跳到主要內容

臺灣博碩士論文加值系統

(3.235.56.11) 您好!臺灣時間:2021/07/29 04:04
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:林建鑫
研究生(外文):Jian-Sin Lin
論文名稱:篩選 ω-transaminase 俾應用於具光學活性去甲麻黃鹼之生合成
論文名稱(外文):Screening of ω-Transaminase for Optically Active Norephedrine Biosynthesis
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:100
語文別:中文
論文頁數:72
中文關鍵詞:轉氨酶去甲麻黃鹼轉胺作用生合成
外文關鍵詞:transaminaseNorephedrineTransaminationBiosynthesis
相關次數:
  • 被引用被引用:1
  • 點閱點閱:167
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
(1R, 2S)-norephedrine (去甲麻黃鹼) 是一種天然的生物鹼,在醫藥用途上屬於類交感神經製劑。除了藥用之外,(1R, 2S)-norephedrine可做為許多對掌化合物 (chiral compounds) 合成的起始原料。此外,(1R, 2S)-norephedrine 及其衍生物在許多不對稱合成中是相當重要的對掌輔助劑,參與 HIV 反轉錄酶抑制劑 Efavirenz 特定立體結構合成的關鍵步驟。因此,開發簡單又方便的新製程來合成具特定光學活性的 norephedrine 已為相關產業積極投入的目標。
本研究建立一新穎性之生合成產程,利用 (S)-selective ω-transaminase 將 L-phenylacetylcarbinol (L-PAC) 與 L-alanine 進行轉胺作用生成具高光學純度之 (1R, 2S)-norephedrine。藉由基因序列比對與轉換活性篩選,我們成功選殖到 3 個具有轉換生合成 (1R, 2S)-norephedrine 能力的ω-transaminase 基因,其基因來源分別為 Chromobacterium violaceum, Paracoccus denitrificans and Bradyrhizobium japonicum,其中帶有 C. violaceum ω-transaminase基因的轉型株 E. coli (pQE-CvTA) 轉換能力最強。以 E. coli (pQE-CvTA) 轉型株進行生物轉換條件的分析,發現當 L-PAC 與 L-alanine 的濃度比例為1:10 時轉換效率最好;而依此比例下,將菌體於pH 7,溫度 37 ℃ 中進行轉換反應,轉換產率最高可達 62.2%。轉型株菌體轉換能力於 35℃ 與 potassium phosphate (pH 6) 緩衝液條件下最為穩定,在此條件下放置 120 小時後,其相對轉換產率仍有 95.1%。在菌體再利用率上,回收菌體重複反應 20 個循環後,其相對轉換產率仍有 67.7 %。目前本實驗已成功建立 (1R, 2S)-norephedrine 的生合成系統,此為全球第一個實現 (1R, 2S)-norephedrine 的生合成技術,且反應產物不需再進行光學異構物的分離與純化,在工業應用上有相當的潛力。

(1R, 2S)-norephedrine is a naturally occurring alkaloid which is used as therapeutic sympathomimetic agents. In addition, (1R, 2S)-norephedrine is widely used as very
useful starting material for preparation of many important chiral compounds. Moreover, (1R, 2S)-norephedrine and its derivatives are of great importance as chiral auxiliaries in
a variety of asymmetric reaction, especially in the synthesis of the HIV reverse transcriptase inhibitor, Efavirenz. Accordingly, the development of a simple and
convenient synthetic method for the optically active norephedrine is of great interest.
In this study, a novel biocatalytic process for production of (1R, 2S)-norephedrine from L-phenylacetylcarbinol (L-PAC) and L-alanine has been developed by using (S)-selective ω-transaminase, which has never been reported in the world. Based on sequence alignment and activity study, we have successfully cloned the ω-transaminases from Chromobacterium violaceum, Paracoccus denitrificans and
Bradyrhizobium japonicum, they were verified to be a candidate for (1R, 2S)-norephedrine biosynthesis. Among them, the gene from C. violaceum showed the highest conversion activity and has been cloned and functionally expressed in E. coli [E. coli (pQE-CvTA)]. The highest conversion yield of 62.2% was obtained in 1 h incubation at pH 7 and 37℃, with the ratio of the amino acceptor (L-PAC) and donor (L-alanine) equals to 1:10. The E. coli (pQE-CvTA) transformant was more stable under the potassium phosphate (pH 6) at 35℃, after 120 h incubation the relative conversion yield was remained about 95%. The cells can be reused for at least 20 cycles with a relactive conversion yield of about 67.7%. This process has higher applied potential for optical pure (1R, 2S)-norephedrine biosynthesis.
摘要........................................................................................................................I
Abstract................................................................................................................ II
目錄.....................................................................................................................III
表目錄 .................................................................................................................. V
圖目錄 ................................................................................................................. VI
前言................................................................................................................................. 1
一、 轉胺酶簡介及其應用............................................................................... 1
二、 研究動機與策略...................................................................................... 4
材料與方法..................................................................................................................... 7
一、化學藥物、菌種、培養基............................................................................... 7
二、一般通用實驗方法.......................................................................................... 7
三、分析方法及條件.............................................................................................11
四、L-PAC 的生合成及回收................................................................................. 12
五、ω-Transaminase 基因選殖及與活性篩選 ...................................................... 13
六、以全細胞轉換 L-PAC 生產 (1R, 2S)-norephedrine 之分析......................... 14
結果............................................................................................................................... 18
一、反應受質與產物分析方法之建立................................................................. 18
二、L-PAC 生合成方法及純化 ............................................................................ 19
三、Transaminase 基因選殖與表現...................................................................... 20
四、Transaminase 活性篩選及酵素特性分析 ..................................................... 21
五、全細胞全細胞轉換 L-PAC 生產 (1R, 2S)-norephedrine .............................. 22
討論............................................................................................................................... 27
一、HPLC 分析方法建立.................................................................................... 27
二、L-PAC 生合成方法及純化............................................................................. 28
三、ω-Transaminase 基因選殖與表現.................................................................. 29 四、酵素特性分析................................................................................................ 30
參考文獻...................................................................................................................... 33
表目錄
表一、 本實驗所使用的菌株與質體..................................................................................... 38
表二、本研究所使用之引子................................................................................................... 40
表三、微生物 transaminase 的特性 ...................................................................................... 42
表四、Vibrio fluvialis JS17 ω-transaminase 胺基酸序列比對分析表 .......................... 43
表五、轉型株活性分析 ....................................................................................................... 44
表六、胺基提供者特異性分析........................................................................................... 45
表 七 、 分 析 利 用 E. coli (pQE-CvTA) 轉 型 株 做 為 生 物 催 化 劑 生 產 (1R,
2S)-norephedrine ............................................................................................................ 46

圖目錄
圖一、Transminase 的作用機制圖。..................................................................................... 47
圖二、Phenylpropanolamine (PPA) 的四種光學異構物。............................................. 48
圖三、(1R, 2S)-norephedrine 的化學合成流程。 ........................................................... 49
圖四、利用 (S)-selective ω-transaminase 生合成 (1R, 2S)-norephedrine 之流程。 50
圖五、不同菌株中 ω-transaminase 的樹枝狀演化關係圖。 ....................................... 51
圖六、LC/MS/MS 分析 (1R, 2S)-norephedrine 及利用轉型株將 L-PAC 轉化生成
(1R, 2S)- norephedrine 之結果。................................................................................ 52
圖七、利 Vercopak inertsil 10 ODS (3.2 × 250 mm) 管柱分析 (1R, 2S)-norephedrine
與 L-PAC。 ................................................................................................................... 53
圖八、利用 Vercopak inertsil 10 ODS (3.2 × 250 mm) 管柱分析 benzylamine、(1R,
2S)-norephedrine 與 L-PAC。.................................................................................... 54
圖九、利用 Vercopak inertsil 10 ODS (3.2 × 250 mm) 管柱分析 benzylamine、(1R,
2S)-norephedrine 與 L-PAC。.................................................................................... 55
圖十、利用 Vercopak inertsil 10 ODS (3.2 × 250 mm) 管柱分析 norephedrine 與
norpseudoephedrine。................................................................................................... 56
圖十一、利用 Crownpak CR (+) column 分析 (1R, 2S)-norephedrine 與 (1S, 2R)-
norephedrine 標準品及利用轉型株將 L-PAC 轉化生成 (1R, 2S)- norephedrine
的光學活性。 ................................................................................................................ 57
圖十二、HPLC 分析利用 AccuBond II SPE ODS-C18 Cartridges 分離 L-PAC 反應液
中的 DMSO 及 L-PAC。........................................................................................... 58
圖十三、質體 pQE-CvTA 的構築 (A) 及以 SDS-PAGE 分析蛋白表現 (B)。..... 59
圖十四、質體 pQE-PdTA 的構築 (A) 及以 SDS-PAGE 分析蛋白表現 (B)。 ..... 60
圖十五、質體 pQE-BjTA 的構築 (A) 及以 SDS-PAGE 分析蛋白表現 (B)。...... 61
圖十六、利用 E. coli (pQE-COBN) 轉形株作為生物催化劑生產 L-PAC。............ 62
圖十七、L-Alanine 濃度對於 E.coli (pQE-CvTA) 轉換率之影響。.......................... 63 圖十八、L-PAC 濃度對於 E.coli (pQE-CvTA) 轉換率之影響。 ............................... 64
圖十九、(1R, 2S)–norephedrine 濃度對於 E.coli (pQE-CvTA) 轉換率之影響。..... 65
圖二十、Pyruvate 濃度對於 E.coli (pQE-CvTA) 轉換率之影響。............................ 66
圖二十一、pH 值對於 E.coli (pQE-CvTA) 轉換率之影響。...................................... 67
圖二十二、pH 值對於 E. coli (pQE-CvTA) 穩定性之影響。..................................... 68
圖二十三、不同溫度對於 E.coli (pQE-CvTA) 轉換率之影響。................................. 69
圖二十四、溫度對於 E. coli (pQE-CvTA) 穩定性之影響。........................................ 70
圖 二 十 五 、 利 用 E. coli (pQE-CvTA) 轉 型 株 做 為 生 物 催 化 劑 生 產 (1R,
2S)-norephedrin。.......................................................................................................... 71
圖二十六、E.coli (pQE- CvTA) 轉形株之菌體再利用率。.......................................... 72
參考文獻
1. Stirling, D.I. 1992. ChemInform Abstract: The Use of Aminotransferases for the Production of Chiral Amino Acids and Amines. Wiley.
2. Chao, Y.P., Lai, Z. J.,Chen, P.,Chern, J. T. 1999. Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12. Biotechnol. Prog. 15(3): p. 453-458.
3. Tufvesson, P., J. Lima-Ramos, J.S. Jensen, N. Al-Haque, W. Neto, and J.M. Woodley. 2011. Process considerations for the asymmetric synthesis of chiral amines using transaminases. Biotechnol. Bioeng. 108(7): p. 1479-1493.
4. Taylor, P.P., D.P. Pantaleone, R.F. Senkpeil, and I.G. Fotheringham. 1998. Novel biosynthetic approaches to the production of unnatural amino acids using transaminases. Trends Biotechnol. 16(10): p. 412-418.
5. D.J. Ager, G.F. 2001. Methods for the synthesis of unnatural amino acids. Curr. Opin. Drug Discov. Dev. (4): p. 800-807.
6. Stewart, J.D. 2001. Dehydrogenases and transaminases in asymmetric synthesis. Curr. Opin. Chem. Biol. (5): p. 120-129.
7. Patel, R.N. 2008. Synthesis of chiral pharmaceutical intermediates by biocatalysis. Coord. Chem. Rev. 252 659–701: p. 659-701.
8. Kaulmann U., S.K., Smith MEB., HaileS HC., Ward JM. 2007. Substrate spectrum of omega-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis. Enzyme Microb. Technol. 41(5): p. 628-637.
9. Park, E.S., M. Kim, and J.S. Shin. 2012. Molecular determinants for substrate selectivity of omega-transaminases. Appl. Microbiol. Biotechnol. 93(6): p. 2425-2435.
10. Alexander, F.W., E. Sandmeier, P.K. Mehta, and P. Christen. 1994. Evolutionary relationships among pyridoxal-5'-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families. Eur. J. Biochem. 219(3): p. 953-960.
11. Bum-Yeol Hwang, B.-K.C., Hyungdon Yun, Kinera Koteshwar, and Byung-Gee Kim. 2005. Revisit of aminotransferase in the genomic era and its application to biocatalysis. J. Mol. Catal. B: Enzym. (37): p. 47-55.
12. Hwang. B. Y., K., S. H., Park, H. Y., Seo, J. H., Lee, B. S., and Kim, B. G. 2008. Identification of omega-aminotransferase from Caulobacter crescentus and site-directed mutagenesis to broaden substrate specificity. J. Microbiol. Biotechnol. 18(1): p. 48-54.
13. Owen, O.E., S.C. Kalhan, and R.W. Hanson. 2002. The key role of anaplerosis and cataplerosis for citric acid cycle function. J. Biol. Chem. 277(34): p. 30409-30412.
14. Chao, Y., T. Lo, and N. Luo. 2000. Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme Microb. Technol. 27(1-2): p. 19-25.
15. Xu, H., P. Wei, H. Zhou, W.P. Fan, and P.K. Ouyang. 2003. Efficient production of L-phenylalanine catalyzed by a coupled enzymatic system of transaminase and aspartase. Enzyme Microb. Technol. 33(5): p. 537-543.
16. Cho, B.K., J.H. Seo, T.W. Kang, and B.G. Kim. 2003. Asymmetric synthesis of L-homophenylalanine by equilibrium-shift using recombinant aromatic L-amino acid transaminase. Biotechnol. Bioeng. 83(2): p. 226-234.
17. Krapcho, J., C. Turk, D.W. Cushman, J.R. Powell, J.M. DeForrest, E.R. Spitzmiller, D.S. Karanewsky, M. Duggan, G. Rovnyak and J. Schwartz. 1988. Angiotensin-converting enzyme inhibitors. Mercaptan, carboxyalkyl dipeptide, and phosphinic acid inhibitors incorporating 4-substituted prolines. J. Med. Chem. 31(6): p. 1148-1160.
18. Li, T., A.B. Kootstra, and I.G. Fotheringham. 2002. Nonproteinogenic alpha-amino acid preparation using equilibrium shifted transamination. Org. Process Res. Dev. 6(4): p. 533-538.
19. Gollapudy, S. and S.V. JOSHI. 2005. Process for preparation of optically active 1-erythro-2-amino-1- phenyl-1-propanol. patent WO/2005/100299.
20. Blumberg, P.M. and J.L. Strominger. 1974. Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes. Bacteriol. Rev. 38(3): p. 291-335.
21. Yoshimura, T., K. Nishimura, J. Ito, N. Esakik, H. Kagamiyama, J.M. Manning, and K. Soda. 1993. Unique stereospecificity of D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase for C-4' hydrogen transfer of the coenzyme. J. Am. Chem. Soc. 115: p. 3897-3900.
22. Tanizawa, K., Y. Masu, S. Asano, H. Tanaka, and K. Soda. 1989. Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination. J. Biol. Chem. 264(5): p. 2445-2449.
23. Bae, H.S., S.P. Hong, S.G. Lee, M.S. Kwak, N. Esaki, and M.H. Sung. 2002. Application of a thermostable glutamate racemase from Bacillus sp SK-1 for the production of D-phenylalanine in a multi-enzyme system. J. Mol. Catal. B-Enzym. 17(6): p. 223-233.
24. Koszelewski, D., K. Tauber, K. Faber, and W. Kroutil. 2010. omega-Transaminases for the synthesis of non-racemic alpha-chiral primary amines. Trends Biotechnol. 28(6): p. 324-332.
25. Stirling, D.I., A.L. Zeitlin, and G.W. Matcham. 1990. Enantiomeric enrichment and stereoselective synthesis of chiral amines. patent US4950606.
26. Malik, M.S., E.S. Park, and J.S. Shin. 2012. Features and technical applications of omega-transaminases. Appl. Microbiol. Biotechnol. 94(5): p. 1163-1171.
27. Shin. J. S., Y.H., Jang. J. W., Park. I., Kim. B. G. 2003. Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17. Appl. Microbiol. Biotechnol. 61(5-6): p. 463-471.
28. Park, E.S. and J.S. Shin. 2011. Free energy analysis of omega-transaminase reactions to dissect how the enzyme controls the substrate selectivity. Enzyme Microb. Technol. 49(4): p. 380-387.
29. Ito, N., S. Kawano, J. Hasegawa, and Y. Yasohara. 2011. Purification and characterization of a novel (S)-enantioselective transaminase from Pseudomonas fluorescens KNK08-18 for the synthesis of optically active amines. Biosci. Biotechnol. Biochem. 75(11): p. 2093-2098.
30. Park, E., M. Kim, and J.-S. Shin. 2010. One-pot conversion of L-threonine into L-homoalanine: biocatalytic production of an unnatural amino acid from a natural one. Adv. Syn. Catal.. 352(18): p. 3391-3398.
31. Koszelewski, D., D. Pressnitz, D. Clay, and W. Kroutil. 2009. Deracemization of mexiletine biocatalyzed by omega-transaminases. Org. Lett. 11(21): p. 4810-4812.
32. Savile, C.K., J.M. Janey, E.C. Mundorff, J.C. Moore, S. Tam, W.R. Jarvis, J.C. Colbeck, A. Krebber, F.J. Fleitz, J. Brands, P.N. Devine, G.W. Huisman, and G.J. Hughes. 2010. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science. 329(5989): p. 305-309.
33. Grue-Sorensen, G. and I.D. Spenser. 1994. Biosynthetic route to the Ephedra alkaloids. J. Am. Chem. Soc. 116: p. 6195-6200.
34. Engel, S., M. Vyazmensky, S. Geresh, Z. Barak, and D.M. Chipman. 2003. Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol. Biotechnol. Bioeng. 83(7): p. 833-840.
35. 陳怡妏. 2010. 以具有乙醯羥酸合成酶 I 活性之全細胞生物轉換系統應用於 L-苯基乙酰基甲醇的生合成。碩士論文,生物科技研究所,弘光科技大學,台中。
36. Koszelewski, D., M. Goritzer, D. Clay, B. Seisser, and W. Kroutil. 2010. Synthesis of optically active amines employing recombinant omega-transaminases in E. coli cells. Chemcatchem. 2(1): p. 73-77.
37. Mandwal, A.K., C.K. Tripathi, P.D. Trivedi, A.K. Joshi, S.C. Agarwal, and V. Bihari. 2004. Production of L-phenylacetyl carbinol by immobilized cells of Saccharomyces cerevisiae. Biotechnol. Lett. 26(3): p. 217-221.
38. Nishiyama, A., N. Kishimoto, and N. Nagashima. 2008. Process for producing optically active β-amino alcohol. patent US7408084.
39. Ellaiah, P. and K.T. Krishna. 1987. Studies on the production of phenyl acetyl carbinol from benzaldehyde by Saccharomyces cerevisiae. Indian Drugs. 24: p. 192-195.
40. Satianegara, G., M. Breuer, B. Hauer, P.L. Rogers, and B. Rosche. 2006. Enzymatic (R)-phenylacetylcarbinol production in a benzaldehyde emulsion system with Candida utilis cells. Appl. Microbiol. Biotechnol. 70(2): p. 170-5.
41. Vinogradov, V., M. Vyazmensky, S. Engel, I. Belenky, A. Kaplun, O. Kryukov, Z. Barak, and D.M. Chipman. 2006. Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties. Biochim. Biophys. Acta. 1760(3): p. 356-363.
42. Long, A., P. James, and O.P. Ward. 1989. Aromatic aldehydes as substrates for yeast and yeast alcohol dehydrogenase. Biotechnol. Bioeng. 33(5): p. 657-660.
43. Dan, L., L. Yan-liang, L. Hui-bin, and L. Jian-qiang. 2009. Biosynthesis, separation, purification and identification of L-phenylacetyl carbinol. J. Food Sci. Biotechnol. 28(5): p. 716-720.
44. Shin, J.S. and B.G. Kim. 2002. Substrate inhibition mode of omega-transaminase from Vibrio fluvialis JS17 is dependent on the chirality of substrate. Biotechnol. Bioeng. 77(7): p. 832-837.
45. Shin, J.S., B.G. Kim, and D.H. Shin. 2001. Kinetic resolution of chiral amines using packed-bed reactor. Enzyme Microb. Technol. 29(4-5): p. 232-239.
46. Schell, U., R. Wohlgemuth, and J.M. Ward. 2009. Synthesis of pyridoxamine 5'-phosphate using an MBA: pyruvate transaminase as biocatalyst. J. Mol. Catal. B-Enzym. 59(4): p. 279-285.


連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top