【作者】 郭正彦；

【导师】 吴文君；

【作者基本信息】 西北农林科技大学 ， 农药学， 2009， 博士

【摘要】 微生物是筛选天然先导化合物的重点来源之一,放线菌又是微生物代谢产物中具有生物活性物质的主要产生菌。随着大量常见链霉菌的发现,遵照传统的分离模式筛选到新菌种或具有新颖结构抗生素的机率越来越小,研究人员转向改进分离培养方法和从特殊环境采集样品等手段来提高筛选新型活性物质的效率,目前已取得显著的成效。本文以通过改进分离培养方法得到的放线菌No.313菌株为材料,从菌株分类鉴定,主要活性代谢产物分离纯化和结构鉴定方面进行了系统研究,并对活性化合物的生物活性和分离工艺的优化也进行了初步研究,主要研究结果如下:1.利用多相分类法,经形态特征观察、生理生化指标测定和16S rDNA的序列分析,No.313菌株与模式菌株Streptomyces alboflavus NBRC-13196T具有高度的相似性(99.5%),除仅牛奶凝固这一特征表现明显差异外,其他指标大部分相同或相似。因此将No.313菌株命名为Streptomyces alboflavus 313。2.采用活性追踪,经过HPD400大孔吸附树脂富集、硅胶柱层析和反相高效液相制备,分离得到了8个活性化合物,4个为新的环六肽化合物,分别命名为NW-G01、NW-G03、NW-G06和NW-G08。根据IR、HR-MS、1D-NMR和2D-NMR等波谱数据分析,鉴定了其中三个化合物的结构:NW-G01由缬氨酸、三个哌嗪-3-羧酸、2-甲基氨基丙酸和6-氯-3a-羟基-1,2,3,3a,8,8a-六氢吡咯[2,3-b]吲哚-2-羧酸组成,X-ray衍射确定了其立体构型[0]; NW-G03是由缬氨酸、两个哌嗪-3-羧酸、2,3,4,5-四氢哒嗪-3-羧酸,2-甲基氨基丙酸和6-氯-3a-羟基-1,2,3,3a,8,8a-六氢吡咯[2,3-b]吲哚-2-羧酸组成;NW-G06是由缬氨酸、两个哌嗪-3-羧酸、4-羟基-6-甲氧基-1,2,3,4-四氢哒嗪-3-羧酸,2-甲基氨基丙酸和6-氯-3a-羟基-1,2,3,3a,8,8a-六氢吡咯[2,3-b]吲哚-2-羧酸组成。3.室内生测结果表明,NW-G01对番茄灰霉病菌(Botrytis cinerea)、葡萄白腐病菌(Coniothyrium diplodiella)和油菜菌核病菌(Sclerotinia sclerotiorum)菌丝生长具有很强抑制的作用,其EC50分别为24.91、11.90和4.01μg·mL-1;对玉米大斑病菌(Exserohlum turcium)和番茄灰霉病菌(Botrytis cinerea)的孢子萌发也有较强的抑制作用,EC50分别为63.52和29.44μg·mL-1;对蜡状芽孢杆菌(Bacillus cereus)、枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)和耐甲氧西林金葡菌(methicillin-resistant Staphylococcus aureus, MRSA)的MIC值为3.90、3.90、7.81和7.81μg·mL-1(阳性对照氨苄青霉素的MIC值为25、12.5、25和大于100μg·mL-1)。NW-G03和NW-G06对上述4种细菌MIC值分别为12.25和50、25和50、3.07和6.13、6.13和12.25μg·mL-1(同比阳性对照氨苄青霉素的MIC值分别为50、50、6.13和大于100μg·mL-1)。三个环肽对大肠埃氏菌(Escherichia coli)和铜绿假单胞菌(Pseudomonas aeruginosa)的MIC值均大于250μg·mL-1。4.采用反相高效液相色谱建立了三个环肽的分析方法。色谱条件为:色谱柱为Sinochrom ODS-BP (5μm, 4.6mm×200mm),检测波长为210 nm,流动相为甲醇:水(70:30,v/v),流速为1.00mL·min-1,进样量为10μL。分析方法的有效性评价结果表明,化合物NW-G01、NW-G03和NW-G06在0.3~10μg·mL-1范围内具有良好的线性关系。化合物NW-G01、NW-G03和NW-G06的相对标准偏差(RSD)分别为0.09%,1.38%和1.24%。当添加10μg·mL-1时,NW-G01、G03和G06的回收率分别为98.9～100.8%,97.9～101.6%和98.1～102.6%;当添加1.0μg·mL-1时,回收率分别为99.6～101.5%,97.7～101.4%和98.1～103.6%。该方法可用于发酵液中三个环肽的定性定量分析。5.从6种不同型号的HPD系列树脂中筛选出HPD400树脂为吸附剂,最大动态吸附量为4300mL原始发酵液(20mL湿体积树脂)。以甲醇为解吸剂,优化后的洗脱条件:先用40%的甲醇洗脱,除去大量的色素等杂质,然后用100%的甲醇将活性成分洗脱下来。在发酵过程中添加树脂试验结果说明,在发酵起始期添加1%的HPD400树脂,不仅可以除去大量杂质,还能提高NW-G03和NW-G06的产量,而且发现了两个在原始发酵液中未检测出的物质。更多还原

【Abstract】 Microorganism is one of the focus sources in the screening for natural lead compounds. Actinomyces are the important producing strains that maybe produce new bioactive secondary metabolites.With the discovery of a large number of common streptomyces from natural environment, the probability of screening news strains or new structural antibiotics has become smaller and smaller following the traditional isolation modle, so the researchers have taken steps to modify the methods of isolation and culture or to collect samples from special circumstances[0] to improve the capacity of screening new bioactive substances, the significant achievement has been obtained.This paper is about the research work based on one actinomycete strains No.313, which was screened through modified methods of isolation and culture, including strain identification, purification of the main active metabolite, structural identification, and evaluation of antimicrobial activity and optimization of separation processes. The primary results are as follows:(1) According to the results of morphological characteristics, physiological and biochemical measurement and analysis of 16S rDNA sequence, No.313 strain was highly homological (99.5%) with the type strain Streptomyces alboflavus NBRC-13196T, and both were similar features in majority except the milk coagulation property. Therefore, the No.313 strain was identified and designated as Streptomyces alboflavus 313.(2) Guided by the results of the bioassay and through enrichment of HPD400 macroporous resin, silica gel column chromatography and preparation of reversed-phase High Performance Liquid Chromatography, eight active compounds have been separated, four of them were cyclic hexadepsipeptide antibiotics named as NW-G01, NW-G03, NW-G06 and NW-G08. According to analysis of IR, HR-MS, 1D-NMR and 2D-NMR spectroscopy datas, the molecular structure of three compounds have been elucidated. Compound NW-G01 was comprised of Valine, three piperazine-3-carboxylic acid, 2-(methylamino)propanoic acid and 6-chlorl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid, the stereoscopic configuration of NW-G01 was determined by X-ray crystallographic analysis. NW-G03 was constited of Valine, two piperazine-3-carboxylic acid, 2,3,4,5-tetrahydro- pyridazine-3-carboxylic acid, 2-(methylamino)propanoic acid and 6-chlorl-3a-hydroxy- 1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. NW-G06 was made of Valine, two piperazine-3-carboxylic acid, 4-hydroxy-6-methoxy-1,2,3,4-tetrahydropyridazine -3-carboxylic acid, 2-(methylamino)propanoic acid and 6-chlorl-3a-hydroxy-1,2,3,3a,8,8a -hexahydropyrrolo[2,3-b]indole-2-carboxylic acid.(3) Bioassay results in vitro showed that NW-G01 exhibited strong inhibitory effect on mycelial growth against Botrytis cinerea, Coniothyrium diplodiella and Sclerotinia sclerotiorum, the values of EC50 were 24.91,11.90 and 4.01μg?mL-1, respectively. It also had inhibitory effect on spore germination against Exserohlum turcium and Botrytis cinerea, the values of EC50 were 63.52 and 29.44μg?mL-1, respectively. Besides, compound NW-G01 showed strong antibacterial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus(MRSA), with MIC values of 3.90, 3.90, 7.81 and 7.81μg?mL-1 (the MICs of ampicillin control were 25,12.5,25 and more than 100μg?mL-1), respectively. The MICs of compound NW-G03 against the four bacteria above were 12.25, 25, 3.07 and 6.13μg?mL-1 and those of NW-G06 were 50, 50, 6.13 and 12.25μg?mL-1, respectively. ( the MICs of ampicillin control were 50, 50, 6.13 and more than 100μg?mL-1). The MICs of three cyclopeptides against Escherichia coli and Pseudomonas aeruginosa were more than 250μg?mL-1.(4) A reversed-phase HPLC method for analyzing three cyclic hexadepsipeptide compounds was established. (Chromatographic parameter: Sinochrom ODS-BP column (5μm, 4.6mm×200mm), detection wavelength of 210 nm, the mobile phase of methanol:water (70:30, v/v), flowing rate of 1.00mL?min-1, injection volume of 10μL). The developed method was validated in terms of linearity, precision and accuracy. The linear regression data showed a good linear relationship for NW-G01, NW-G03 and NW-G06 over a concentration range of 0.3～10μg·mL-1. It showed a good precision that the RSD of NW-G01, NW-G03 and NW-G06 were 0.09%, 1.38% and 1.24%, respectively. When adding 10μg?mL-1, the recovery rates of NW-G01, NW-G03 and NW-G06 were 98.9～100.8%, 97.9～101.6% and 98.1～102.6%, respectively. When adding 1.0μg?mL-1, the recovery rates were 99.6～101.5%, 97.7～101.4% and 98.1～103.6%, respectively.(5) The studies on optimization of macroporous adsorption resin separation process were carried out. From six kinds of different models of HPD Series, HPD400 was selected as the adsorbent resin, the maximum dynamic adsorption capacity was 4300mL original fermentation broth (20mL wet resin volume). After optimization of elution conditions, use 40% methanol to elute firstly to clear most impurities and pigment and then with 100% methanol so that the active ingredient would be eluted down. From preliminary studies on the adding resin[0] into fermentation process, it was found that not only a large number of impurities were removed, but also the output of NW-G03 and NW-G06 were improved while adding 1% HPD400 resin in the initial fermentation period. Moreover,two substances, not been found from fermentation broth without adding resin, were obviously found from methanol eluate.更多还原