Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3742
標題: 酵素法生產N-乙醯甘露醣胺之研究
Enzymatic process for the synthesis of N-Acetyl-D-Mannosamine
作者: 謝孟鋼
Hsieh, Meng-Kang
關鍵字: 差向異構酶葡萄醣胺
Sialic acid
異構化
固定化
N-acetyl-D-mannosamine
N-actyl-D-glucosamine 2-epimerase
epimerization
immobilization.
出版社: 化學工程學系所
引用: 呂鋒洲 (1991)。”基礎酵素學”,聯經出版事業公司。1-4 David L. Nelson, Michael M. Cox (2003) ” Lehninger生物化學原理3rd ”,合記書局有限公司。Chapter 8 賴奕成 (2002)。 固定化HRP 於工業廢水處理之應用。碩士論文,化學工程研究所,中興大學,台中。 拜永孝(2005)。固定化酶技術及其應用。化學通报第68 卷 w027,大陸。 吳冠毅 (2006) 。 雙硫鍵對差向異構酶熱穩定性之影響。碩士論文,化學工程研究所,中興大學,台中。 王豐寶(2006) 。 雙標誌融合酵素蛋白質之基因表現與固定化。碩士論文,化學工程研究所,中正大學,嘉義。 顏敏智(2007) 。以固定化酵素生產L-同苯丙胺酸。碩士論文,化學工程研究所,中興大學,台中。 李晏忠 (2007) 。N-acetyl-D-glucosamine 2-epimerase 之生化及結構分析俾應用於酵素法生產N-acetyl-D-neuraminic acid。博士論文,分 子生物學研究所,中興大學,台中。 Ghosh, S., and S. Roseman, “The sialic acids, V. N-acyl-D-glucosamine 2-epimerase,” J. Biol. Chem., 240, 1531-1536 (1965). Maru, I., J. Ohnishi, Y. Ohta, and Y. Tsukada, “Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosarnine 2-epimerase and N- acetylneuraminate lyase,”J. Biosci. Bioen ., 306, 575-578 (1998) . Von Itzstein, M., W. Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason, B. Jin, T. Van Phan, M. L. Smythe, H. F. White, S. W. Oliver, P.M. Colman, J. N. Varghese, D. M. Ryan, J. M. Woods, R. C. Bethell, V.J. Hotham, J. M. Cameron, and C. R. Renn, “Rational design of potent sialidase-based inhibitors of influenza virus replication.” Nature., 363 418-423 (1993). De Ninno, M. P. “The synthesis and glycosidation of N- Acetyl- D- neuraminic acid.” Synthesis., 8, 583-593 (1991). Kragl, U., Gygax, D., Ghisalba, O. and Wandrey, C. “Enzymatic two-step synthesis of N-acetyl-neuraminic acid in the enzyme membrane reactor.” Angewandte Chemie ., 30, 827-828 (1991). Lee, J. O., J. K. Yi, S. G. Lee, S. Takahashi, and B. G. Kim, “Production of N-acetylneuraminic acid from N-acetylglucosamine and pyruvate using recombinant human rennin binding protein and sialic acid aldolase in one pot.” Enzyme and Microbial Technolog ., 35, 121-125 (2004). Farinas, E. T., T. Bulter,and F. H., 2001. Arnold, “Directed enzyme evolution”, Biotechnology., 12, 545-551. Bull, A. T., A. W. Bunch, and G. K. Robinson, "Biocatalysts for clean industrial products and process", Microbiol., 2, 246-251 (1999). Schauer, R.,and A. P. Cprfield, In:Schauer R. (ed) Sialic Acids, Chemistry, Metabolism and Functions. New York: Springer Verlag, (1982) . Maru I., Y. Ohta, K. Murata & Y. Tsukada , "Molecular cloning and identification of of N-acyl-D-glucosamine 2-epimerase from porcine kidney as a renin-binding protein, " J. Biol. Chem. 271,16294~16299 (1996). Comb, D. G. and S. Roseman, “The sialic acid,” The Journal of Biological Chemistry, 235, No. 9 (1960). Shuler, M. L. and, F. Kargi, “Bioprocess Engineering,” Prentic-Hall Press ,Inc, 59, 78 ( 1992.) Hartmeier, W., "Immobilized Biocatalysts", Heidelberg New York., 82-102 (1988). Messing, R. A., , "Immobilized Enzymes For Industrial Reactors", Academic press, 2-3 (1975.). Bickerstaff, G. F., "Immobilization of Enzymes and Cells", Humana press, 1-11 (1997). Chaplin, M. F., and, C. Bucke, "Enzyme technology," Cambridge university press, 81-90 (1990). Kennedy, J. F. and E. H. M. Melo, "Immobilization enzymes and cells, "Chem. Emg. Prog, 81-89 (1990). Bautista, F. M., M. C. Bravo, J. M. Campelo, A. Garcia, D. Luna , J. M. Marinas, and A. A. Romero, "Covalent immobilization of acid phosphatase on amorphous AlPO4 support," Journal of Molecular CatalysisB: Enzymatic, 6, 473-481 (1999). Kallenberg, A. I., F. van Rantwijk, R. A. Sheldon, "Adv. Synth. Catal," 347, 905–926 (2005). Ephraim, K. K.,and D. M. Kraemer, “Eupergitw C, a carrier for immobilization of enzymes of industrial potential, “Journal of Molecular Catalysis B: Enzymatic, 10, 157–176 (2000). Veliky, I. A., and R. J. C. Mclean, "Immobilization biosystems," 1-128 (1994). Ballesteros, A., L. Boross, K. Buchholz, J. M. S. Cabral, and V. Kasche, "Biocatalyst performance," Applied Biocatalysis, Harwood academic publisher, 237-278 (1994). Rong-Huay Juang 09/06/25 http://juang.bst.ntu.edu.tw/BC2008/Enzyme3.htm
摘要: 唾液酸(sialic acid;NeuAc)在許多生化功能中扮演著重要的角色,它是合成許多藥物的重要前驅物,如: 抗流感病毒藥物、治療糖尿病、癲癇及消炎用藥等。在過去的報告指出以N-acetyl-D-mannosamine (ManNAc)和pyruvate為反應物,藉由N-acetyl-D-neuraminic acid (NeuAc) aldolase來催化來生成唾液酸之酵素合成法已經被認定為較理想,且可大量生產唾液酸的方法。不過,為了節省反應物成本,衍生出一個二階段式的生產方法。此種方法即利用價格較低廉的N-acetyl-D-glucosamine (GlcNAc)為起始反應物,經GlcNAc 2-epimerase催化生成ManNAc。本研究主要是將豬的基因重組蛋白GlcNAc 2-epmerase以共價鍵結的方式固定於商用膠體上,並探討最適固定化條件及固定化後的酵素性質。其中,酵素在固定化過程中有33 %的活性損失;膠體所能吸附的最大酵素量為1.0 ,但在吸附量達0.41 即有最佳的比活性2.87 。在反應溫度的研究中,自由態與固定化酵素皆在60 oC下得到最高的活性,並未隨著酵素作固定化操作而改變。不過,經由固定化操作的酵素在高溫上確實展現出較好的溫度耐抗性,在反應溫度為80 oC時,固定化的酵素相較於自由態(14.7 %)能保有較高的殘餘活性47.8 %。固定化酵素的熱穩定性也有明顯的提升;將酵素存放在80 oC下長達5個小時後取出,並於37 oC下進行反應,自由態酵素已完全失去其活性,而固定化後的酵素仍保有20.9 %的初始活性 。至於在反應液pH方面,自由態與固定化酵素的整體趨勢相似,皆在pH 9有最高的活性表現。對於固定化後批次反應的『可重複使用性』也有作相關的研究;固定化酵素在重複24個循環操作下依然能保有63.8 %的初始活性。在連續式操作方面,將進料流速控制在0.1ml/min可得到最大的平衡轉化率,以此流速進行長時間的連續操作,在連續使用了6.3天之後還能擁有59.0%的初始活性。
Sialic acid, which plays a significant role in numerous biological functions, is an important precursor for the synthesis of many pharmaceutical drugs such as anti-influenza virus agents. Enzymatic process employing N-acetyl-D-neuraminic acid (NeuAc) aldolase for the synthesis of sialic acid from N-acetyl-D-mannosamine (ManNAc) and pyruvate has been reported. To reduce substrate costs, an alternative process with N-acetyl-D-glucosamine (GlcNAc) and GlcNAc 2-epimerase as the catalysts has been proposed, enabling the use of relatively inexpensive GlcNAc instead of ManNAc as the starting reactant. In this study, we report the enzymatic production of ManAc from GlcNAc with immobilized recombinant porcine kidney GlcNAc 2-epmerase. GlcNAc 2-epimerse, a homo-dimeric protein with a molecular weight of 84 kDa, was immobilized onto Eupergit C, a commercial oxirane-based support with reactive amino groups. A maximal enzyme load of 1.0 mg/g gel was achieved. The effect of enzyme load on the specific activity of the immobilized 2-epimerase was investigated. At an optimal enzyme load of 0.41 mg/g gel, a specific activity of 2.87 IU/mg epimerase, approximately 33 % lower than that of the free enzyme, was obtained. Further increase in enzyme load led to a decline in enzyme specific activity. Surprisingly, the optimal reaction temperature of the enzyme, 60 oC, was not increased upon immobilization. Nevertheless, the immobilized activity did exhibit a higher activity at elevated temperature. A higher residual activity of 47.8 % was observed for the immobilized epimerase at 80 oC than that for the free enzyme, 14.7 %. The thermal stability of the enzyme was also significantly enhanced upon immobilization. While incubating at 80 oC for 5 h completely inactivated the free enzyme, a residual activity of 20.9 % was obtained for the immobilized enzyme. The pH profile of the immobilized enzyme is similar to that of the free enzyme. The reusability of the immobilized enzyme in a batch reactor was also studied. A residual activity of 63.8 % was obtained after 24 cycles. The effect of space time on conversion in a continuous packed-bed reactor will also be discussed
URI: http://hdl.handle.net/11455/3742
其他識別: U0005-1408200914065600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1408200914065600
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