放线菌CD5872产驱虫抗生素的研究

发布时间：2018-03-24 10:39

本文选题：橄榄网状链霉菌　切入点：分类鉴定　出处：《成都学院》2017年硕士论文

【摘要】：寄生虫病一直是制约畜牧业发展的重要因素,尤其是集约化养殖业的发展导致寄生虫病的流行迫切需要人们从自然界中筛选具有驱虫效果的化合物。实验室发现分离自四川阿坝州土壤样品的一株放线菌CD5872的发酵液具有驱虫活性。本文通过对该菌株及其代谢产物的研究,分离纯化得到2个氨基糖苷类驱虫抗生素并进行了结构鉴定。通过菌种诱变、培养基优化和发酵条件的优化进一步提高了目标产物的发酵水平。1.采用培养特征、形态特征、生理生化和16S rRNA序列分析等多种分类技术对菌株CD5872进行了分类鉴定,初步鉴定为橄榄网状链霉菌(Streptomyces olivoreticuli)。2.目标产物的分离纯化和结构鉴定研究:通过发酵液过滤、732强酸性阳离子交换树脂富集、HD-2弱酸性阳离子交换树脂层析、正相硅胶柱层析等方法获得两种活性次级代谢产物,分别命名为CD-1、CD-2。利用红外光谱、高分辨质谱、核磁共振、旋光度比对和高效液相分析等方法对2种化合物进行结构解析,最终确定这两种化合物分别与越霉素A和越霉素C同质。3.菌种诱变:对野生菌株CD5872进行自然分离筛选出产量较高且遗传性状稳定的菌株CD5872-08,以菌株CD5872-08作出发菌株,分别采用紫外诱变和亚硝基胍诱变育种方式,筛选出高产菌株CD5872-08-02、CD5872-08-01A和CD5872-08-13A,产量分别达到664.6U/mL、625.1U/m L、612.0U/mL,较出发菌株分别提高了52.3%、43.6%和40.1%。4.发酵工艺的初步优化:对紫外诱变株CD5872-08-02进行培养条件和培养基的单因素优化实验,再通过响应面法优化确定组份及比例:可溶性淀粉50.0g/L,葡萄糖10.0g/L,黄豆粉39.80g/L,酵母粉10.15g/L,硝酸钾0.50g/L,氯化钠2.28g/L,磷酸氢二钾0.50g/L,硫酸镁0.50g/L,碳酸钙2.00g/L,蛋氨酸5.00g/L。发酵液效价提高到1151.4U/mL,相较于优化前摇瓶发酵产量664.6U/mL,提高了73.2%。为越霉素A的进一步工业化打下基础。
[Abstract]:Parasitosis has been an important factor restricting the development of animal husbandry. In particular, the development of intensive aquaculture has led to the epidemic of parasitic diseases. Screening of insecticide-repellent compounds from nature is urgently needed. Laboratory results showed that an actinomycetes CD5872 isolated from soil samples of Aba Prefecture, Sichuan Province. The fermentation broth has insecticidal activity. In this paper, the strain and its metabolites were studied. Two aminoglycoside repellent antibiotics were isolated and purified and their structures were identified. The fermentation level of the target product was further improved by mutagenesis, optimization of culture medium and optimization of fermentation conditions. The strain CD5872 was classified and identified by physiological and biochemical techniques and 16s rRNA sequence analysis. It was identified as Streptomyces olivoreticulium. 2. Purification and structure identification of the target product: enrichment of HD-2 weak acid cation exchange resin by fermentation broth filtration of 732 strong acid cation exchange resin. Two kinds of active secondary metabolites were obtained by normal phase silica gel column chromatography. The structures of the two compounds were analyzed by infrared spectroscopy, high resolution mass spectrometry, nuclear magnetic resonance spectroscopy, optical rotation ratio and high performance liquid phase analysis. Finally, the two compounds were identified to be homogenous with viramycin A and V. viramycin C. mutagenesis: the wild strain CD5872 was naturally isolated and screened out with high yield and stable hereditary character, and strain CD5872-08 was used as the hair strain. UV mutagenesis and nitrosoguanidine mutagenesis were used respectively. The high yield strains CD5872-08-02CCD5872-08-01A and CD5872-08-13A were screened, and the yield reached 664.6U / mLX 625.1U / mL, which increased 52.30.63.6% and 40.1.4than the original strain, respectively. The primary optimization of fermentation technology: the single factor optimization experiment on the culture conditions and culture medium of UV induced strain CD5872-08-02 was carried out. Then the components and ratios were optimized by response surface method: soluble starch 50.0 g / L, glucose 10.0 g / L, soybean powder 39.80 g / L, yeast powder 10.15 g / L, potassium nitrate 0.50 g / L, sodium chloride 2.28 g / L, potassium hydrogen phosphate 0.50 g / L, magnesium sulfate 0.50 g / L, calcium carbonate 2.00 g / L, methionine 5.00 g / L. Compared with the optimized fermentation yield of 664.6 U / mL before shaking flask, the yield increased by 73.2%, which laid the foundation for further industrialization of viramycin A.
【学位授予单位】：成都学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.796

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