南极来源真菌Oidiodendron truncatum GW3-13生产HDN-1发酵工艺研究

发布时间：2018-05-01 00:32

  本文选题：Oidiodendron + truncatum　； 参考：《中国海洋大学》2014年硕士论文

【摘要】：HDN-1是本研究室从一株南极来源树粉胞属真菌Oidiodendron truncatum GW3-13次级代谢产物分离得到的,属于含有新颖三硫桥结构的epipolythiodioxopiperazine (ETP)类化合物。五株人癌细胞活性测试表明,该化合物对肿瘤细胞具有较强的细胞毒活性。特别是在20nM浓度下,可以诱导M2型白血病细胞HL-60分化与凋亡,且对正常白细胞活力和小鼠体重无明显影响；分子机制研究表明,HDN-1是一种作用于HSP90 C端的抑制剂。目前,HDN-1已作为新型靶向抗肿瘤药物做新药研究开发。本研究内容重在通过新型高产培养基开发、发酵条件优化及5L罐体优化等工艺研究提高HDN-1的产量,并建立HDN-1制备工艺,为后续药理学研究、毒理学研究及动物实验奠定基础。我们首先考察了培养温度及发酵初pH、摇瓶装液量和摇床转速对该菌株生产HDN-1产量的影响。摇瓶实验提示,25℃为最适发酵温度。摇瓶发酵初始pH实验表明菌株GW3-13最适生长pH为5.0。在500m1三角瓶中装液100m1有利于菌株产HDN-1,转速为180r/min较为适宜菌株生长。通过单因素实验确定了三种较优碳源(葡萄糖、麦芽糖、可溶性淀粉)和氮源(酵母膏、蛋白胨、玉米粉)。采用八因素十水平的均匀设计实验确定了培养基成分之间的配比,得到了一个可用模型及培养基组成。在均匀设计的基础上,利用Plackett-Burman设计筛选了三个显著因子(葡萄糖、酵母膏、MgSO4·7H2O)。通过响应面设计(Response surface methodology)的Box-Behnken实验优化了三因子之间的配比,开发了一种新型高产HDN-1培养基组成和配比,在此条件下,摇瓶培养HDN-1最大产量为54.7mg/L,比初始产量提高了9.6倍。模拟剪切力及消泡剂对HDN。1的影响实验表明,该菌株对剪切力不敏感：实验采用的消泡剂(体积分数在2%之内)不影响产物产量。结合以上结果,5L发酵罐重点研究了不同通气量、补料、间歇控制pH、不同桨叶组合等因素,结果表明通气1vvm条件下,产物产量和菌体干重均高于通气0.5wm,提示较高的通气量可以促进菌体生长及产物形成；间歇控制pH实验表明,整个发酵过程中不调整pH,保持初始pH为5.0对该菌株产HDN-1有利；一次性补加碳源的补料方式,产物最大产量为39.4mg/L,对产物产量稍有促进作用；不同桨叶组合实验表明,下层四斜叶桨上层六平叶涡轮桨叶的组合对菌体生长和HDN-1的产量无显著影响,菌体形态观察表明松散稀疏状态有利于菌体生物量积累和产物合成,HDN-1的最大产量为42.1mg/L,菌体最大干重达到15.29g/L。与上下均为六平叶涡轮桨叶不同的是,氧消耗的速度增加,提示可以通过这种组合提高气液混合度。开发了一套完整、高效的HDN-1制备分离流程,提高了化合物分离纯化的效率。
[Abstract]:HDN-1 was isolated from a secondary metabolite of the genus Oidiodendron truncatum GW3-13 from an Antarctic fungus. It belongs to a new epipolythiodioxopiperazine compound with a novel trisulfide bridge structure. The activity test of five human cancer cells showed that the compound had strong cytotoxicity to tumor cells. Especially at the concentration of 20nM, HL-60 differentiation and apoptosis could be induced in M2 leukemia cells, and the activity of normal leukocytes and the body weight of mice were not significantly affected, and the molecular mechanism showed that HHDN-1 was a kind of inhibitor acting on the C-terminal of HSP90. At present, HDN-1 has been developed as a new anti-tumor drug. In this study, the production of HDN-1 was studied through the development of new high-yield medium, the optimization of fermentation conditions and the optimization of 5L tank. The preparation process of HDN-1 was established, which laid a foundation for the follow-up pharmacological study, toxicological study and animal experiment. At first, the effects of culture temperature, initial pH of fermentation, volume of shaking liquid and rotating speed of shaking table on the production of HDN-1 from the strain were investigated. The shaking flask experiment indicated that 25 鈩，

本文编号：1826953