研究生: |
葛麗絲 Grace-Marlina Winata |
---|---|
論文名稱: |
Expression of 5-Aminolevulinic Acid Synthase (ALAS) for 5-Aminolevulinic Acid (ALA) Production in Recombinant Escherichia coli Expression of 5-Aminolevulinic Acid Synthase (ALAS) for 5-Aminoelvulinic Acid (ALA) Production in Recombinant Escherichia coli |
指導教授: |
李振綱
Cheng-Kang Lee |
口試委員: |
陳秀美
Chen, Hsiu-Mei 陳志成 C-Will Chen |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2006 |
畢業學年度: | 94 |
語文別: | 英文 |
論文頁數: | 120 |
中文關鍵詞: | 5-Aminolevulinic acid synthase |
外文關鍵詞: | 5-Aminolevulinic acid synthase |
相關次數: | 點閱:209 下載:2 |
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5-Aminolevulinic acid (ALA) is the precursor of protoporphyrin IX (PP IX) in the heme biosynthesis pathway and has been suggested for photodiagnosis and photodynamic therapy of epithelial cancer. The Rhodobacter sphaeroides hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned into the expression vector pWHmA and transformed into Escherichia coli BL21(DE3) for its heterologous expression under the control of Bacillus xylA promoter. By introducing sopABC gene into pWHmA to make the expression plasmid become more stable, pGHmA plasmid was generated so that higher ALAS expression level and higher amount of ALA production was achieved. With the expression of ALAS and addition of glycine and succinate as substrates, the recombinant E. coli could produce ALA extracellularly. In shaker-flask cultures at 37oC for 36 h with addition of D(+)-xylose as inducer enhanced the ALA concentration produced.
The expression level of ALAS under the control of xylA promoter was also compared with that under the control of T7 promoter. It was found that the basal expression of ALAS driven by xylA promoter is higher than that of T7 promoter and with no inclusion body formation based on Western Blot analysis.
It was observed that recombinant E. coli BL21(DE3) harboring pGHmA cultivated under dark condition, with the repeated addition of ALA precursors, succinate (90 mM) and glycine (30 mM), and LA (inhibititor of ALA dehydratase (ALAD) in heme biosynthesis), can increase the ALA accumulation to 15.5 mM. On the other hand, in the culture under light illumination, the more ALA produced was converted into prophyrins and resulted in a lower ALA concentration. Besides, the cell concentration obtained was also lower in the light culture.
5-Aminolevulinic acid (ALA) is the precursor of protoporphyrin IX (PP IX) in the heme biosynthesis pathway and has been suggested for photodiagnosis and photodynamic therapy of epithelial cancer. The Rhodobacter sphaeroides hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned into the expression vector pWHmA and transformed into Escherichia coli BL21(DE3) for its heterologous expression under the control of Bacillus xylA promoter. By introducing sopABC gene into pWHmA to make the expression plasmid become more stable, pGHmA plasmid was generated so that higher ALAS expression level and higher amount of ALA production was achieved. With the expression of ALAS and addition of glycine and succinate as substrates, the recombinant E. coli could produce ALA extracellularly. In shaker-flask cultures at 37oC for 36 h with addition of D(+)-xylose as inducer enhanced the ALA concentration produced.
The expression level of ALAS under the control of xylA promoter was also compared with that under the control of T7 promoter. It was found that the basal expression of ALAS driven by xylA promoter is higher than that of T7 promoter and with no inclusion body formation based on Western Blot analysis.
It was observed that recombinant E. coli BL21(DE3) harboring pGHmA cultivated under dark condition, with the repeated addition of ALA precursors, succinate (90 mM) and glycine (30 mM), and LA (inhibititor of ALA dehydratase (ALAD) in heme biosynthesis), can increase the ALA accumulation to 15.5 mM. On the other hand, in the culture under light illumination, the more ALA produced was converted into prophyrins and resulted in a lower ALA concentration. Besides, the cell concentration obtained was also lower in the light culture.
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