本文選題：Oidiodendron + truncatum�。� 參考：《中國海洋大學(xué)》2014年碩士論文

【摘要】：HDN-1是本研究室從一株南極來源樹粉胞屬真菌Oidiodendron truncatum GW3-13次級(jí)代謝產(chǎn)物分離得到的,屬于含有新穎三硫橋結(jié)構(gòu)的epipolythiodioxopiperazine (ETP)類化合物。五株人癌細(xì)胞活性測試表明,該化合物對(duì)腫瘤細(xì)胞具有較強(qiáng)的細(xì)胞毒活性。特別是在20nM濃度下,可以誘導(dǎo)M2型白血病細(xì)胞HL-60分化與凋亡,且對(duì)正常白細(xì)胞活力和小鼠體重?zé)o明顯影響；分子機(jī)制研究表明,HDN-1是一種作用于HSP90 C端的抑制劑。目前,HDN-1已作為新型靶向抗腫瘤藥物做新藥研究開發(fā)。本研究內(nèi)容重在通過新型高產(chǎn)培養(yǎng)基開發(fā)、發(fā)酵條件優(yōu)化及5L罐體優(yōu)化等工藝研究提高HDN-1的產(chǎn)量,并建立HDN-1制備工藝,為后續(xù)藥理學(xué)研究、毒理學(xué)研究及動(dòng)物實(shí)驗(yàn)奠定基礎(chǔ)。我們首先考察了培養(yǎng)溫度及發(fā)酵初pH、搖瓶裝液量和搖床轉(zhuǎn)速對(duì)該菌株生產(chǎn)HDN-1產(chǎn)量的影響。搖瓶實(shí)驗(yàn)提示,25℃為最適發(fā)酵溫度。搖瓶發(fā)酵初始pH實(shí)驗(yàn)表明菌株GW3-13最適生長pH為5.0。在500m1三角瓶中裝液100m1有利于菌株產(chǎn)HDN-1,轉(zhuǎn)速為180r/min較為適宜菌株生長。通過單因素實(shí)驗(yàn)確定了三種較優(yōu)碳源(葡萄糖、麥芽糖、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勻設(shè)計(jì)實(shí)驗(yàn)確定了培養(yǎng)基成分之間的配比,得到了一個(gè)可用模型及培養(yǎng)基組成。在均勻設(shè)計(jì)的基礎(chǔ)上,利用Plackett-Burman設(shè)計(jì)篩選了三個(gè)顯著因子(葡萄糖、酵母膏、MgSO4·7H2O)。通過響應(yīng)面設(shè)計(jì)(Response surface methodology)的Box-Behnken實(shí)驗(yàn)優(yōu)化了三因子之間的配比,開發(fā)了一種新型高產(chǎn)HDN-1培養(yǎng)基組成和配比,在此條件下,搖瓶培養(yǎng)HDN-1最大產(chǎn)量為54.7mg/L,比初始產(chǎn)量提高了9.6倍。模擬剪切力及消泡劑對(duì)HDN。1的影響實(shí)驗(yàn)表明,該菌株對(duì)剪切力不敏感：實(shí)驗(yàn)采用的消泡劑(體積分?jǐn)?shù)在2%之內(nèi))不影響產(chǎn)物產(chǎn)量。結(jié)合以上結(jié)果,5L發(fā)酵罐重點(diǎn)研究了不同通氣量、補(bǔ)料、間歇控制pH、不同槳葉組合等因素,結(jié)果表明通氣1vvm條件下,產(chǎn)物產(chǎn)量和菌體干重均高于通氣0.5wm,提示較高的通氣量可以促進(jìn)菌體生長及產(chǎn)物形成；間歇控制pH實(shí)驗(yàn)表明,整個(gè)發(fā)酵過程中不調(diào)整pH,保持初始pH為5.0對(duì)該菌株產(chǎn)HDN-1有利；一次性補(bǔ)加碳源的補(bǔ)料方式,產(chǎn)物最大產(chǎn)量為39.4mg/L,對(duì)產(chǎn)物產(chǎn)量稍有促進(jìn)作用；不同槳葉組合實(shí)驗(yàn)表明,下層四斜葉槳上層六平葉渦輪槳葉的組合對(duì)菌體生長和HDN-1的產(chǎn)量無顯著影響,菌體形態(tài)觀察表明松散稀疏狀態(tài)有利于菌體生物量積累和產(chǎn)物合成,HDN-1的最大產(chǎn)量為42.1mg/L,菌體最大干重達(dá)到15.29g/L。與上下均為六平葉渦輪槳葉不同的是,氧消耗的速度增加,提示可以通過這種組合提高氣液混合度。開發(fā)了一套完整、高效的HDN-1制備分離流程,提高了化合物分離純化的效率。
[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，

本文編號(hào)：1826953