| Summary: | 碩士 === 弘光科技大學 === 生物科技研究所 === 101 === Duloxetine is a new-generation popular antidepressant drug; it is a dual inhibitor of serotonin and norepinephrine reuptake. It has been approved for psychiatric and metabolic conditions, such as diabetic peripheral neuropathic pain, fibromyalgia, stress urinary incontinence, major depressive disorder, and generalized anxiety disorder. Duloxetine contains 1 chiral center and only the (S)-enantiomer is pharmaceutically active. Therefore, development of its specific optically active (S)-enantiomer by using a new process has high market value. In this study, a novel bioreduction process for the production of optically active (S)-B, a chiral intermediate of duloxetine, from A has been developed. Using HPLC screening methods, we successfully screened five N422 strains that showed A-reduction activity. Among them, N422-5 showed the highest reduction activity and good stereoselectivity. Under the reaction conditions used, the highest conversion yield of 1.3% was obtained when the cells collected were incubated at pH 8.5 and 30°C with 10 mM A for 72 h. On the basis of the sequenced genomes, 21 putative reductase genes from N422-5, N422-1, and N422-9 were cloned and expressed in Escherichia coli. Of these, the n42242, n42237, and n42285 genes showed sequence homology and possessed enzymatic activity for converting A to (S)-B with a conversion yield of approximately 2.6%. In conclusion, we successfully screened N422 strains that could act as a whole-cell biocatalyst for the bioreduction process. Moreover, genes that might be involved in the bioreduction of A to (S)-B were also cloned. Further research on enhancing the conversion yield is important for improving the industrial application of this bioreduction process.
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