【摘要】：布雷菲德菌素A(Brefeldin A,BFA)是首次從Penicillium decumben發(fā)酵液中發(fā)現(xiàn)的大環(huán)內(nèi)酯類抗生素,能誘導(dǎo)高爾基體的分解、抑制蛋白質(zhì)從內(nèi)質(zhì)網(wǎng)中轉(zhuǎn)運(yùn)為高爾基體復(fù)合物,從而阻斷高爾基體復(fù)合物的蛋白質(zhì)運(yùn)輸,具有抗真菌、抗病毒、抗有絲分裂、抗腫瘤等生物學(xué)活性。美國(guó)NCI研究發(fā)現(xiàn)布雷菲德菌素A能夠誘導(dǎo)腫瘤細(xì)胞分化和凋亡,作為化學(xué)治療試劑布雷菲德菌素A用于腫瘤治療具有巨大的應(yīng)用前景,受到產(chǎn)學(xué)界的高度重視。 我們從采集的植物內(nèi)生真菌樣本中分離得到一株絲狀真菌A1163,其發(fā)酵液表現(xiàn)出抗真菌活性。利用紅外光譜、質(zhì)譜、核磁共振、X一射線衍射分析等技術(shù)對(duì)真菌A1163次級(jí)代謝產(chǎn)物活性組分進(jìn)行結(jié)構(gòu)鑒定,確定其活性物質(zhì)結(jié)構(gòu)為大環(huán)內(nèi)酯類抗生素布雷菲德菌素A。結(jié)合形態(tài)學(xué)特征和分子生物學(xué)鑒定,菌株A1163被鑒定為Eupenicillium brefeldianum。結(jié)合自然選育和誘變育種方法,進(jìn)行多輪選育,獲得布雷菲德菌素A高產(chǎn)菌株ZJB082702,保藏號(hào)為CCTCC M208113。 論文建立了快速定量和定性檢測(cè)E. brefeldianum CCTCC M208113發(fā)酵液中布雷菲德菌素A含量的HPLC、TLC分析方法。采用單因素試驗(yàn)設(shè)計(jì)、響應(yīng)面設(shè)計(jì)系統(tǒng)優(yōu)化了E. brefeldianum CCTCC M208113的發(fā)酵條件,獲得最佳的培養(yǎng)基配方(g/l):淀粉13.3,葡萄糖26.7,酵母膏9.7×10~(-1),玉米漿1.0,黃豆餅粉4.6×10~(-1),麥芽浸出物2.5,硫酸鎂3.0,磷酸二氫鉀4.0,碳酸鈣6.0,硝酸鈉7.4×10~(-1),硫酸銅1.0×10~(-2);最佳發(fā)酵條件:搖瓶選擇帶尖頭擋板的三角瓶,起始發(fā)酵液pH 6.0,接種量3.0%(v/v),裝液量140:500(ml/ml),敞開(kāi)式迥轉(zhuǎn)搖床轉(zhuǎn)速180 r/min,發(fā)酵溫度28℃,培養(yǎng)時(shí)間6 d。在上述最優(yōu)培養(yǎng)條件下,布雷菲德菌素A最高累積濃度達(dá)到1301.45 mg/l,較優(yōu)化前提高了約17.0倍。發(fā)酵實(shí)驗(yàn)結(jié)果揭示,預(yù)培養(yǎng)72 h后添加麥芽糖促進(jìn)E. brefeldianum CCTCC M 208113合成布雷菲德菌素A,0.7%麥芽糖提高布雷菲德菌素A濃度10.9%;正丙醇和異丙醇對(duì)布雷菲德菌素A的生物合成有促進(jìn)作用,預(yù)培養(yǎng)96 h后,添加0.67g/l異丙醇較對(duì)照組提高了21.7%;吐溫80對(duì)布雷菲德菌素A的生物合成有促進(jìn)作用,接種后0 h添加1.54g/l吐溫80,產(chǎn)量比對(duì)照提高了22.3%左右,最高布雷菲德菌素A濃度達(dá)1586.0 mg/l。 基于搖瓶小試優(yōu)化結(jié)果,分別在51、151機(jī)械攪拌罐上考察了E.brefeldianum CCTCC M 208113的發(fā)酵動(dòng)力學(xué)。在51機(jī)械攪拌罐規(guī)模上,布雷菲德菌素A最高發(fā)酵單位達(dá)到375.2 mg/l;151機(jī)械攪拌罐規(guī)模上,布雷菲德菌素A最高發(fā)酵單位達(dá)到648.17 mg/l。 基于搖瓶分批發(fā)酵實(shí)驗(yàn)結(jié)果,建立了E. brefeldianum CCTCC M208113發(fā)酵生產(chǎn)布雷菲德菌素A的發(fā)酵動(dòng)力學(xué)模型。結(jié)合模型參數(shù)估計(jì)和非線性曲線擬合,建立E. brefeldianum CCTCC M 208113生長(zhǎng)動(dòng)力學(xué)模型: 菌體生長(zhǎng)動(dòng)力學(xué)模型: dX/dt=1.7×10~(-1)X(1-X/(17.0)) 產(chǎn)物生長(zhǎng)動(dòng)力學(xué)模型: dP/dt=8.5×10~(-1)X 底物消耗動(dòng)力學(xué)模型: -dS/dt=9.8×10~(-1)dX/dt+3.4×10~(-3)dP/dt
[Abstract]:BFA is the first macrolide antibiotic found in Penicillium decumben fermentation broth, which can induce the decomposition of Golgi apparatus and inhibit the transport of protein from endoplasmic reticulum to Golgi complex, thus blocking the protein transport of Golgi complex, and has antifungal, antiviral, anti-mitotic, anti-tumor and other biological activities. NCI studies in the United States have found that Brefedixin A can induce tumor cell differentiation and apoptosis. As a chemotherapy reagent, Brefedixin A has a great application prospect in tumor therapy, and has been highly valued by the field of production. A filamentous fungus A1163 was isolated from plant endophytic fungus samples, and its fermentation broth showed antifungal activity. The structure of active components of secondary metabolites of fungi A1163 was identified by infrared spectroscopy, mass spectrometry, nuclear magnetic resonance and X-ray diffraction. The structure of active substances was determined to be macrolide antibiotic Brefedixin A. Combined with morphological characteristics and molecular biological identification, strain A1163 was identified as Eupenicillium brefeldianum.. Combined with natural breeding and mutagenesis breeding methods, multiple rounds of breeding were carried out, and the high yield strain ZJB082702, of Brefedixin A was obtained as CCTCC M208113. In this paper, a rapid HPLC,TLC method for the quantitative and qualitative determination of Brefedixin A in E. brefeldianum CCTCC M208113 fermentation broth was established. The fermentation conditions of E. brefeldianum CCTCC M208113 were optimized by single factor design and response surface design system. the optimum medium formula (g 鈮，

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