摘要
埃博霉素与紫杉醇一样具有促微管聚合作用从而抑制肿瘤细胞的增殖,并且与紫杉醇相比,埃博霉素具有毒性更小,水溶性更好,对耐药性细胞作用更强之优点,因而引起医药界广泛关注。本文进行了埃博霉素菌种的改良和发酵工艺的优化研究,同时对改良菌株发酵液提取物的抗肿瘤活性进行了研究。
菌种改良方面分别采用紫外诱变,紫外-氯化锂诱变,硫酸二乙酯(DES)诱变,亚硝酸-紫外复合诱变,原生质体紫外-氯化锂诱变对埃博霉素原始菌株进行诱变,然后分别采用原始菌株的碳氮源(葡萄糖和硝酸钾)抗性培养基、埃博霉素合成前体物质(丙酸钠和乙酸钠)抗性培养基和埃博霉素结构类似物(红霉素)抗性培养基对诱变株进行筛选,得到一株遗传稳定的埃博霉素高产菌株——纤维堆囊菌UNH127,其埃博霉素(EPOs)总产量是原始菌株的28.2倍。
本文对该诱变株的发酵条件进行了探索,首先对其发酵培养基进行优化,在筛选合适的碳氮源种类的基础上,应用均匀设计方法结合逐步回归方法建立多元高次回归数学模型,优化纤维堆囊菌UNH127的发酵培养基,EPOs总产量达到2.36 mg/L,比优化前提高了778%。采用中心组合设计方法优化埃博霉素合成前体物质培养基方案,EPOs产量提高了22%,在此基础上采用中心组合响应面分析方法优化纤维堆囊菌UNH127的摇瓶发酵条件,EPOs产量达到3.18 mg/L,EPOs总产量提高了10%。同时采用HPLC法分析了纤维堆囊菌发酵液中组分,并考察了纤维堆囊菌发酵液粗提物的抗乳腺癌细胞MCF-7的活性和Hela细胞的活性,采用线性回归分析方法建立HPLC法分析纤维堆囊菌发酵液中组分与其抗癌细胞活性间的相关模型,并采用t检验的方法分析各组分的抗癌活性,为筛选纤维堆囊菌发酵液中活性代谢产物提供了实验依据。
Epothilones are macrolides antibiotics that were candidate drug as inhibitors of the tubulin system as Taxol mimetics. The epothilones, like Taxol and other taxoids, could stabilize microtubules, leading to inhibit the cell cycle and eventually to apoptosis at the concentration of 10-120 ng/ml. The epothilones is regarded as antitumor drugs because of low toxic, water soluble and high activity against cell lines resistant to Taxol. Though the epothilones could be got by chemical synthesis, it is not widely acceptable because of condition, expensive cost, longer synthesis route, lower productivity and environment pollution. The epothilones are usually produced by biosynthesis way while there are many reports on fermentation, however, the low productivity and genetic instability restrain its industrialization. There are two ways to improve the productivity, one is to improve the strain and the other is to optimize the fermentation process.
In this study, the analysis and purification ways were optimized. The content of epothilones in the fermentation broth of Sorangium cellulosum was analyzed with HPLC. The factors were optimized with uniform design combined with neural network. Based on the neural network model, the optimal HPLC conditions were obtained genetic algorithm as follow, acetonitrile volume fraction 29.2% , column temperature 34℃and pH of mobile phase 4.23. The effectivity of the epothilone A and B increased significantly after the optimization.
The desorption conditions of epothilone were optimized by response surface methodology and the optimal conditions were that the ratio of solvent to broth was 10.5, desorption time was 11.5 hours and desorption was 3 times.
Sorangium cellulosum ATCC15384 was mutated by UV, UV-LiCl, DES, UV-HNO2 and protoplast-UV- LiCl. Then , the mutants were screened with medium containing high concentration carbon and nitrogen source, precursor of epothilone or epothilone mimetics. The Sorangium cellulosum UNH127 with high genetic stability was obtained, whose productivity increased 28.2 times as that of the original strain.
The medium to culture Sorangium cellulosum UNH127 was optimized with uniform design combined with stepwise regression and the multiple regression model was got. The optimal medium was as follow, KNO3 8, soybean peptide 17.6, beef extract 6.46, yeast extract 1.0, CaCl2 0.25, K2HPO41, NaCl 1 and FeCl3 0.02 (g/L). The predicted max EPOs productivity is 2.48mg/L, and the actual EPOs productivity is 2.36mg/L through validation experiment, which is 7.78 time of that before optimization.
The central composite design was employed to optimize the medium containing the precursor of epothilone. The optimal medium is as follow, the sodium propionate concentration 0.48g/L, cysteine concentration 0.06g/L and the opportunity to add cysteine 1.6 h. The predicted optimal productivity is 2.91mg/L, while the actual productivity is 2.88mg/L, which is 22% higher than that without the precursor.
The central composite design was employed to optimize the fermentation conditions in flask. The optimal conditions were as follow, temperature 31℃, rotational speed 160 r/min, inculum 9.24%, medium volumn 117ml, pH 6.6, strain age 22.6h and fermentation time 4 days. The predicted optimal productivity of EPOs is 3.22mg/L while the actual productivity is 3.18mg/L, which increase 10%.
The anti-tumor activity of Sorangium cellulosum UNH127 fermentation broth was studied. The components of fermentation broth were analyzed by HPLC, and the activity to inhibit the breast cancer cell and hela cell were studied. The linear regression model was got to describe the relationship between components of Sorangium cellulosum UNH127 fermentation broth and anti-tumor activity. The significance was analyzed with t test, which is an important base to screen the active metabolites.
引文
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