Yield of the pharmaceutical enzyme L-N-Carbamoylase in transgenic rice for the production of L-Homophenylalanine

• Main Authors: Chih-Wei Tzeng, 曾智偉
• Other Authors: Liang-Jwu Chen
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/37540937336331008912

碩士 === 國立中興大學 === 分子生物學研究所 === 93 === Abstract 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.