【作者】 张文娟；

【导师】 吴文君；

【作者基本信息】 西北农林科技大学 ， 化学生物学， 2013， 博士

【摘要】 放线菌是一类重要的微生物资源，它是抗生素及其他生物活性物质的主要生产者，其中又以链霉菌属产生的最多。随着大量具有生物活性菌株被分离，再用传统的筛选思路往往造成重复研究，从中发现新生物活性化合物的几率也越来越低，所以寻找稀有放线菌进一步发现新的活性物质成为科学研究的重要方向。本论文以从农药污染源土壤中分离到的放线菌NW35菌株为研究对象，系统研究该菌株的分类地位、发酵液抑菌活性、发酵液中活性成分的分离鉴定。此外，还对作用机理及发酵条件优化进行了初步研究。主要研究结果如下：1.形态特征和培养特征观察、生理生化特性测定和16S rDNA序列分析的结果表明，NW35菌株与模式菌株Streptomyces djakartensis NBRC15409的同源相似性为99%，NW35菌株除在葡萄糖天冬酰胺琼脂培养基上的培养特征，在马铃薯块上的生长情况、明胶液化、淀粉水解这些指标与模式菌株略不相同外，NW35菌株与模式菌株其他指标大体一致。因此采用多相分类法，将NW35菌株鉴定为Streptomyces djakartensis NW35。该菌株已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏日期为2012年11月13日，保藏登记号为CGMCC No.6817。2.测定了NW35菌株发酵液的抑菌活性。离体活性生测结果表明，NW35菌株发酵液对供试9种细菌和8种植物病原真菌都有一定的抑制作用。发酵液对革兰氏阳性细菌蜡状芽孢杆菌、枯草芽孢杆菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌有较强的抑制作用，抑菌圈透明；对革兰氏阴性细菌大肠埃氏菌、铜绿假单胞菌、白菜软腐病菌、猕猴桃溃疡病菌、青枯病病菌也有一定的抑制作用，但抑菌圈透明度较差。发酵液对供试植物病原真菌的抑制作用较弱，只对油菜菌核病菌的菌丝生长抑制率达到73.68%，其余都在70%以下。盆栽试验结果表明，菌株发酵液对小麦白粉病的保护和治疗作用都较差，分别为41.89%和38.41%。3.采用活性追踪法，将NW35菌株发酵液通过HPD100大孔吸附树脂富集、硅胶柱层析和反相高效液相色谱制备，分离到4个化合物。根据波谱数据分析确定了它们的化学结构和立体构型，其中3个为新化合物，分别为（E）-2-甲氧基-1,4萘醌-1-肟（Z-4-1）、（R）-2-羟甲基-3,4-二氢-1,4-苯并氮氧杂卓-5（1H）酮（Z-4-2）及2-（2-羟基-1-羟甲基-乙氨基）苯甲酸（Z-8-2）；1个已知化合物，即N-乙酰色胺（Z-9-2）。其中化合物Z-4-2为发酵液的主要抑菌活性成分，为一新抗生素，命名为西农霉素（Xinongmycin），并申请了国家发明专利（专利申请号：201310028444.1）。采用微量稀释法，测得西农霉素对革兰氏阳性细菌和革兰氏阴性细菌均有较强的抑制作用，对猕猴桃溃疡病菌的最小抑菌浓度（MIC）为7.81μg/mL，对蜡状芽孢杆菌、枯草芽孢杆菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、青枯病病菌最小抑菌浓度均为15.63μg/mL，对大肠埃氏菌、铜绿假单胞菌、白菜软腐病菌的最小抑菌浓度为31.25μg/mL，比阳性对照氨苄青霉素抑菌活性强。西农霉素对4种植物病原真菌的孢子萌发都有一定的抑制作用，当浓度为500μg/mL时，对棉花枯萎病菌、玉米弯孢叶斑病菌、黄瓜炭疽病菌的孢子萌发抑制率均在90%以上。Z-4-1、Z-9-2在剂量为10μg/纸碟时对蜡状芽孢杆菌、枯草芽孢杆菌、金黄色葡萄球菌、大肠埃氏细菌、猕猴桃溃疡病菌、青枯病病菌、耐甲氧西林金黄色葡萄球菌均有一定抑制作用，其中对蜡状芽孢杆菌和枯草芽孢杆菌有明显的抑制作用，抑菌圈透明。4.初步研究了西农霉素对蜡状芽孢杆菌的作用机理。西农霉素以MIC浓度处理不同时间后细胞内还原糖、丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）都有泄露。经西农霉素处理后，和对照相比，细胞膜相对渗透率升高。这些结果说明西农霉素破坏了细胞膜。此外，蜡状芽孢杆菌经西农霉素处理后其细胞蛋白含量、琥珀酸脱氢酶（SDH）和苹果酸脱氢酶（MDH）活性均降低。结合扫描电镜症状学观察，初步认为西农霉素可能破坏细胞膜，改变细胞膜相对渗透率，抑制蛋白合成进而导致细胞代谢紊乱，最后细胞裂解死亡。5.采用响应面分析法和正交试验设计，对菌株NW35发酵条件进行了优化，确定了最佳发酵培养基及最佳摇瓶发酵条件。最佳发酵培养基为：蔗糖10g/L，乳糖10g/L，麦芽糖10g/L，麦麸27.68g/L，蛋白胨4.5g/L，NH₄Cl3g/L，（NH₄）₂SO₄3g/L，CaCO₃0.87g/L、FeSO40.0073g/L。最佳发酵条件：250mL三角瓶装入60mL发酵培养基，接入6×10⁷cfu/mL菌量，28℃培养144h。在最优培养基、最优发酵条件下对NW35菌株进行发酵培养，得到的西农霉素含量为12.86μg/mL，而原始发酵条件西农霉素含量仅为1.6021μg/mL，是原始含量的8.03倍。研究了前体添加对NW35菌株发酵液中西农霉素含量的影响。结果表明，无论是单一前体添加还是复合前体添加，均未显著提高NW35菌株发酵液中西农霉素的含量，但在添加0.5g/L缩水甘油实验中，HPLC谱图中出现了一个在原始发酵液中未曾出现的新的波谱峰。更多还原

【Abstract】 Acyinomyces is one of the most important microorganism resources, which can produceantibiotics and other bioactive substances, the discovery of novel bioactive compounds isbecoming difficult with a large number of bioactive strain have been isolated by traditionaltactics. Exploration of rare actinomycete has become an important direction to discover newactive substance.This paper is about the research work on the actinomycete strain NW35, which wasisolated from a pesticide-polluted soil, including strain identification, antimicrobial activity offermentation broth, purification and structural identification, mechanism and optimizationfermentation conditions. The primary results are as follows:1. According to the results of morphological and culture characteristics, physiology andbiochemical measurement and16S rDNA sequence analysis, NW35strain was highlyhomological（up to99%） with Streptomyces djakartensis NBRC15409, except for culturecharacteristics on glucose asparagines agar and potato, gelatin liquefaction and starchhydrolysis properties. The NW35strain was identified and designated as Streptomycesdjakartensis NW35. The strain is deposited in the center of the China GeneralMicrobiological Culture Collection Center, November13,2012, with the registration numberof CGMCC No.6817.2. The antimicrobial activity of fermentation broth from strain NW35was tested.Bioassay results in vitro showed that the fermentation broth exhibited different antimicrobialactivity against9species of tested bacteria and8species of tested plant pathogens. Thefermentation broth had strong inhibition against Bacillus cereus, Bacillus subtilis,Staphylococcus aureus, methicillin-resistant Staphylococcus aureus with transparentinhibition zone, and inhibition against Escherichia coil, Pseudomonas aeruginosa, Erwiniacarotovora, Pseudomonas syringae pv.actinidiae, Ralstonia solanacearum with clearinhibition zone. The fermentation broth possessed good inhibition against Sclerotiniasclerotiorum（73.68%） and certain degree inhibition against the other tested plant pathogens,The protective efficacy and curative efficacy of the fermentation broth against Blumeria giamiais were41.89%and38.41%, respectively.3.Four compounds have been separated through bioassay guided fraction, includingenrichment by HPD100macroporous resin, and separation on silica gel column chroma-tography and preparation reversed-phase high performance liquid chromatography. Accordingto spectroscopy datas, the separated four compounds have been elucidated as:（E）-2-methoxy1-4-naphthoquinone1-oxime（Z-4-1）,2-（hydroxymethyl）-2,3-dihydrobenzo[e][1,4]oxazepin-5-（1H）-one（Z-4-2）,2-（2-Hydroxy-1-hydroxymethyl-ethylamino）-benzoic acid （Z-8-2） arenovel compounds. N-Acetyl-tryptamine （Z-9-2） are known compounds. Compound Z-4-2isthe main bacteriostatic active ingredients of the fermentation broth, which is a new antibiotic,and named as Xinongmycin.According to microdilution method, the bioassay results in vitro showed that Xinongmycinexhibited antibacterial activity against Pseudomonas syringae pv.actinidiae with MIC valuesof7.8μg/mL, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, methicillin-resistantStaphylococcus aureus, Ralstonia solanacearum with MIC values of15.6μg/mL, Escherichiacoil, Pseudomonas aeruginosa, Erwinia carotovora with MIC values of31.3μg/mL. Sporegermination demonstrated Xinongmycin showed strong inhibition against Fusariumoxysporum f.sp.vasinfectum, Curvularia lunata, Colletotrichum orbiculare, Colletotrichumgloesporioides, with the inhibition rate of above90%at the concentration of500μg/mL.Z-4-1，Z-9-2also showed certain degree antibiotic activity against Bacillus cereus, Bacillussubtilis, Erwinia carotovora, Staphylococcus aureus, Escherichia coil, Pseudomonasaeruginosa, methicillin-resistant Staphylococcus aureus, Pseudomonas syringae pv. actinidiae,Ralstonia solanacearum.4. The preliminary mechanism of Xinongmycin against Bacillus cereus was investigated.When the bacteria was treated with Xinongmycin at MIC with different time, the releasementof reducing sugar, alanine aminotransferase（ALT）, and aspartate aminotransferase （AST）were detected and the relative permeability of cell membrane was higher than control.Together with scanning electron microscopy, further detection of the decrement, in activitiesof of succinate dehydrogenase （SDH） and malate dehydrogenase （MDH） demonstratedXinongmycin might damage the cell membrane, enhance the cell membrane relativepermeability, and inhibit protein synthesis leading to cell metabolism disorder and cell deathfinally.5. The optimized fermentation for strain NW35was obtained by response surfacemethodology and the orthogonal experiment design. The best formula is: sucrose10g/L,lactose,10g/L, maltose,10g/L, wheat bran,27.68g/L, peptone4.5g/L, NH₄Cl3g/L and （NH₄）₂SO₄3g/L, CaCO₃,0.87g/L, FeSO40.0073g/L. Best fermentation condition:60mLmedium in250mL flask,6×10⁷cfu/mL bacteria amount,28℃,144h. In the optimal culturemedium and fermentation conditions, the content of Xinongmycin（12.86μg/mL） is8.03timesof the original content（1.60μg/mL）. The influence of feeding precursor on the production ofXinongmycin showed that neither single precursor nor composite precursor can significantlyimprove the content of Xinongmycin.更多还原