重组大肠杆菌产L-色氨酸发酵生产工艺

生物工程论文编号:SW139  论文字数:12151,页数:37

目  录
中文摘要 I
英文摘要 III
目录 Ⅴ
1. 绪论 1
 1.1 L-TRP的理化性质 1
 1.2 L-TRP的用途 1
 1.3 L-TRP的测定方法 2 
 1.4 L-TRP的生产方法 2
 1.5 L-TRP的代谢途径和代谢工程的应用 4
 1.6 L-TRP的发酵 5
 1.6 重组大肠杆菌的高密度发酵 6
2. 批次发酵实验部分 6
 2.1 材料和设备  6
 2.2 实验内容和实验方法 7
 2.3 实验结果和讨论 9
3. 批次流加发酵实验部分 13
 3.1 材料和器材   13
 3.2 实验内容和实验方法   15
 3.3实验结果和讨论 19
4．总结与展望 31
致谢 32
参考文献 33

摘 要：L-色氨酸（L-Trp）是一种人体必需的氨基酸，在食品添加剂、医药和饲料添加剂方面有着广泛的应用。全世界的年需求量在一万吨以上，但是实际的产能只有三千吨，远远没有满足市场的需求。在国内，只有武汉上海北京三地有小规模生产L-Trp的厂家，年产量也只有一百吨左右，因此L-Trp的应用被限制在了医药方面。本文利用产L-Trp的重组大肠杆菌菌株，主要进行发酵流加葡萄糖模式的探索，以期能够提高L-Trp产量到一定的水平。
 首先，我们利用摇瓶发酵的方式考察了葡萄糖浓度、NH4+ 和PO43-浓度对菌体生长和L-Trp的累积的影响。结果表明，在高浓度的葡萄糖条件下，发酵液中累积大量的乙酸，并且菌体生物量和L-Trp的累积浓度都不高。NH4+ 浓度在50mM和PO43-浓度在7.5g/L～15g/L时，发酵的色氨酸产量最高。同时我们也看到，NH4+ 对发酵最终的生物量和色氨酸产量影响比PO43-更大。
 在发酵罐上发酵时，我们进行了葡萄糖分批补料实验。葡萄糖的流加模式包括μset为0.10、0.15、0.20的指数流加和根据经验值脉冲流加。最终L-Trp的产量在8g/L～12g/L之间，相对于其他文献报道并不高。分析其主要的原因是发酵过程中累积了大量的乙酸，同时在发酵的稳定期内，L-Trp的浓度并未增加。所以减少乙酸累积和让大肠杆菌在稳定期继续产L-Trp成为研究的重点。乙酸一般都是在细菌比生长速率最快的时候开始累积。所以以后发酵过程中当细菌生长快接近稳定期的时候，流加的速度应该更加慢。

关键词：L-色氨酸；重组大肠杆菌；发酵；葡萄糖指数流加

Abstract: L-Trp is an essential amino acid, which is widely used in food supplement, medicine and feed-stocks. Annual demand in the world is more than 10,000 tons, but the actual capacity all over the world is only 3000 tons, which is far from satisfying the needs of market. In China, there is only small-scale production of L-Trp in Wuhan, Shanghai and Beijing, about 100 tons a year. This makes the application of L-Trp limit in the medicine. In this paper, we used a recombinant E.coli strain to develop a mode of glucose-feeding to enhance the production of L-Trp to a certain level
 Firstly, we studied the impact of glucose, NH4+ and PO43-concentrations on cell growth and L-Trp production. Result shows that, under the condition of high glucose concentration, the broth accumulates a lot of Acetic acid, which made cell and L-Trp concentration is low. 50mM NH4+ and 7.5g/L~15g/L PO43- made the fermentation process have the highest yield of L-Trp. At the same time we could see that NH4+ concentration have a stronger impact on the L-Trp production than PO43-.
 In the fermentor, we performed glucose-fed batch fermentation. Glucose fed mode includes the index fed of µset=0.10, 0.15, 0.20and pulse fed mode according to the experience. The final output of L-Trp is between 8g/L~11g/L, lower than data reported by others. The main reason for the low output is that the fermentation process accumulates excessive acetic acid and there is no production in the stability period. Therefore, to reduce the acetic acid accumulation and to allow E. coli produce L-Trp in the stable period has become the focus of future study. The time when the acetic acid began to accumulate generally is the time when bacteria has fastest growth rate. Therefore, it is necessary to reduce the fed rate of glucose, especially near the period of stability.
Key words：L-Tryptophan; recombinant Escherichia coli; fermentation; glucose index fed