Asymmetric synthesis of a fluoxetine precursor with an artificial fusion protein of a ketoreductase and a formate dehydrogenase |
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| Affiliation: | 1. Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany;2. Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany;1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China;2. Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, PR China;1. Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;2. Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan;3. Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China;2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China;1. Enzyme and Fermentation Technology Laboratory, College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China;2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China;3. Department of Chemical & Biomolecular Engineering, National University of Singapore, 117585 Singapore |
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| Abstract: | Herein we describe the kinetic characterization of a fusion protein from the 3-ketoacyl-[acyl-carrier-protein]-reductase (KR) from Synechococcus PCC 7942 and a mutant formate dehydrogenase from Mycobacterium vaccae N10 (MycFDH). Upon purification, a specific proteolytic cleavage of the MycFDH was observed. The cleavage site was elucidated, which is ubiquitously spread among prokaryotic FDHs. After depletion of the cleavage site the correct, full length fusion protein was obtained. In asymmetric reductions of ethylbenzoyl acetate (EBA) this fusion protein performed equal or even better than the free enzymes, yielding up to 39% more of the fluoxetine precursor ethyl-(S)-3-hydroxy-3-phenylpropanoate ((S)-HPPE). The rate acceleration is due to an improved Km,EBA of the KR subunit. |
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| Keywords: | Fusion protein Formate dehydrogenase Ketoreductase Asymmetric reduction Proteolytic degradation Chiral alcohol |
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