Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst |
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Authors: | Qi Ye Hou Cao Guanglou Zang Lan Mi Ming Yan Yan Wang Yueyuan Zhang Ximu Li Jian Li Lin Xu Jian Xiong Pingkai Ouyang Hanjie Ying |
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Institution: | 1. State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, 210009, People’s Republic of China 2. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 210009, People’s Republic of China 3. National Engineering Technique Research Center for biotechnology, Nanjing, 211816, People’s Republic of China 4. National Center for Quality Supervision and Test of Chinese Liquor, Suqian, 223800, People’s Republic of China 5. College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Xin mofan Road 5, Nanjing, 210009, People’s Republic of China
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Abstract: | A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester (S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980. |
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