【作者】 王芳；

【导师】 黄建新；

【作者基本信息】 西北大学 ， 微生物学， 2008， 硕士

【摘要】 鬼臼毒素类物质因具有显著的抗病毒、抗肿瘤活性,在医药领域具有重要的应用价值。目前该类物质主要从植物中提取获得,但天然资源有限,无法满足市场需求。由于植物内生真菌具有产生与宿主相同或相似代谢物的特性,且易培养、易控制,并可通过诱变育种等手段来提高其有效成分产量,真菌发酵为获得鬼臼毒素类药物资源提供了新途径。目前这一领域的研究多集中于对有价值菌株的筛选,在菌种改造、发酵条件等方面有待进一步深入研究。目的对可产生鬼臼类物质的内生真菌采取复合诱变处理,进一步对选育获得的高产株进行发酵条件的研究,以期为解决鬼臼类抗癌药物短缺提供新途径;通过研究获得微生物发酵生产鬼臼类物质的工艺数据,为实现工业化生产奠定基础。方法以药用真菌Ty为研究对象,采用紫外线及He-Ne激光复合诱变处理,获得优势变异菌株;利用薄层层析、薄层色谱扫描、高效液相色谱分析确定高产菌株的发酵培养物中含有目的产物;设计适于该菌株生长的半合成发酵培养基,并优化其组分。结果紫外诱变选育获得的高产菌株Ty4-16,较同批发酵培养的出发菌株生物量提高31%,产物量提高约25%。在此基础上进行激光诱变,最终获得高产株Ty4-16-26,较Ty4-16菌株的生物量提高17%,产物量提高约20%。确定该诱变菌株可产生鬼臼毒素。经优化的半合成发酵培养基配方为:2%淀粉、2%蔗糖、0.1%蛋白胨、0.1%NH₄（SO₄）₂、0.1%酵母膏、0.03%K₂HPO₄、0.03%Nacl、0.03%MgSO₄,水100mL,pH6.5。采用该发酵培养基,28℃培养7天,菌株生物量提高约20.3%,鬼臼毒素产量提高约9.7%。菌株5L发酵罐放大试验表明菌体生长较好,产物合成稳定。结论得到鬼臼毒素高产菌株的诱变选育方法以及用微生物发酵法获得鬼臼毒素类物质的技术路线,该研究具有实现工业化生产的潜力。更多还原

【Abstract】 Podophyllotoxin is of great important in the field of medicine because of its antivirus and anti-cancer activities. It was derived mainly from the Podophyllotoxin plants. But the natural sources were limited. Endophytic fungi have the activity that can produce same or similar compounds to the plant. They are easy to be raised and controlled and their effective component output may be raised through mutation breeding. It is a new method by fermentation of endophytic fungui. So far,many research focused on separating valuable endophytic fungi which can produce Podophyllotoxin. Through compound mutation to producing-srains,then sudy on the optimizational fermentation conditions of high production strains,in the hope of providing some theoretic reference on producing botanic activity compouds by microbiology fermentation.Objectives:Compound mutation to producing-srains,then sudy on the optimizational fermentation conditions of high production strains in order to provide a new method obtaining anti-cancer material——podophyllotoxin and the technical data obtained from research on producing botantic activity compounds by microbiology fermentation. This will be lay the foundation for industrialization. Methods:High producing strain is attained by UV and He-Ne laser .The components were identified by thin layer chromatographic（TLC）,Thin layer chromatographic scanning,high performance liquid chromatographic（HPLC） that consist in fermentation of high producing strain;The thesis designed semi-synthesized medium compatibhe to the strain, and optimized composition respectively. Results:Ty4-16-26 was attained after UV mutation with its biomass enhancing 31% and production-biomass enhancing 25%. Laser mutation based on this carries obtained high production Ty4-16-26, with biomass enhancing 17% and production-biomass enhancing 20%. This theis confirm that the mutated strain can produce podophyllotoxin. The optimizational ingredient of the semi-synthesized medium were as follows: 2% soluble srarch, 2 %sucrose, 0.1% peptone, 0.1% yeast extract, 0.1% NH₄（SO4）₂, 0.03% K₂HPO₄, 0.03% Nacl , 0.03% MgSO₄, pH6.5. The biomass increase 20.3% and the production-biomass ratio increase 9.7% by using the optimizational medium at temperature of 28℃for 7d. The scale-up trial in 5L fermentor showed that mycelium grew well and products produced steadily. Conclusions:The method that obtain high-producing with mutation breeding;The technics that obtain podophyllotoxin with fermentation and the tenique for extraction were gained. This research has the potential of industrialization.更多还原