【作者】 田威；

【导师】 何建勇； 白秀峰；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2002， 硕士

【摘要】 本文以尼拉霉素单一组分—安普霉素产生菌S.tenebrarius A04(发酵单位为2847u／ml)为出发菌株，通过对单孢子悬液和超声波破碎菌体的诱变处理并复合筛选模型，获得了遗传特性稳定的单组分高产菌株：A2-23、A2-30、ASM6、A1-16，摇瓶发酵单位达4800-5200u／ml。证明了UV处理复合耐受自身产物、UV处理复合耐受链霉素能获得较好的筛选结果。 以菌株A1-16为实验菌株，运用均匀设计方法，对发酵培养基、通气量、接种量等进行了考察，得到发酵培养基和种子培养基的优化配方。对三级发酵工艺、补料工艺等发酵工艺条件进行了优化，进一步确定了中试发酵工艺条件，4m~3发酵罐上平均发酵单位达4478u／ml。 通过氨基酸添加实验研究了安普霉素生物合成途径中的氮的来源及调节机制。Glu、Gln及α—酮戊二酸添加实验结果表明Glu、Gln对菌体的生长无明显的影响，但能强烈促进安普霉素的生物合成，且相同浓度条件下Gln的促进作用明显高于Glu，而α—酮戊二酸则对安普霉素的生物合成有抑制作用。因而我们认为Gln可能是安普霉素生物合成氮元素的供体。Arg添加实验结果表明，Arg可能通过两种途径影响黑暗链霉菌体内的氮代谢：(1)Arg可能影响胞外蛋白酶的活性，进而促进含氮大分子物质的分解代谢，补充发酵过程中的氮素来源。(2)Arg可能在体内转化为Glu，同时增加了游离氨的含量，进而促进安普霉素的生物合成。更多还原

【Abstract】 Streptomyces tenebrarius A04, the producer (with a titer of 2874u/ml) of single component of nebramycin ?apramycin was studied in this paper. After treatment of spore suspension and mycelia shivered by supersonic with mutagen ,combined with the application of screening models, some stable high yield apramycin-producing strains (with a fermentation titer of 4800-5200u/ml by shaking flask) such as A2-23, A2-30, ASM6 and Al-16 were obtained.This result proved that UV treatment combined with tolerating apramycin or with tolerating streptomycin could be effective way to obtain high yield apramycin-producing strains.With strain Al-16, the fermentation medium, aeration and inoculation volume etc. were investigated and the optimized vegetative medium and fermentation medium were obtained by uniform design.The tertiary fermentation techniques and the FBC (fed-batch culture) and other fermentation techniques were also optimized. A scale-up fermentation technique was set up and the apramycin productivity reached 4478u/ml in a 4m3 fermentator.Amino acid feeding experiments were also performed in this study in order to investigate the pathway, by which nitrogen incoperated into apramycin molecule, and the regulatory mechanism of nitrogen metabolism.The feeding of glutamic acid, glutamine and a -keto- glutaric acid respectively showed that glutamic acid and glutamine had no obvious effectson cell growth , but stimulated apramycin production greatly.With the same concentration of amino acids supplemented ,glutamine showed a stronger stimulation effect than glutamic acid, while a -keto-glutaric acid showed a repression effect on apramycin production. It could be deduced from above results that glutamine possibly is the donor of nitrogen element for the biosynthesis of apramycin. Arginine feeding experiment showed that nitrogen metabolism in the S.tenebraius was obviously affected by arginine through two possible ways:(l)pronase activity in vitro could be influnced by arginine, as a result, the catabolism of nitrogen-containing macro-molecule was promoted and the nitrogen element in the broth was increased.(2)arginine could be transformed into glutamic acid, so that the biosynthesis of apramycin was promoted.更多还原