重組大腸桿菌產L-色氨酸發酵生產工藝

生物工程論文編號:SW139  論文字數:12151,頁數:37

目  錄
中文摘要 I
英文摘要 III
目錄 Ⅴ
1. 緒論 1
 1.1 L-TRP的理化性質 1
 1.2 L-TRP的用途 1
 1.3 L-TRP的測定方法 2 
 1.4 L-TRP的生產方法 2
 1.5 L-TRP的代謝途徑和代謝工程的應用 4
 1.6 L-TRP的發酵 5
 1.6 重組大腸桿菌的高密度發酵 6
2. 批次發酵實驗部分 6
 2.1 材料和設備  6
 2.2 實驗內容和實驗方法 7
 2.3 實驗結果和討論 9
3. 批次流加發酵實驗部分 13
 3.1 材料和器材   13
 3.2 實驗內容和實驗方法   15
 3.3實驗結果和討論 19
4．總結與展望 31
致謝 32
參考文獻 33

摘 要：L-色氨酸（L-Trp）是一種人體必需的氨基酸，在食品添加劑、醫藥和飼料添加劑方面有著廣泛的應用。全世界的年需求量在一萬噸以上，但是實際的產能只有三千噸，遠遠沒有滿足市場的需求。在國內，只有武漢上海北京三地有小規模生產L-Trp的廠家，年產量也只有一百噸左右，因此L-Trp的應用被限制在了醫藥方面。本文利用產L-Trp的重組大腸桿菌菌株，主要進行發酵流加葡萄糖模式的探索，以期能夠提高L-Trp產量到一定的水平。
 首先，我們利用搖瓶發酵的方式考察了葡萄糖濃度、NH4+ 和PO43-濃度對菌體生長和L-Trp的累積的影響。結果表明，在高濃度的葡萄糖條件下，發酵液中累積大量的乙酸，并且菌體生物量和L-Trp的累積濃度都不高。NH4+ 濃度在50mM和PO43-濃度在7.5g/L～15g/L時，發酵的色氨酸產量最高。同時我們也看到，NH4+ 對發酵最終的生物量和色氨酸產量影響比PO43-更大。
 在發酵罐上發酵時，我們進行了葡萄糖分批補料實驗。葡萄糖的流加模式包括μset為0.10、0.15、0.20的指數流加和根據經驗值脈沖流加。最終L-Trp的產量在8g/L～12g/L之間，相對于其他文獻報道并不高。分析其主要的原因是發酵過程中累積了大量的乙酸，同時在發酵的穩定期內，L-Trp的濃度并未增加。所以減少乙酸累積和讓大腸桿菌在穩定期繼續產L-Trp成為研究的重點。乙酸一般都是在細菌比生長速率最快的時候開始累積。所以以后發酵過程中當細菌生長快接近穩定期的時候，流加的速度應該更加慢。

關鍵詞：L-色氨酸；重組大腸桿菌；發酵；葡萄糖指數流加

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