肌苷育种MicrosoftWord文档介绍.docVIP

天津科技大学硕士学位论史 摘要 本文以代谢控制发酵理论为指导，重点研究了肌苷高产菌的选育及其发 酵条件。主要研究内容和结果如下： 1．建立了发酵液中肌苷定量测定的方法——离子交换法，对其最佳分离 条件进行了研究，并与纸层析法进行比较，发现该方法准确度和精确度比纸 层析方法高，可用于简单条件下对肌苷发酵液中目的产物的测定。 2．以枯草芽孢杆菌TY一21为出发菌株，通过硫酸二乙酯(DES)诱变定 向选育出一株肌苷产生菌TX一3，其遗传标记为腺嘌呤缺陷、硫胺素缺陷、8． 氮鸟嘌呤抗性和磺胺胍抗性(Ade。+TH+8．AG。+SG。)。在未经优化的摇瓶发酵 条件下可产肌苷12．41dL。 3．研究了枯草芽孢杆菌(Bacillus subtilis)TX一3摇瓶发酵生产肌苷的最 佳条件。通过均匀设计和模式识别方法分别研究了肌苷种子和发酵培养基的 最佳配比。在最佳条件下，摇瓶分批发酵产量为18．94∥L。同时采用流加方 式研究了摇瓶补料分批发酵条件，该条件下肌苷产量可达到24．79∥L。 4．利用5 L罐分批发酵数据，对发酵过程进行了代谢流分析。基于物流 平衡原理，初步进行了发酵过程的代谢流分析；结合发酵过程曲线分析了肌 苷发酵全程代谢行为；应用TX一3菌株的代谢流量平衡模型计算出了肌苷发酵 中后期的代谢流分布并通过MATLAB软件线性规划得到理想代谢流分布，结’ 果表明尽量降低EMP途径的代谢通量有利于肌苷的合成，提高肌苷合成途径 中各关键酶的活力可有效增加肌昔的生成量，减少副产物的生成和提高发酵 过程溶氧水平均可增加肌苷合成代谢流。 关键词：肌苷，枯草芽孢杆菌，诱变育种，发酵，代谢流分析天津科技大学硕士学位论文 Abstract According to the theory of metabolic control fermentation，the dissertation focuses on the breeding and fermentation condition of inosine producing strain． The main research contents and results are as follows： (1)The method of ion exchange chromatography was defined to determinate inosine concentration quantitatively in the fermentation broth．The separation conditions of this method was optimized and compared with paper chromatography method．Both accuracy and precision are better than that of paper chromatography． (2)One inosine high—producing strain TX一3(Ade一+Thi++8一AG‘+SG’)was derived from Bacillus subtilis TY-21 with DES treatment，which could produce inosine 12．41 g／L under the fermentation condition without being optimized． (3)The batch fermentation condition of strain TX一3 in shake flask was studied in this dissertation．The proportions of seed medium and fermentation medium were optimized by pattern recognition and uniform design experiment．The seed culture conditions and fermentation conditions were also studied．Under the optimum condition，strain TX-3 could produce inosine 18．949／L by shaking flask batch fermentation．Meanwhile the fed batch fermentation conditions were studied． The production of inosine under optimized fed batch fermentation was 24．79