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標題: Yield of the pharmaceutical enzyme L-N-Carbamoylase in transgenic rice for the production of L-Homophenylalanine
利用轉基因水稻表現醫療工業用酵素L-N-Carbamoylase以生產L-Homophenylalanine 之研究
作者: 曾智偉
Tzeng, Chih-Wei
關鍵字: L-NCA;轉基因水稻;transgenic rice;L-homophenylalanine;醫療工業用酵素
出版社: 分子生物學研究所
A thermostable L-N-carbamoylase ( L-NCA ) is a kind of enantioselective amidohydrolase. In the combination of racemase ( or L-hydantoinase ) and L-NCA, D,L-homophenylalanyl hydantoins was converted to L-homophenylalanine ( L-HPA ), an angiotensin-converting enzyme inhibitor ( ACEI ) precursor for the synthesis of many antihypertensive drugs. According to IMS HEALTH report, the global market of ACEI reached total of $ 7.3 billion in the year of 2000. This in dicated that the production of L-NCA has a great impact in the market. The purpose of this research is to analyze the enzymetic activity of a pharmaceutical enzyme L-NCA produced in transgenic rice, and to evaluate the potential for future production of L-NCA in transgenic plant. This study have obtained the homozygous transgenic rice lines 705 L-NCA and have cultivated it to the 3th generation. The existence of L-NCA gene in transgenic rice has been confirmed by polymerase chain reaction ( PCR ) and Southern blot assay. The expression of L-NCA has been confirmed by Western blot and its purification has also been worked out by using His-tag affinity chromatography.
High performance liquid chromatography ( HPLC ) with C18 column could separate N-carbamoyl L-homophenylalanine ( NC-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 L-NCA required the divalent metal ions Mn2+, Ni2+, Co2+ for increasing activity. The pH and temperature optima of the enzyme were pH 8.0 and 60 ℃, respectively. This enzyme was completely thermostable at 50 ℃ for 14 days in the presence of L-specific substrates. In the results we confirmed the transgenic rice as a bioreactor to yield pharmaceutical enzyme L-NCA for the production of L-HPA.

耐熱 L-N-Carbamoylase ( L-NCA ) 屬於旋光異構物水解酶的一種,配合 racemase 或是 L-hydantoinase 能將 D,L-homophenylalanyl hydantoins 水解形成 L-homophenylalanine ( L-HPA ) ,而 L-HPA 目前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,根據 IMS HEALTH 的資料,西元 2000年全球 ACEI 市場約為 73 億美金,因此L-NCA 具有非常高的經濟價值。本研究的目的在於評估及分析利用轉殖基因水稻表現來自原核生物之 L-NCA 蛋白之酵素活性,及未來增加國產水稻附加價值之潛力。目前經由抗抗生素篩選、 PCR 、 Southern blot 、 Western blot 確認,已得到台農 67 號 705 L-NCA 之 T3 同質品系轉殖株,且已大量萃取及純化穀粒中的 L-NCA 蛋白。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 ( NC-L-HPA ) 與產物 ( L-HPA ) 解析。在 Mn2+ 、 Ni2+ 、 Co2+ 等二價金屬離子存在時,可以增加轉殖基因水稻所生產之L-NCA 其酵素活性。轉殖基因水稻所生產之 L-NCA 其酵素最佳反應 pH 值為 8.0 ,及最佳反應溫度為 60 ℃ 。另外利用轉基因水稻所生產之 L-NCA 酵素,可以在長達 14 天 50 ℃ 環境下,維持原始的酵素活性,證明其為熱穩定性極佳之酵素。從以上實驗的結果,證實可以利用轉殖基因水稻做為一生物反應器,來生產醫療工業用酵素 L-NCA 。
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