Development of a substrate-coupled biocatalytic process driven by an NADPH-dependent sorbose reductase from Candida albi
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ORIGINAL RESEARCH PAPER
Development of a substrate-coupled biocatalytic process driven by an NADPH-dependent sorbose reductase from Candida albicans for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate Ping Cai • Mingdong An • Lin Xu • Sheng Xu Ning Hao • Yan Li • Kai Guo • Ming Yan
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Received: 30 May 2012 / Accepted: 15 August 2012 / Published online: 24 August 2012 Ó Springer Science+Business Media B.V. 2012
Abstract A substrate-coupled biocatalytic process was developed based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which ethyl 4-chloro-3-oxobutanoate (COBE) was reduced to (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols. (S)-CHBE yields of 1,140, 1,150, and 780 mM were obtained from 1,220 mM COBE when sorbitol, P. Cai M. An L. Xu S. Xu N. Hao Y. Li K. Guo M. Yan (&) State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, People’s Republic of China e-mail: [email protected] P. Cai e-mail: [email protected] M. An e-mail: [email protected] L. Xu e-mail: [email protected] S. Xu e-mail: [email protected] N. Hao e-mail: [email protected] Y. Li e-mail: [email protected] K. Guo e-mail: [email protected]
mannitol, and xylitol were used as co-substrates, respectively. Optimization of COBE and sorbitol proportions resulted in a maximum yield of (S)-CHBE (2,340 mM) from 2,500 mM COBE, and the enantiomeric excess was 99.6 %. The substrate-coupled system driven by SOU1 maintained a stable pH and a robust intracellular NADPH circulation; thus, pH adjustment and addition of extra coenzymes were unnecessary. Keywords Candida albicans Ethyl (S)-4-chloro-3hydroxybutanoate NADPH-dependent sorbose reductase Sorbose reductase Substrate-coupled biocatalysis
Introduction The biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], a chiral intermediate in the synthesis of pharmacologically active compounds, such as hydroxymethylglutaryl-CoA reductase inhibitors and 4-hydroxypyrrolidone (Karanewsky et al. 1990), has attracted significant attention. The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) by enantioselective oxidoreductases is a practical method for the production of (S)-CHBE (Ye et al. 2011). However, this method requires an efficient regeneration for the coenzyme NAD(P)H. To overcome this problem, enzyme-coupled systems were proposed and many coenzyme regeneration systems, including formate/formate dehydrogenase
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(FDH) (Yamamoto et al. 2004, 2005), glucose 6-phosphate (G6P)/glucose-6-phosphate dehydrogenase (G6PDH) (Yu et al. 2007), and glucose/glucose dehydrogenase (GDH) (Kizaki et al. 2001; Ye et al. 2010a), have been widely used to provide NAD(P)H coupling with oxidoreductase-catalyzed synthesis systems. The substrate-coupled method, using NAD(P)Hdependent alcohol dehydrogenase (ADH) (Wang et
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