抗真菌活性放线菌的多相分类鉴定及代谢产物的初步分析

发布时间：2018-05-20 01:20

本文选题：放线菌 + 16S　；参考：《沈阳药科大学》2008年硕士论文

【摘要】： 本文采用琼脂扩散法从29株土壤放线菌中筛选出了2株具有较强抗真菌活性的菌株(P-127及1043)。采用多相分类方法对放线菌P-127及1043进行了鉴定,结果表明：菌株P-127的16SrDNA序列(获得的gene bank登记号为EU797798)与Streptomyces padanus有着最相近的进化关系,序列完全相同(同源性达100%),因此确定菌株P-127为Streptomyces padanus;菌株1043的16S rDNA(获得的gene bank登记号为EU864307)与许多链霉菌的16S rDNA序列有着高度同源性,确定该菌株为一株链霉菌。为了提高活性代谢产物的产量,对P-127及1043的基本发酵条件进行了考察。通过杯碟法检测不同条件下发酵液中抗生素的产量,确立了最适摇瓶发酵条件：(1)菌株P-127为种龄34h,接种量10%,摇瓶装液量40ml/250ml三角瓶,发酵周期为4天。(2)菌株1043为种龄40h,接种量10%,摇瓶装液量50ml/250ml三角瓶,发酵周期为6天。对菌株P-127及1043发酵液中的活性物质进行了热、酸碱稳定性研究；针对P-127菌体的活性代谢产物进行了pH纸层析、八种溶剂系统纸色谱试验,为初步确定该活性物质的理化特性提供了依据。采用大孔吸附树脂对P-127菌体的活性代谢产物进行分离,得到粗品。再利用制备型高效液相进一步分离纯化,获得了抗真菌活性物质的结晶,纯度较高,经分析型高效液相色谱法检测含量超过97%,质谱显示该活性物质的分子量为670,紫外吸收波谱显示该活性物质具有多烯大环内酯类抗生素的特征吸收峰,结合该菌株的多相分类鉴定和文献检索,提示该活性物质可能是fungichromin。
[Abstract]:In this paper, two strains of soil actinomycetes with strong antifungal activity were screened by Agar diffusion method. The polyphase classification method was used to identify the actinomycetes P-127 and 1043. The results showed that the 16SrDNA sequence of the strain P-127 (gene bank registration number EU797798) had the closest evolutionary relationship with Streptomyces padanus. The strain P-127 was identified as Streptomyces padanus.The 16s rDNA of strain 1043 (gene bank registration number EU864307) was highly homologous with the 16s rDNA sequence of many Streptomyces, and the strain was identified as Streptomyces. In order to increase the yield of active metabolites, the basic fermentation conditions of P-127 and 1043 were investigated. The yield of antibiotics in fermentation broth under different conditions was determined by the method of cup and dish. The optimum fermentation condition of strain P-127 was established as the seed age of 34 h, the inoculation amount of 10% and the quantity of 40ml/250ml triangle bottle. The fermentation period of strain 1043 was 40 h, the inoculation amount was 10%, and the quantity of liquid in shake bottle was 50ml/250ml triangle bottle. The fermentation period was 6 days. The thermal, acid-base stability of the active substances in the fermentation broth of strain P-127 and 1043 were studied, and the pH paper chromatography and eight solvent system paper chromatography tests were carried out for the active metabolites of P-127 bacteria. It provides a basis for the determination of the physical and chemical properties of the active substance. The active metabolites of P-127 were separated by macroporous adsorption resin and the crude products were obtained. The antifungal active substances were obtained by further separation and purification by using the preparative high performance liquid phase, and the purity of the antifungal active substances was high. The mass spectrometry showed that the molecular weight of the active substance was 670. The UV absorption spectrum showed that the active substance had the characteristic absorption peak of polyene-macrolide antibiotics. Combined with the multiphasic taxonomy and literature retrieval of the strain, it was suggested that the active substance might be fungichromin.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R379

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