Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23287
標題: 利用轉基因水稻表現 醫藥工業用酵素L-aminoacylase之研究
Expression of the pharmaceutical enzyme L-aminoacylase in transgenic rice
作者: 王怡雯
Wang, I-Wen
關鍵字: L-aminoacylase
L-aminoacylase
transgenic rice
轉基因水稻
出版社: 生命科學院碩士在職專班
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摘要: 隨著植物生物科技的進步,使得植物生產重組蛋白質的策略可以實現。加上植物易於轉殖且能提供廉價的蛋白質來源,因此認為植物具有生產重組蛋白質的潛力。水稻是重要的榖類之ㄧ,它可以作為安全且廉價的工廠來生產具商業價值的蛋白質,因此,本研究的目的在於評估及分析利用轉基因水稻表現來自原核生物之 L-aminoacylase 蛋白之酵素活性。L㏄-aminoacylase (N-acyl-amino-acid amino-hydrolase,EC3.5.1.14)屬於旋光異構物水解酶的一種,配合 racemase 能將 D,L-homophenylalanyl hydantoins 水解形成 L-homophenylalanine (L-HPA)。L-HPA是一種非尋常的胺基酸,廣泛被使用在降血壓藥物血管收縮素轉化酶抑制劑ACE-inhibitors (ACEI)的合成上,例如:Enalapril、linsinopril、delapril、imidapril、benezepril、tempocapril等。經由農桿菌轉殖的方式,獲得含有目標基因L-aminoacylase (LAA)的轉殖株。經過抗生素篩選、PCR、Southern blot、Western blot 的確認,已得到705 LAA及ActLAA 之 T3 同質品系轉殖株,且已大量萃取及利用親合性管柱純化轉基因水稻穀粒中的 LAA 蛋白。這些蛋白利用Ninhydrin呈色法偵測證實具有酵素活性,進一步利用高效能液相層析儀(HPLC)搭配 Reversed Phase-C18 管柱,於移動相 acetonitrile / 0.01% H3PO4 = 50/50,流速0.7 ml/min 時,可以將受質(NAc-L-HPA)與產物(L-HPA)解析。在 Mn2+、Co2+等二價金屬離子存在時,可以增加轉基因水稻所生產之LAA 的酵素活性。轉殖基因水稻所生產之 LAA 其最佳反應之 pH 值為 8.0,最佳反應溫度為 45℃,在此反應環境下,LAA活性在36小時之後,仍然還有37%。以上的實驗結果,證實可以利用轉基因水稻做為生物反應器,來生產醫療工業用酵素LAA。
Advances in plant biotechnology have made the production of recombinant proteins in plants feasible. Plants have considerable potential for the production of recombinant proteins because they are easily transformed and provide a cheaper source of proteins. Rice is one of the most important crops. It can potentially provide a safe and inexpensive factory for the mass production of commercially valuable proteins. The purpose of this research is to analyze the enzymatic activity of the pharmaceutical enzyme L-aminoacylase (LAA) produced in transgenic rice, and to evaluate the potential for future mass production of LAA by transgenic plant. The LAA is a kind of enantioselective amidohydrolase that can be used to convert L-homophenylalanyl hydantoins to L-homophenylalanine (L-HPA). It is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors, such as enalapril, linsinpril, delapril, benezepril, and tempocapril et al. LAA transgenic rice plants were obtained by using agrobacterium-mediated transformation and confirmed by using hygromycin selection, PCR analysis, Southern blot and western blot analysis. Homozygous transgenic rice lines of 705 LAA and Act LAA have been cultivated up to the third generation. Extraction and purification of LAA protein from rice grain was made successful by using His-tag affinity chromatography. Activity of LAA was demonstrated through high performance liquid chromatography (HPLC) with C18 column which can separate N-Acetyl-L-homophenylalanine (NAc-L-HPA) and L-homophenylalanine (L-HPA) using acetonitrile / 0.01% H3PO4 = 50/50 as mobile phase at a flow-rate of 0.7 ml/min. The transgenic rice LAA required the divalent metal ions Mn2+, Co2+ to increase the enzyme activity. The pH and temperature optima of the enzyme were pH 8.0 and 45℃, respectively. This LAA remained 37% activity after 36 hours of incubation under the optimum reaction condition. This study concluded that the transgenic rice can be an useful source to produce the pharmaceutical enzyme LAA for the production of L-HPA.
URI: http://hdl.handle.net/11455/23287
其他識別: U0005-2008200716500400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2008200716500400
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