Screening of ω-Transaminase for Optically Active Norephedrine Biosynthesis

• Main Authors: Jian-Sin Lin, 林建鑫
• Other Authors: Chao-Hung Kao
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/00666694176846794124

碩士 === 弘光科技大學 === 生物科技研究所 === 100 === (1R, 2S)-norephedrine is a naturally occurring alkaloid which is used as therapeutic sympathomimetic agents. In addition, (1R, 2S)-norephedrine is widely used as very useful starting material for preparation of many important chiral compounds. Moreover, (1R, 2S)-norephedrine and its derivatives are of great importance as chiral auxiliaries in a variety of asymmetric reaction, especially in the synthesis of the HIV reverse transcriptase inhibitor, Efavirenz. Accordingly, the development of a simple and convenient synthetic method for the optically active norephedrine is of great interest. In this study, a novel biocatalytic process for production of (1R, 2S)-norephedrine from L-phenylacetylcarbinol (L-PAC) and L-alanine has been developed by using (S)-selective ω-transaminase, which has never been reported in the world. Based on sequence alignment and activity study, we have successfully cloned the ω-transaminases from Chromobacterium violaceum, Paracoccus denitrificans and Bradyrhizobium japonicum, they were verified to be a candidate for (1R, 2S)-norephedrine biosynthesis. Among them, the gene from C. violaceum showed the highest conversion activity and has been cloned and functionally expressed in E. coli [E. coli (pQE-CvTA)]. The highest conversion yield of 62.2% was obtained in 1 h incubation at pH 7 and 37℃, with the ratio of the amino acceptor (L-PAC) and donor (L-alanine) equals to 1:10. The E. coli (pQE-CvTA) transformant was more stable under the potassium phosphate (pH 6) at 35℃, after 120 h incubation the relative conversion yield was remained about 95%. The cells can be reused for at least 20 cycles with a relactive conversion yield of about 67.7%. This process has higher applied potential for optical pure (1R, 2S)-norephedrine biosynthesis.