Abstract
A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg−1. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K m and V max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min−1 mg−1, respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous–organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP+ to (S)-CHBE were 26.5 mmol L−1 h−1 g−1 DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.
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Acknowledgments
The supports of this work by the National Basic Research Program of China (973 Program) (No. 2011CB710800), Natural Science Foundation of Zhejiang Province (No. R3110155) and Qianjiang Talent Project of Zhejiang Province are gratefully acknowledged.
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You, ZY., Liu, ZQ. & Zheng, YG. Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield. Appl Microbiol Biotechnol 98, 1671–1680 (2014). https://doi.org/10.1007/s00253-013-5042-3
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DOI: https://doi.org/10.1007/s00253-013-5042-3