| Summary: | 碩士 === 弘光科技大學 === 生物科技研究所 === 105 === (1R, 2S)-Norephedrine is a naturally occurring alkaloid which is used as sympathomimetic drugs. The pharmacological can be treatment of urinary incontinence in dogs. Moreover, (1R, 2S)-norephedrine and its derivatives are of great significance as chiral auxiliaries in a variety of asymmetric reaction, especially in the synthesis of the HIV reverse transcriptase inhibitor, Efavirenz, and development of antineoplastic agents, Mivobulin isethionate. Accordingly, we want to develop a simple and convenient biosynthetic method for the optically active norephedrine that is meaningfully. According to previous research, we found two enzyme which had conversion acticity from Chromobacterium violaceum, and we found that (1R, 2S)-norephedrine were limited conversion activity. We used the way of protein structure and protein docking to predicting amino acid residues that may be involved in an active reaction or product inhibitiong, and used mutation to improve this problem.
Using the above method, we have found amino acid residues predicted from the ω-transaminase (ω-TA) of C. violaceum (Cv) which may increase the activity of the enzyme or decrease the product inhibiton, and two high-throughput screen methods, TTC assay and thin layer chromatography were searched active strains. From the results of structural modeling, it was found that the amino acid residues, Cv W188, Cv D372, and Cv L393 will interact with the product and which may affect the conversion activity. In the part of increased the conversion activity, the mutant of Cv ω-TA had higher conversion rate of Cv D372E and Cv L393M, the conversion rate was 55.4% and 54.0%, respectively. And in the part of improved product inhibition, Cv ω-TA mutant strain showed the most improvement with Cv L393M, IC50 value from 11.3 mM to 16.2 mM. It was concluded that the mutant strains were successfully cloned, which could improve the inhibition of the products, and found that the binding position of the non-competitive products was inhibited. It is expected to be helpful to the future research.
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