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  • 學位論文

遺傳工程Escherichia coli BL21(DE3)W/ pET20-LacZ連續式醱酵特性及代謝物之探討

Studies on the Fermentation Properties and the Metabolic products during continuous Fermentation by Escherichia coli BL21(DE3)W/ pET20-LacZ

指導教授 : 段國仁
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摘要


本研究探討E. coli BL21(DE3)及遺傳工程E. coli BL21(DE3)w/ pET20-LacZ的連續培養中胞內胞外的中間代謝產物,以了解其醱酵代謝碳源之能量流。在四種不同的稀釋率(0.57,0.4,0.23,0.13 h-1)之下。就E. coli BL21(DE3)及遺傳工程E. coli BL21(DE3)w/ pET20-LacZ之間,相同的稀釋率E. coli BL21(DE3)的菌體濃度及殘餘葡萄糖濃度比E. coli BL21(DE3)w/ pET20-LacZ高,可能是E. coli BL21(DE3)w/ pET20-LacZ生長需要負擔plasmid所需的能量,因此需要較多的碳源,也因此菌體濃度也較E. coli BL21(DE3)低。就質體穩定度的測試,將E. coli BL21(DE3)w/ pET20-LacZ培養在含有ampicillin 的M9培養基中,培養4倍體積後,改用不含ampicillin 的M9培養基進行連續式培養25倍體積,發現其質體穩定性相當高。以0.57 h-1和0.4 h-1的稀釋率來培養E. coli BL21(DE3)w/ pET20-LacZ並在穩定後4倍體積加入IPTG進行誘導生產酵素並比較沒有加入IPTG誘導時的菌體濃度及殘餘葡萄糖濃度,稀釋率0.4 h-1時加入IPTG的菌體濃度分別為從OD600 3.14降為3.02。而稀釋率0.57 h-1時加入IPTG使菌體濃度從2.85降為2.12。加入IPTG後,菌體因為要負擔一些蛋白質表現所需要的能量,而使生長速率下降。有機酸代謝中,在糖解(glycolysis)和檸檬酸循環(TCA cycle)的中間代謝物,胞內及胞外oxaloacetate的累積,隨著稀釋率的提高而累積量增加,推測OAA的累積是因為能量過剩所造成的一種形式的累積。胺基酸代謝中,細胞主要利用的胺基酸是來自培養基,而Phenylalanine、Valine、Serine、Threonine是因為培養基裡缺乏,因此需要自行合成,隨著稀釋率的提高,胞外的胺基酸會被菌體大量直接攝取利用,這在Tryptophan、Alanine、Tyrosine、Histidine更為明顯。

並列摘要


The present study employed E. coli BL21 (DE3) and recombinant E. coli BL21 (DE3) w/pET20-LacZ to investigate extra-cellular and intra-cellular metabolic products in the continuous fermentation. The results could be used for understanding the metabolic flux of nutrients in the medium. Four dilution rates (0.57, 0.4, 0.23, 0.13 h-1) were performed in the continuous fermentation. The results revealed that glucose consumption rates (g/DCW/h) in the steady state for E. coli BL21 (DE3) w/pET20-LacZ were higher than those for E. coli BL21 (DE3). This indicated recombinant E. coli BL21 (DE3) w/pET20-LacZ consumed more glucose because of the burden of the plasmid. The plasmid of E. coli BL21 (DE3) w/pET20-LacZ was stable after performing continuous fermentation for 25 working volume without ampicillin. IPTG was introduced in the steady state of the continuous fermentation for E. coli BL21 (DE3) w/pET20-LacZ. The cell densities in terms of OD600 decreased from 2.85 and 3.14 to 2.12 and 3.02 respectively for the dilution rate of 0.4 and 0.57. The results revealed that the specific growth rate decreased because of nutrient was consumed for the production of recombinant protein. Metabolites from glycolysis and TCA cycle are analysed. Accumulation of oxaloacetate in-cell and medium at high dilution rate suggest over-supply of nutrient. For the amino acids, large amount of tryptophan, alanine, tyrosine, and histidine were consumed from the medium. However, the cell needed to convert nutrients to phenylalanine, valine, serine, and threonine that were not exited in the medium.

並列關鍵字

continuous fermentation E. coli

參考文獻


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