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研究生:詹馨嬅
研究生(外文):Hsin-Hua Chan
論文名稱:篩選具轉換生成度洛西汀藥物光學活性中間體之菌株
論文名稱(外文):Screening of microorganisms in biosynthesis of optically active intermediate for Duloxetine
指導教授:高肇鴻
指導教授(外文):Chao-Hung Kao
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:101
語文別:中文
論文頁數:66
中文關鍵詞:度洛西汀生合成(S)-對映體生物還原
外文關鍵詞:Duloxetinebiosynthesis(S)-enantiomerbioreduction
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度洛西汀 (Duloxetine,Cymbalta®) 為新一代暢銷的抗憂鬱藥物,用於 5-羥色胺和去甲腎上腺素雙重的再攝取抑制劑。此藥物也通過驗證可用於許多精神及代謝相關疾病的治療藥物如:糖尿病周邊神經痛、纖維肌痛、壓力性尿失禁、重度憂鬱症及廣泛性焦慮症等疾病。Duloxetine 包含有一種立體結構,僅有 (S)-對映體,具有藥學活性。因此,開發其特定光學活性 (S)-對映體的新製程具有高度的市場價值。本研究開發一新穎性生合成流程,利用全細胞催化不對稱還原 A 製備 (S)-Duloxetine 的關鍵藥物中間體 (S)-B。利用 HPLC 進行篩選,我們成功的篩選到 5 株 N422 菌屬對於 A 具有不對稱還原的能力。其中 N422-5 的轉換率較強且具有高度立體選擇性。將回收的菌體於 30℃,pH8.5 的條件下反應 72 小時,其最高轉換率可達約 1.3%。根據以發表的基因體序列,在 N422-5、N422-1 和 N422-9 中也成功選殖到 21 組可能參與此還原反應的酵素基因並表現於 Escherichia coli。其中 n42242、n42237 與 n42285 這三組序列具有同源性,皆具有將 A 專一性還原為 (S)-B 的能力,目前轉換率最高約為 2.6%。本研究已成功篩選到 N422 菌株可以在生物還原反應的過程中作為生物催化劑。另外,也選殖到可能參與 A 還原成 (S)-B 反應的酵素基因。未來將進一步探討如何改善其轉換效率以提升其在工業上應用的價值。
Duloxetine is a new-generation popular antidepressant drug; it is a dual inhibitor of serotonin and norepinephrine reuptake. It has been approved for psychiatric and metabolic conditions, such as diabetic peripheral neuropathic pain, fibromyalgia, stress urinary incontinence, major depressive disorder, and generalized anxiety disorder. Duloxetine contains 1 chiral center and only the (S)-enantiomer is pharmaceutically active. Therefore, development of its specific optically active (S)-enantiomer by using a new process has high market value. In this study, a novel bioreduction process for the production of optically active (S)-B, a chiral intermediate of duloxetine, from A has been developed. Using HPLC screening methods, we successfully screened five N422 strains that showed A-reduction activity. Among them, N422-5 showed the highest reduction activity and good stereoselectivity. Under the reaction conditions used, the highest conversion yield of 1.3% was obtained when the cells collected were incubated at pH 8.5 and 30°C with 10 mM A for 72 h. On the basis of the sequenced genomes, 21 putative reductase genes from N422-5, N422-1, and N422-9 were cloned and expressed in Escherichia coli. Of these, the n42242, n42237, and n42285 genes showed sequence homology and possessed enzymatic activity for converting A to (S)-B with a conversion yield of approximately 2.6%. In conclusion, we successfully screened N422 strains that could act as a whole-cell biocatalyst for the bioreduction process. Moreover, genes that might be involved in the bioreduction of A to (S)-B were also cloned. Further research on enhancing the conversion yield is important for improving the industrial application of this bioreduction process.
摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
前言 1
一、 憂鬱症及其藥物 Duloxetine 1
二、 (S)-Duloxetine 現今資源及合成方法 2
三、 Carbonyl reductases 簡介及其應用 4
四、 研究動機與策略 7
材料與方法 9
一、 化學藥品、菌種及培養基 9
二、 一般實驗通用方法 9
三、 Carbonyl reductase 活性篩選 12
四、 全細胞轉換A生產(S)-B 13
結果 18
一、 反應受質與產物分析方法之建立 18
二、 具 A 還原能力之微生物菌株篩選 18
三、 利用全細胞轉換 A 生產 (S)-B 19
四、 Carbonyl reductase 選殖、表現與活性分析 23
討論 26
一、 分析方法之建立 26
二、 具 A 還原能力之微生物菌株篩選 26
三、 酵素特性分析 27
四、 Carbonyl reductase 選殖 27
結論 30
參考文獻 31
表目錄
表一、本研究所使用菌株及質體 37
表二、以微生物全菌體 A 的生物還原反應 41
表三、輔因子對N422-5生物還原反應活性的影響 44
表四、不同誘導劑對N422-5轉換A為B的影響 45
表五、以表現 sdr 基因之 E.coli 全菌體進行 A 之生物轉換 46
表六、以表現 N422-5之 carbonyl reductase 基因選殖於 E.coli 全菌體進行 A 之生物轉換 47
表七、以表現 N422-1之 carbonyl reductase 基因選殖於 E.coli 全菌體進行 A 之生物轉換 48
表八、以表現 N422-8 和 N422-9 之 carbonyl reductase 基因選殖於 E.coli 全菌體進行 A 之生物轉換 49
圖目錄
圖一、禮來公司公佈2007-2012年度業績。 50
圖二、(S)-度洛西汀的化學合成法。 51
圖三、HPLC (C18 column) 分析不同濃度的 A 標準品,依據每個濃度所屬的 peak 面積製作 A 標準檢量線。 52
圖四、HPLC (C18 column) 分析不同濃度的 B 標準品,依據每個濃度所屬的 peak 面積製作 B 標準檢量線。 53
圖五、HPLC (C18 column) 分析 A 與 B。 54
圖六、HPLC (Chiral OJ-H column) 分析 A 與 B。 55
圖七、N422-5生長階段對轉換能力之影響。 56
圖八、N422-5菌體濃度對轉換能力之影響。 57
圖九、A 的濃度對於 N422-5對轉換能力 (A) 和轉換率 (B) 的影響。 58
圖十、Glucose濃度對於對轉換能力的影響。 59
圖十一、(±)-B 於不同溫度 (A) 及於 N422-5 菌體中不同時間 (B) 的穩定性。 60
圖十二、不同濃度的 (S)-B 對於 N422-5 轉換能力的影響。 61
圖十三、pH 值對於 N422-5 對轉換能力之影響。 62
圖十四、溫度對於 N422-5對轉換能力之影響。 63
圖十五、本研究中用來當誘導劑的化合物。 64
圖十六、利用 N422-5做為生物催化劑生產 (S)-B。 65
圖十七、以SDS-PAGE分析carbonyl reductase基因經過IPTG誘導的E.coli NovaBlue 66



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