跳到主要內容

臺灣博碩士論文加值系統

(3.236.23.193) 您好!臺灣時間:2021/07/26 07:45
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:陳建宇
研究生(外文):Chien-Yu Chen
論文名稱:Rhodococcusspp.aminoketonereductase之基因選殖、特性分析及其應用於phenylephrine之生產
論文名稱(外文):Cloning and characterization of aminoketone reductase from Rhodococcus spp. and its application to phenylephrine production
指導教授:許文輝許文輝引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:47
中文關鍵詞:酮基還原酵素生物轉換麻黃素phenylephrine
外文關鍵詞:ketone reductasebioconversionephedrinephenylephrine
相關次數:
  • 被引用被引用:3
  • 點閱點閱:100
  • 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:0
L-Phenylephrine(L-PE)是化學合成的藥物,廣泛應用在抗過敏藥、感冒藥及血管加壓劑之中。由於目前化學合成生產L-PE牽涉到複雜危險的反應過程,本研究的主要目標在於建立一全細胞轉換(whole cell conversion)產程以取代化學合成中,將L-PE之前驅物1-(3-hydroxyphenyl)-2-(methylamino)ethanone (HPMAE)還原成為L-PE的步驟。以Rhodococcus菌株進行微生物酵素的篩選,利用高效能液相層析儀解析HPMAE及PE,沒有發現產生PE的酵素活性。以基因選殖方式,在6株Rhodococcus之中選殖到aminoketone asymmetric reductase (akr)酵素基因。將E. coli NovaBlue轉型株攜帶akr基因作為生物催化劑(biocatalyst)可以轉換HPMAE產生D-PE。加入20 mM HPMAE 在45℃ pH 7.0中反應4小時可得到17.30 mM D-PE (>99% e.e.),轉換率(conversion yield) 86.5%及生產率(productivity) 877.98 mg l-1 h-1。利用immobilized metal affinity chromatography純化回收AKR酵素蛋白並分析in vitro AKR酵素活性。顯示AKR為對NADPH專一性酵素,催化1-amino-2-propanol的比活性(specific activity)為116 mU/mg,酵素動力學分析結果,Km值為49.36 mM、Kcat值0.87 min-1及Kcat/Km值17.63 min-1M-1。
L-Phenylephrine (L-PE) is a pharmaceutical compound, synthesized by chemical method and used in anti-allergic drug, cold medicine and vasopressin. Beacause chemical synthesis of L-PE involved multifarious and dangerous processes, the main purpose of this study is to establish a whole cell conversion process to convert 1-(3-hydroxyphenyl)-2-(methylamino)ethanone (HPMAE) to L-PE instead of chemical methods. Aminoketone asymmetric reductase (akr) genes were cloned from 6 strains of Rhodococcus genus. Use E. coli NovaBlue harboring akr gene as a biocatalyst to convert 20 mM HPMAE to D-PE (>99% e.e.) with conversion yield 86.5% and productivity 877.98 mg l-1 h-1 at 45℃ pH 7.0 for 4 h reaction. Using immobilized metal affinity chromatography to recover AKR, in vitro AKR activity assay demonstrated that AKR protein was a NADPH-dependent enzyme, which exhibit a specific activity of 116 mU/mg to 1-amino-2-propanol. Analyze AKR kinetic for the Km, Kcat and Kcat/Km, the value are 49.36 mM, 0.87 min-1 and 17 .63 min-1M-1 respectively.
目錄------------------------------------------------------------------Ⅰ
表目錄---------------------------------------------------------------III
圖目錄--------------------------------------------------------------IV
摘要------------------------------------------------------------------V
Abstract------------------------------------------------------------VI
縮寫對照表-------------------------------------------------------VII

前言
一、 L-Phenylephrine之臨床應用與生理作用機制---1
二、 L-PE之化學合成與市場狀況-------------------------2
三、 不對稱性還原反應與生物催化-----------------------2
四、 Rhodococcus之特性與應用--------------------------4
五、 研究動機----------------------------------------------------5

材料與方法
一、 菌株、質體、酵素與化學藥品-----------------------6
二、 Rhodococcus染色體DNA的抽取-------------------6
三、 DNA的洋菜膠體電泳分析與回收純化-------------6
四、 聚合酵素連鎖反應---------------------------------------7
五、 DNA黏合反應、E. coli質體抽取與轉型作用----7
六、 AKR酵素之基因選殖與表現--------------------------7
七、 全細胞轉換HPMAE生產PE --------------------------8
八、 蛋白質電泳分析-------------------------------------------8
九、 AKR酵素之純化-------------------------------------------9
十、 AKR酵素之活性分析-----------------------------------10
十一、 蛋白質濃度測定---------------------------------------10
十二、 利用HPLC分析HPMAE及PE---------------------10

結果
一、 篩選能將HPMAE轉換成PE之菌株-------------------12
二、 akr基因的選殖-----------------------------------------------12
三、 akr基因於E. coli菌體之表現及純化------------------12
四、 利用基因重組E. coli之菌體轉換HPMAE成PE --15
五、 AKR之生化特性--------------------------------------------17
六、 AKR酵素之alcohol dehydrogenase活性-----------18

討論---------------------------------------------------------------------19

總結---------------------------------------------------------------------22

參考文獻---------------------------------------------------------------43

表 目 錄
表一、------------------------------------------------------------------23
表二、------------------------------------------------------------------24
表三、------------------------------------------------------------------25
表四、------------------------------------------------------------------26
表五、------------------------------------------------------------------27
表六、------------------------------------------------------------------28
表七、------------------------------------------------------------------29
表八、------------------------------------------------------------------30
表九、------------------------------------------------------------------31

圖 目 錄
圖一、------------------------------------------------------------------32
圖二、------------------------------------------------------------------33
圖三、------------------------------------------------------------------34
圖四、------------------------------------------------------------------35
圖五、------------------------------------------------------------------36
圖六、------------------------------------------------------------------37
圖七、------------------------------------------------------------------38
圖八、------------------------------------------------------------------39
圖九、------------------------------------------------------------------40
圖十、------------------------------------------------------------------41
圖十一、---------------------------------------------------------------42
1.中華民國行政院衛生署。 2009。 http://www.doh.gov.tw/。
2.李澤維。 1981。 最新藥物化學,第五版。 南山堂。台北市,台灣。
3.陳慧中。 2006。 酮基還原酵素之篩選俾應用於L-phenylephrine之生合成。碩士論文,中興大學分子生物學研究所。 台中市,台灣。
4.潘志龍。 1997。 Corynebacterium glutamicum DHAO synthase與prephenate dehydratase之間的蛋白質交互作用。碩士論文,中興大學分子生物學研究所。 台中市,台灣。
5.Adam, Z. W. and Jon, D. S. 2004. Understanding and Improving NADPH-dependent reactions by nongrowing Escherichia coli cells. Biotechnol. Prog. 20:403-411
6.Akamatsu, H. 2003. Processes for producing optically active 2-amino-1-phenylethanol derivatives. US patent 6528686 B1.
7.Amemura, A., Chakraborty, R., Fujita, M., Noumi, T., and Futai, M. 1988. Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15. J Biol Chem. 263:9271-5.
8.Atsuko, U., Nomoto, F., Sakoda, A., Nishimoto, Y., Kataoka, M., and Shimizu, S. 2009. Stereoselective synthesis of (R)-3-quinuclidinol through asymmetric reduction of 3-quinuclidinone with 3-quinuclidinone reductase of Rhodotorula rubra. Appl Microbiol Biotechnol. 83:617-26.
9.Barksdale, L. 1970. Corynebacterium diphtheriae and its relatives. Bacteriol. Rev. 34:378-422
10.Banerjee, A., Sharma, R., and Banerjee, U. C. 2002. The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44.
11.Burkovski, A. 2008. Corynebacteria: Genomics and Molecular Biology.
12.Dorokhova, M. I., Somelina, N. E., Tikhonova, O. Ya. and Mikahalev, V. A. 1974. Inversion of configuration of optically active 1-m-nitrophenyl-2-methylamino ethanol. Pharm. Chem. J. 8:209-211
13.Faber, K. 2000. Biotransformations in organic chemistry. Springer-Verlag, Heidelberg, Berlin.
14.Fessner, W. D. 2000. Biocatalysis- from discovery to application. Springer-Verlag, Heidelberg, Berlin.
15.Gilman, A. G., Goodman, L. S., Hardman, J. G., and Limbird, L. E. 2001. Goodman and Gilman''s thepharmacological basis of therapeutics, 10th ed. McGraw-Hill, New York.
16.Gotor, V. 2000. Pharmaceuticals through enzymatic transesterification and enzymatic aminolysis reactions. Biocat Biotrans. 18:87-103.
17.Gurtler, V., Mayall, B. C., and Seviour, R. 2004. Can whole genome analysis refine the taxonomy of the genus Rhodococcus? FEMS Microbiol Rev. 28:377-403.
18.Jan, W., Frank, H., Ursula, R. 2002. Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 2. redirection of metabolic fluxes. Biotechnol Bioeng. 80:320-30.
19.Judith, B., Corinna, K., Oskar Z., Elmar, H., and Christoph, W. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol. 71:8587-8596.
20.Katja. G., Kirsten, S., Stephan, L., and Andreas, L. 2007. Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part II: whole-cell reductions. Appl Microbiol Biotechnol. 76:249-255
21.Katzung, B. G. 2001. Basic and clinical pharmacology, 8th ed. McGraw-Hill, New York.
22.Krebs Biochems Ltd. 2003. Annual Report 2002-2003.
23.Loyd, V., Allen, Jr., and Berardi, R. R. 2002. Handbook of nonprescription drugs, 13th ed. Alpha Publications, Washington, DC.
24.Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685.
25.Mahmoudian, M. 2000. Biocatalytic production of chiral pharmaceutical intermediates. Biocat Biotrans. 18:105-16.
26.McLeod, M. P., Warren, R. L., Hsiao, W. W., Araki, N., Myhre, M., Fernandes, C., Miyazawa, D., Wong, W., Lillquist, A. L., Wang, D., Dosanjh, M., Hara, H., Petrescu, A., Morin, R. D., Yang, G., Stott, J. M., Schein, J. E., Shin, H., Smailus, D., Siddiqui, A. S., Marra, M. A., Jones, S. J., Holt, R., Brinkman, F. S., Miyauchi, K., Fukuda, M., Davies, J. E., Mohn, W. W., and Eltis, L. D. 2006. The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. PNAS. 103:15582-15587.
27.Meadows, M. 2001. FDA issues public health advisory on phenylpropanolamine in drug products. FDA Consum. 35:9.
28.Michael, S., and Clarence, I. Kado. 2002. Horizontal gene transfer. 2nd ed. Academic press. New York.
29.News-register.com. 2005. Drug companies consider substitute for cold medicine, McMinnville, Oregon.
30.O’Brien, M. K., and Vanasse, B. 2000. Asymmetric processes in the large-scale preparation of chiral drug candidates. Curr Opin Drug Discov Dev. 3:793-806.
31.Patel, R. N. 2000. Microbial/enzymatic synthesis of chiral drug intermediates. Adv Appl Microbiol. 47:33-78.
32.Sambrook, J., Fritsh, E. F., and Maniatis, E. F. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold spring harbor laboratory press, Cold spring harbor, N. Y.
33.Shaw, N. M., Robins, K. T., and Kiener, A. 2003. Lonza: 20 years of biotransformations. Adv Synth Catal. 345:425-435.
34.Singh, M., Scrutton, N. S., and Scrutton, M. C. 1988. NADPH generation in Aspergillus nidulans: is the mannitol cycle involved? J Gen Microbiol. 134:643-54.
35.Sohail, M. 1998. A simple and rapid method for preparing genomic DNA from gram-positive bacteria. Mol Biotechnol. 10:191-3.
36.Stewart, D. 2001. Dehydrogenases and transaminases in asymmetric synthesis. Curr Opin Chem Biol. 5:120-29.
37.Treadway, S.L., Yanagimachi, K. S., Lankenau, E., Lessard, P. A., Stephanopoulos, G., and Sinskey, A. J. 1999. Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24. Appl. Microbiol. Biotechnol. 51:786-793.
38.Vaseghi, S., Baumeister, A., Rizzi, M., and Reuss, M. 1999. In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae. Metab Eng. 1:128-40.
39.Van der Geize, R., and Dijkhuizen, L. 2004. Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications. Microbiology 7:255-261
40.Verduyn, C., Van Kleef, R., Frank, J., Schreuder, H., Van Dijken, J. P., and Scheffers, W. A. 1985. Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis. Biochem J. 226: 669-77.
41.Zak, A. 2001. Industrial biocatalysis. Curr Opin Chem Biol. 5:130-36.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊