大环内酯类抗生素布雷菲德菌素A生产菌种选育及其发酵工艺研究
发布时间：2019-06-24 17:42
【摘要】：布雷菲德菌素A(Brefeldin A,BFA)是首次从Penicillium decumben发酵液中发现的大环内酯类抗生素,能诱导高尔基体的分解、抑制蛋白质从内质网中转运为高尔基体复合物,从而阻断高尔基体复合物的蛋白质运输,具有抗真菌、抗病毒、抗有丝分裂、抗肿瘤等生物学活性。美国NCI研究发现布雷菲德菌素A能够诱导肿瘤细胞分化和凋亡,作为化学治疗试剂布雷菲德菌素A用于肿瘤治疗具有巨大的应用前景,受到产学界的高度重视。 我们从采集的植物内生真菌样本中分离得到一株丝状真菌A1163,其发酵液表现出抗真菌活性。利用红外光谱、质谱、核磁共振、X一射线衍射分析等技术对真菌A1163次级代谢产物活性组分进行结构鉴定,确定其活性物质结构为大环内酯类抗生素布雷菲德菌素A。结合形态学特征和分子生物学鉴定,菌株A1163被鉴定为Eupenicillium brefeldianum。结合自然选育和诱变育种方法,进行多轮选育,获得布雷菲德菌素A高产菌株ZJB082702,保藏号为CCTCC M208113。 论文建立了快速定量和定性检测E. brefeldianum CCTCC M208113发酵液中布雷菲德菌素A含量的HPLC、TLC分析方法。采用单因素试验设计、响应面设计系统优化了E. brefeldianum CCTCC M208113的发酵条件,获得最佳的培养基配方(g/l):淀粉13.3,葡萄糖26.7,酵母膏9.7×10~(-1),玉米浆1.0,黄豆饼粉4.6×10~(-1),麦芽浸出物2.5,硫酸镁3.0,磷酸二氢钾4.0,碳酸钙6.0,硝酸钠7.4×10~(-1),硫酸铜1.0×10~(-2);最佳发酵条件:摇瓶选择带尖头挡板的三角瓶,起始发酵液pH 6.0,接种量3.0%(v/v),装液量140:500(ml/ml),敞开式迥转摇床转速180 r/min,发酵温度28℃,培养时间6 d。在上述最优培养条件下,布雷菲德菌素A最高累积浓度达到1301.45 mg/l,较优化前提高了约17.0倍。发酵实验结果揭示,预培养72 h后添加麦芽糖促进E. brefeldianum CCTCC M 208113合成布雷菲德菌素A,0.7%麦芽糖提高布雷菲德菌素A浓度10.9%;正丙醇和异丙醇对布雷菲德菌素A的生物合成有促进作用,预培养96 h后,添加0.67g/l异丙醇较对照组提高了21.7%;吐温80对布雷菲德菌素A的生物合成有促进作用,接种后0 h添加1.54g/l吐温80,产量比对照提高了22.3%左右,最高布雷菲德菌素A浓度达1586.0 mg/l。 基于摇瓶小试优化结果,分别在51、151机械搅拌罐上考察了E.brefeldianum CCTCC M 208113的发酵动力学。在51机械搅拌罐规模上,布雷菲德菌素A最高发酵单位达到375.2 mg/l;151机械搅拌罐规模上,布雷菲德菌素A最高发酵单位达到648.17 mg/l。 基于摇瓶分批发酵实验结果,建立了E. brefeldianum CCTCC M208113发酵生产布雷菲德菌素A的发酵动力学模型。结合模型参数估计和非线性曲线拟合,建立E. brefeldianum CCTCC M 208113生长动力学模型: 菌体生长动力学模型: dX/dt=1.7×10~(-1)X(1-X/(17.0)) 产物生长动力学模型: dP/dt=8.5×10~(-1)X 底物消耗动力学模型: -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，
本文编号：2505240
[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，
本文编号：2505240
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