抗真菌抗生素的分离纯化及生物活性的初步研究

【作者】 张龙；

【导师】 刘党生； 吴文芳； 纪有海；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2008， 硕士

【摘要】 迄今为止,已发现的抗生素有80%皆来自于放线菌,而放线菌主要存在于土壤中,并在土壤菌种中占有相当大的比例,本实验所用的菌株FL₁是在中科院应用生态研究所微生物室对2000多株土壤放线菌进行初筛,在得到的15个对真菌具有拮抗作用的菌株中,进一步复筛而得到的。实验采用玉米小斑菌为指示菌,使用琼脂块发酵结合摇瓶液体发酵法,对发酵液进行抗菌活性考察,发现其对水稻恶苗菌,玉米丝黑穗菌,玉米大斑菌,番茄灰霉菌,黄瓜枯萎菌,烟草赤星菌,西瓜炭疽菌,茄子褐斑菌等真菌均具有较好的抑菌效果,属广谱抗生素。此外,在对发酵液做的浸种实验中,还发现其对玉米、小麦、黄瓜等作物的种子均具有较好的促生长作用。为了提高抗生素的产量,本实验对发酵培养基及发酵条件进行了优化,在不同条件下测定抗生素对鉴定菌的抑菌效果,初步确定发酵培养条件为:培养温度28℃,摇瓶装液量为70mL/250mL,发酵周期4天。采用经典鉴定方法和分子生物学分类方法对放线菌FL₁进行了鉴定。结果表明,放线菌FL₁属于链霉菌属,烬灰类群,通过在NCBI数据库中比对,与白浅灰链霉菌（Streptomyces albogriseus）的16S ribosomal DNA gene同源性最高,达100.0%,但其形态特征,生理生化特性与该菌属有一定差异,因此初步认定其为Streptomycesalbogriseus的一个变种。对放线菌FL₁的发酵液进行初步分离提取,得到具有生物活性的发酵上清液提取物,并考察了其物理化学性质,在此基础上进行了稳定性实验及纸层析、薄层层析实验,为进一步的分离纯化提供了依据。纸层析试验结果初步显示该抗生素可能属于氨基糖苷类或多烯类,但该抗生素的理化性质与此二类抗生素的性质均存在一定的差异。对于抗生素的分离纯化主要应用硅胶柱分离,活性物质在流动相为甲醇:氯仿=1:5梯度下洗脱出来;全波长紫外扫描发现其最大吸收波长为277nm。HPLC的色谱条件为:检测波长277nm,流动相为乙腈:磷酸二氢钾（0.02mol/L）=40:60,流速1.5mL/min。在t_R=12.723min时出峰,分离度高,峰形良好。经ESI-MS检测,其分子量为495。更多还原

【Abstract】 So far,80%of antibiotics which have been found are from actinomycetes, actinomycetes mainly exist in soil and account for great proportion.The strain FL₁ used in this paper was supplied by the cell engineering group of IEA,who firstly screened 20 active strains from 2000 different actinomycetes,and then we screened again and got one high efficiency strain named FL₁.With the Bipolaris maydis as a test microorganism,applied the agar solid fermentation method combined with shaking flask liquid fermentation method,we investigated the bioactivity of the broth and found that the broth which was produced by FL₁ had good bacteriostasis effect to Fusarium.moniliforme,Sphacelotheca reiliana,Exserohilμm turcicμm,Botrytis cinerea,Fusariμm oxysporum f.sp.cucμmerinμm, Alternaria longipes Tobacco pathotype,Colletotrichum orbicμlare,alternaria leaf spot of egg plant,so it belongs to broad-spectrum antibiotic.Besides,in the experiment of seed soaking of corn,wheat and cucumber,we also found that the broth had good effect to the growth promotion of the crops.For improving the productivity of bioactive substance of strain FL₁,both the fermentation medium and the fermentation conditions were optimized.Determining the bacteriostasis effect to Fusariμm.moniliforme in different conditions,the suitable fermentation conditions were as follows:cultivation temperature,28℃;loading liquid volume,70mL/250mL;fermentation period,4d.Strain FL₁ was identified to be the cinerogriseus group of actinomycetes through the classical and molecular biology categorization method.Blasted in NCBI,Strain FL₁ was most closely related to Streptomyces albogriseus,sharing 16S rDNA similarity value of 100%.However,the morphological,cultural and phy-biochemical characteristics between the two strains revealed some extent difference.Based on the polyphase taxonomica data, Strain FL₁ was primarily identified as Streptomyces albogriseus.n.Var.Bioactive substance was obtained from extracts of fermentation supernatant.We also conducted the preliminary study on the phy-chemical properties of the bioactive products,basing on that,we did the stability experiment,paper chromatography and thin layer chromatography,provided enough evidences for further isolation and purification. The result of paper chromatography showed that the antibiotic may belong to aminoglycosides,or multi-peptid,but there were big differences between them.We applied silica gel column chromatography to separate bioactive substance, bioactive substance was eluted under the condition of mobile phase（CH₃OH:CHCl₃ =1:5）.Through The whole ultraviolet wavelength scanning,it made sure that the maximum absorption wavelength was 277nm.Chromatographic condition:detection wavelength:277nm,mobile phase:acetonitrile:KH₂PO₄ buffer（0.02mol/L）=40:60,flow rate:1.5mL/min.when the retention time came to 12.723,chromatographic peak came out, which had great resolving power and good peak form.By means of ESI-MS,the molecular weight（MW）of active substance was conceived as 495.更多还原