Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21995
標題: Cloning and characterization of aminoketone reductase from Rhodococcus spp. and its application to phenylephrine production
Rhodococcus spp. aminoketone reductase之基因選殖、特性分析及其應用於phenylephrine之生產
作者: 陳建宇
Chen, Chien-Yu
關鍵字: ketone reductase;酮基還原酵素;bioconversion;ephedrine;phenylephrine;生物轉換;麻黃素;phenylephrine
出版社: 分子生物學研究所
引用: 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.
摘要: 
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.

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。
URI: http://hdl.handle.net/11455/21995
其他識別: U0005-0608200917194100
Appears in Collections:分子生物學研究所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.