本文選題：橄欖網(wǎng)狀鏈霉菌　切入點(diǎn)：分類鑒定　出處：《成都學(xué)院》2017年碩士論文

【摘要】：寄生蟲病一直是制約畜牧業(yè)發(fā)展的重要因素,尤其是集約化養(yǎng)殖業(yè)的發(fā)展導(dǎo)致寄生蟲病的流行迫切需要人們從自然界中篩選具有驅(qū)蟲效果的化合物。實(shí)驗(yàn)室發(fā)現(xiàn)分離自四川阿壩州土壤樣品的一株放線菌CD5872的發(fā)酵液具有驅(qū)蟲活性。本文通過(guò)對(duì)該菌株及其代謝產(chǎn)物的研究,分離純化得到2個(gè)氨基糖苷類驅(qū)蟲抗生素并進(jìn)行了結(jié)構(gòu)鑒定。通過(guò)菌種誘變、培養(yǎng)基優(yōu)化和發(fā)酵條件的優(yōu)化進(jìn)一步提高了目標(biāo)產(chǎn)物的發(fā)酵水平。1.采用培養(yǎng)特征、形態(tài)特征、生理生化和16S rRNA序列分析等多種分類技術(shù)對(duì)菌株CD5872進(jìn)行了分類鑒定,初步鑒定為橄欖網(wǎng)狀鏈霉菌(Streptomyces olivoreticuli)。2.目標(biāo)產(chǎn)物的分離純化和結(jié)構(gòu)鑒定研究:通過(guò)發(fā)酵液過(guò)濾、732強(qiáng)酸性陽(yáng)離子交換樹脂富集、HD-2弱酸性陽(yáng)離子交換樹脂層析、正相硅膠柱層析等方法獲得兩種活性次級(jí)代謝產(chǎn)物,分別命名為CD-1、CD-2。利用紅外光譜、高分辨質(zhì)譜、核磁共振、旋光度比對(duì)和高效液相分析等方法對(duì)2種化合物進(jìn)行結(jié)構(gòu)解析,最終確定這兩種化合物分別與越霉素A和越霉素C同質(zhì)。3.菌種誘變:對(duì)野生菌株CD5872進(jìn)行自然分離篩選出產(chǎn)量較高且遺傳性狀穩(wěn)定的菌株CD5872-08,以菌株CD5872-08作出發(fā)菌株,分別采用紫外誘變和亞硝基胍誘變育種方式,篩選出高產(chǎn)菌株CD5872-08-02、CD5872-08-01A和CD5872-08-13A,產(chǎn)量分別達(dá)到664.6U/mL、625.1U/m L、612.0U/mL,較出發(fā)菌株分別提高了52.3%、43.6%和40.1%。4.發(fā)酵工藝的初步優(yōu)化:對(duì)紫外誘變株CD5872-08-02進(jìn)行培養(yǎng)條件和培養(yǎng)基的單因素優(yōu)化實(shí)驗(yàn),再通過(guò)響應(yīng)面法優(yōu)化確定組份及比例:可溶性淀粉50.0g/L,葡萄糖10.0g/L,黃豆粉39.80g/L,酵母粉10.15g/L,硝酸鉀0.50g/L,氯化鈉2.28g/L,磷酸氫二鉀0.50g/L,硫酸鎂0.50g/L,碳酸鈣2.00g/L,蛋氨酸5.00g/L。發(fā)酵液效價(jià)提高到1151.4U/mL,相較于優(yōu)化前搖瓶發(fā)酵產(chǎn)量664.6U/mL,提高了73.2%。為越霉素A的進(jìn)一步工業(yè)化打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：成都學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S859.796

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