臺灣博碩士論文加值系統

　　(1R, 2S)-Norephedrine 是一種天然的生物鹼，在醫學上用作於類交感神經的藥物，並且可以做為治療狗的尿失禁藥物，其衍生物可以做為 HIV 病毒的反轉錄抑制劑 Efavirenz 的合成以及開發中藥物 Mivobulin isethionate 的合成上，因此我們想要以簡單的生物合成方式來生成具特定光學活性的 (1R, 2S)-norephedrine。於先前的研究中本實驗室已由 Chromobacterium violaceum 菌種中篩選到兩個具有轉換能力之基因，並將其構築於 E. coli 表現系統中；然而，研究發現此基因表現之重組酵素於轉換過程中會受到產物 (1R, 2S)-norephedrine 的影響而使轉換活性受限。因此，本實驗利用蛋白質結構模擬 (protein structure modeling) 及蛋白質對接 (protein docking) 等方式，來預測可能參與活性反應或產物抑制之重要胺基酸殘基 (residues)，再輔以突變的方式試圖改善這個問題。
　　利用上述方式我們已由 C. violaceum (Cv) 之 ω-transaminase (ω-TA) 蛋白質中找出預測可能可以增加酵素活性或減少產物抑制性的胺基酸殘基，並建立兩種快速篩選的方法來篩選具有活性的菌株，分別為 TTC assay 及薄層色譜法。由結構模擬的結果中發現發現產物相互作用並可能影響轉換活性的胺基酸殘基，分別為 Cv W188、Cv E372 及 Cv L393。在活性提升部分，發現 Cv ω-TA 的突變株以 Cv D372E 及 Cv L393M 的轉換率較高，轉換率分別為 55.4% 及 54.0%。在產物抑制改善部分，Cv ω-TA 的突變株中以 Cv L393M 改善最多，IC50 從11.3 mM 提高為 16.2 mM。從上述結果得知，本實驗成功選殖不同的突變株，且具改善產物抑制的問題，期望對未來的研究有所幫助。

　　(1R, 2S)-Norephedrine is a naturally occurring alkaloid which is used as sympathomimetic drugs. The pharmacological can be treatment of urinary incontinence in dogs. Moreover, (1R, 2S)-norephedrine and its derivatives are of great significance as chiral auxiliaries in a variety of asymmetric reaction, especially in the synthesis of the HIV reverse transcriptase inhibitor, Efavirenz, and development of antineoplastic agents, Mivobulin isethionate. Accordingly, we want to develop a simple and convenient biosynthetic method for the optically active norephedrine that is meaningfully. According to previous research, we found two enzyme which had conversion acticity from Chromobacterium violaceum, and we found that (1R, 2S)-norephedrine were limited conversion activity. We used the way of protein structure and protein docking to predicting amino acid residues that may be involved in an active reaction or product inhibitiong, and used mutation to improve this problem.
　　Using the above method, we have found amino acid residues predicted from the ω-transaminase (ω-TA) of C. violaceum (Cv) which may increase the activity of the enzyme or decrease the product inhibiton, and two high-throughput screen methods, TTC assay and thin layer chromatography were searched active strains. From the results of structural modeling, it was found that the amino acid residues, Cv W188, Cv D372, and Cv L393 will interact with the product and which may affect the conversion activity. In the part of increased the conversion activity, the mutant of Cv ω-TA had higher conversion rate of Cv D372E and Cv L393M, the conversion rate was 55.4% and 54.0%, respectively. And in the part of improved product inhibition, Cv ω-TA mutant strain showed the most improvement with Cv L393M, IC50 value from 11.3 mM to 16.2 mM. It was concluded that the mutant strains were successfully cloned, which could improve the inhibition of the products, and found that the binding position of the non-competitive products was inhibited. It is expected to be helpful to the future research.

摘要 I
Abstract II
表目錄 V
圖目錄 VI
前言 1
一、Norephedrine 的簡介 1
二、(1R, 2S)-Norephedrine 的應用與市場 2
三、(1R, 2S)-Norephedrine 合成方法 3
四、轉胺酶簡介及其應用 5
五、蛋白質結構 8
六、研究動機 9
材料與方法 11
一、化學藥物、菌種、培養基 11
二、一般通用實驗方法 11
三、分析方法及條件 15
四、L-PAC的生合成及回收 15
五、ω-transaminase突變基因選殖及活性篩選 16
六、以全細胞轉換 L-PAC 生產 (1R, 2S)-norephedrine之分析 17
七. 純化 (1R, 2S)-norephedrine 18
結果 19
一、ω-transaminase 蛋白質結構模擬及突變點篩選 19
二、ω-transaminase 活性篩選及酵素特性分析 20
三、產物濃度對菌體轉換能力之影響 22
四、純化 (1R, 2S)-norephedrine 22
討論 24
一、ω-transaminase 蛋白質結構模擬及突變株篩選 24
二、ω-transaminase 突變株活性快速篩選方法 25
三、產物濃度對菌體轉換活性之影響 26
四、純化 (1R, 2S)-norephedrine 27
結論 28
參考文獻 29

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