2018-11-22 06:42
【摘要】：昆蟲(chóng)病原線蟲(chóng)共生菌是一類代謝產(chǎn)物豐富的藥物資源型細(xì)菌,這類細(xì)菌主要分為兩個(gè)屬即致病桿菌屬和發(fā)光桿菌屬,其中致病桿菌屬的次生代謝產(chǎn)物較多,目前已經(jīng)發(fā)現(xiàn)有10多類40多種化合物。這些結(jié)構(gòu)新穎的化合物擁有著廣泛的生物活性,包括抗細(xì)菌、抗真菌、抗病毒、抗癌、殺農(nóng)業(yè)害蟲(chóng)等,我國(guó)研究工作者也對(duì)這類細(xì)菌進(jìn)行了多年的研究并取得了優(yōu)異的成績(jī)。其發(fā)酵液和發(fā)酵液有機(jī)相粗提物在農(nóng)業(yè)病蟲(chóng)害方面都有較好的開(kāi)發(fā)前景,但國(guó)內(nèi)對(duì)這類菌的基礎(chǔ)研究以及應(yīng)用方面的研究不夠深入和全面,并且對(duì)這類菌次生代謝產(chǎn)物的化學(xué)分離純化研究較少。本文在本實(shí)驗(yàn)室的研究基礎(chǔ)上,通過(guò)利用常規(guī)的色譜柱層析技術(shù)以及高效液相(HPLC)制備技術(shù)對(duì)已被鑒定的一株伯氏致病桿菌SN52菌株的次生代謝產(chǎn)物進(jìn)行了分離純化。最終鑒定了其化學(xué)結(jié)構(gòu)并測(cè)試了它們的生物活性。研究結(jié)果如下:1.對(duì)共生菌SN52的發(fā)酵液有機(jī)相粗提物進(jìn)行了初步的抑菌試驗(yàn)評(píng)價(jià),發(fā)現(xiàn)粗提物對(duì)蠟樣芽胞桿菌和白色念珠菌有較強(qiáng)的抑制作用。利用色譜柱層析技術(shù)和高效液相色譜制備對(duì)發(fā)酵液有機(jī)相粗提物進(jìn)行分離純化,最終得到6個(gè)黃色粉末單體化合物和2個(gè)白色粉末單體化合物。通過(guò)1D和2D核磁技術(shù)、高分辨質(zhì)譜等波譜技術(shù)和比對(duì)文獻(xiàn)數(shù)據(jù)對(duì)分離純化得到的純化合物進(jìn)行了結(jié)構(gòu)解析,其結(jié)構(gòu)分別為:4個(gè)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個(gè)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.利用微量肉湯稀釋法對(duì)二硫吡咯酮化合物1-6的抗菌活性進(jìn)行測(cè)試,結(jié)果發(fā)現(xiàn)化合物3-6對(duì)革蘭氏陽(yáng)性細(xì)菌枯草芽孢桿菌、蠟樣芽孢桿菌、金黃色葡萄球菌具有較強(qiáng)的抑制作用其IC50值為0.8-4.9 μg/mL。而化合物1和2對(duì)這三株病原菌表現(xiàn)出中等的抑制活性。此外發(fā)現(xiàn)化合物3-6對(duì)柞蠶鏈球菌和白色念珠菌也有一定的抑制活性,分析其結(jié)構(gòu)-活性關(guān)系發(fā)現(xiàn)這類化合物的N-7位可變酰基側(cè)鏈對(duì)化合物的抑菌活性有強(qiáng)烈的影響。此外化合物1-4對(duì)番茄灰霉病菌的抑制活性較差可能與其溶解性有關(guān)。3.從昆蟲(chóng)病原線蟲(chóng)共生菌中分離到的含異腈化合物對(duì)昆蟲(chóng)免疫系統(tǒng)的關(guān)鍵酶(酚氧化酶)具有納摩爾級(jí)(IC50)的抑制活性,化合物7和8是這類物質(zhì)的類似物,只是缺少了異腈基。體外測(cè)試其對(duì)昆蟲(chóng)血腔的酚氧化酶的抑制作用,試驗(yàn)結(jié)果支持了異腈基在酶抑制作用中起關(guān)鍵作用的觀點(diǎn)。上述結(jié)果表明二硫吡咯酮N-7位�；勺儌�(cè)鏈對(duì)其抑菌活性有強(qiáng)烈的影響,這為以后合成和衍生這類化合物提供了指向性作用,此外4個(gè)新化合物的發(fā)現(xiàn)豐富了這類細(xì)菌次生代謝產(chǎn)物的多樣性為研究和利用這類細(xì)菌奠定了一定基礎(chǔ)。
[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.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
，
本文編號(hào)：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.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
，
本文編號(hào)：2348394
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