【作者】 叶丹；

【导师】 岑沛霖； 徐志南；

【作者基本信息】 浙江大学 ， 生物化工， 2008， 博士

【摘要】 米多霉素(mildiomycin, MIL)是从放线菌Streptoverticillium rimofaciens的培养液中分离到的一种核苷类次级代谢产物,对多种植物的白粉病具有显著防治作用。本文研究目的在于通过在培养过程中添加米多霉素的理论前体：羟甲基胞嘧啶的结构类似物,利用微生物自身的合成代谢体系合成多种米多霉素的结构类似物,以获得活性更高的生物农药。对其中两种衍生物：米多霉素胞嘧啶衍生物(MIL-C)和米多霉素5-氟胞嘧啶衍生物(MIL-F),开展了发酵条件和分离工艺优化,并进行了分子结构鉴定、生物活性和毒性评价。1.在Streptoverticillium rimofaciens的培养过程中添加5-氟胞嘧啶后,获得了米多霉素5-氟胞嘧啶衍生物(MIL-F)。通过对发酵条件优化,使MIL-F产量提高到了0.6g/l。对从发酵液分离提取MIL-F工艺进行了研究,获得了纯度超过95%的MIL-F产品。采用红外光谱、核磁共振及质谱分析等方法证明了所获得的MIL-F产物的分子结构确实是氟原子取代了MIL中胞嘧啶环上的羟甲基。考察了MIL-F的生物活性及毒理学性质,确定了MIL-F是一种高效、低毒、无刺激的生物农药。2.在Streptoverticillium rimofaciens的培养基中添加胞嘧啶后,获得了米多霉素胞嘧啶衍生物(MIL-C)。考察了MIL-C发酵条件,发现适当控制胞嘧啶加入量可以抑制米多霉素合成,使MIL-C成为唯一产物；通过单因素实验、Plackett-Burman实验和响应面中心组合实验使MIL-C的产量达到1336.5 mg/l,比优化前产量提高了3.6倍。在详细研究了MIL-C在DK110弱酸性阳离子交换树脂上的离子交换特性的基础上,通过优化离子交换条件分离得到了纯度约70%的MIL-C粗产品。经树脂吸附脱色和CM650离子交换凝胶柱精制,使MIL-C纯度达到了95%以上。采用全波段扫描、核磁共振及质谱分析等方法证明了所获得的MIL-F产物的分子结构确实是氢原子取代了MIL中胞嘧啶环上的羟甲基。3.考察了MIL-C的体内、体外生物活性,并开展了一年两地的田间小区实验。实验结果表明MIL-C制剂对南瓜白粉病和黄瓜白粉病都具有显著防效,药力控制良好。MIL-C对南瓜和黄瓜安全,对花和果实生长均无不利影响,对周围环境友好。对MIL-C进行安全性评价确定MIL-C是一种低毒、无刺激的农用抗生素。4.结合chemoffce组件对MIL、MIL-C和MIL-F参数进行分析比较,可以初步推断MIL的主要药效基团是可能是精氨酸上的胍基,该结构如果被破坏会导致MIL失活。通过本论文的研究,获得了两种米多霉素的结构类似物：MIL-F和MIL-C；经发酵工艺优化,大幅度提高了它们的产量；对生物活性的测试结果表明,它们对白粉病的防治效果都高于米多霉素,有可能成为高效、低毒的新型农药。对米多霉素及其类似物结构-功能关系的研究为将来发现新农药打下了良好的基础。更多还原

【Abstract】 Mildiomycin (MIL), a novel nucleoside antibiotic with a specific and strong inhibitory activity against powdery mildews, is the secondary metabolite of Streptoverticillium rimofaciens. The purpose of this work is to produce various mildomycin analogues by adding different precursors, such as structural analogues of hydroxymethyl cytosine, into the fermentation broth, from them, mildiomycin analogues with higher bioactivity may be identified. Two of them: Mildiomycin 5-flucytosine-substituted analogue (MIL-F) and mildiomycin cytosine-substituted analogue (MIL-C) were studied in detail, including the optimization of fermentation conditions and separation process, identification of molecular structure, evaluation of bioactivity and toxicity.1. After the addition of 5-flucytosin into the fermentation process of Streptoverticilliumrimofaciens, 5-flucytosine-substituted analogue (MIL-F) was obtained. The MIL-F yield reached 0.6 g/l after the optimization of fermentation conditions. The separation and purification process of MIL-F from fermentation broth was developed, and the purity of MIL-F product was above 95%. The molecular structure of MIL-F was examined by Infrared, MNR and MS etc. The bioactivity and toxicity of MIL-F were evaluated, and the results indicated that MIL-F should be a new biopesticide with higher efficiency, lower toxicity and less stimulation.2. After the addition of cytosin into the fermentation broth of Streptoverticillium rimofaciens,cytosine-substituted analogue (MIL-F) was obtained. It was found that by controlling the addition of cytosine, the biosynthesis of mildiomycin was inhibited and it is possible that the MIL-C was the only product. The yield of MIL-C was increased from 350 mg/l to 1336.5 mg/1 by the optimization of fermentation conditions with one-factor-in-one experiment, Plackett-Burman design and response surface method. Based on the ion-exchange characteristic of MIL-C from fermentation broth by DK110 weak cationic exchange resin, a separation technology was developed and the crude product of MIL-C with 70% purity was obtained. After decolor and further purification by a CM650 ion-exchange gel column, the purity of the final MIL-C product was higher than 95%. The molecular structure of MIL-C was also examined by UVS, NMR and MS.3. The bioactivity of MIL-C was investigated in vitro and in vivo including the greenhousetests and field tests in two different places in one year. The results showed that MIL-C was remarkable efficiency against the powdery mildew of cucumber and pumpkin, and the potency of drug can be kept stable. MIL-C was safe to cucumber and pumpkin without any negative effect on their flower and fruit growth, and it was also environmental friendly. The safety evaluation of MIL-Cshowed that it was regard as a safe agro-antibiotic with low toxicity and less motivation by the evaluation of toxicological tests.4. Some important parameters of MIL, MIL-C and MIL-F was analyzed and compared by the software of chemoffice. And the main pharmacophore was guessed as the guanazole group in the structure of MIL analogue, which means that destroying this group will make the MIL analogues deactivation.In this work, two kinds of mildiomycin analogues, MIL-F and MIL-C, were produced. The yields were improved greatly because of the optimization of both fermentation and separation processes. The bioactivity of these two MIL-analogues were higher than that of mildiomycin against powdery mildew, which indicated that it is possible to develop MIL-F and MIL-C into new biopesticides. The study in structure-function relation in mildiomycin analogues should be beneficial in discovering new biopesticides.更多还原