Highly Efficient Deracemization of Racemic 2-Hydroxy Acids in a Three-Enzyme Co-Expression System Using a Novel Ketoacid Reductase
- 132 Downloads
Enantiopure 2-hydroxy acids (2-HAs) are important intermediates for the synthesis of pharmaceuticals and fine chemicals. Deracemization of racemic 2-HAs into the corresponding single enantiomers represents an economical and highly efficient approach for synthesizing chiral 2-HAs in industry. In this work, a novel ketoacid reductase from Leuconostoc lactis (LlKAR) with higher activity and substrate tolerance towards aromatic α-ketoacids was discovered by genome mining, and then its enzymatic properties were characterized. Accordingly, an engineered Escherichia coli (HADH-LlKAR-GDH) co-expressing 2-hydroxyacid dehydrogenase, LlKAR, and glucose dehydrogenase was constructed for efficient deracemization of racemic 2-HAs. Most of the racemic 2-HAs were deracemized to their (R)-isomers at high yields and enantiomeric purity. In the case of racemic 2-chloromandelic acid, as much as 300 mM of substrate was completely transformed into the optically pure (R)-2-chloromandelic acid (> 99% enantiomeric excess) with a high productivity of 83.8 g L−1 day−1 without addition of exogenous cofactor, which make this novel whole-cell biocatalyst more promising and competitive in practical application.
KeywordsBiocatalysis 2-Hydroxy acid Deracemization Ketoacid reductase Co-expression (R)-2-Chloromandelic acid
This work was funded by the National Natural Science Foundation of China (No. 21676254).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that there is no conflict of interest.
The authors declare that there are no studies conducted with human participants or animals.
- 2.Ma, B. D., Yu, H. L., Pan, J., Liu, J. Y., Ju, X., & Xu, J. H. (2013). A thermostable and organic-solvent tolerant esterase from Pseudomonas putida ECU1011: catalytic properties and performance in kinetic resolution of α-hydroxy acids. Bioresource Technology, 133(2013), 354–360.CrossRefGoogle Scholar
- 6.Zhang, C. S., Zhang, Z. J., Li, C. X., Yu, H. L., Zheng, G. W., & Xu, J. H. (2012). Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata. Applied Microbiology and Biotechnology, 95(1), 91–99.CrossRefGoogle Scholar
- 14.Xue, Y. P., Shi, C. C., Xu, Z., Jiao, B., Liu, Z. Q., Huang, J. F., Zheng, Y. G., & Shen, Y. C. (2015). Design of nitrilases with superior activity and enantioselectivity towards sterically hindered nitrile by protein engineering. Advanced Synthesis & Catalysis, 357(8), 1741–1750.CrossRefGoogle Scholar
- 15.Norihiro, K., Hiroaki, Y. & C, D (2004). Alpha-keto acid reductase, method for producing the same, and method for producing optically active alpha-hydroxy acids using the same. US Patent Application, 2004086993 A1.Google Scholar
- 17.Adam, W., Lazarus, M., Saha-Moller, C. R., & Schreier, P. (1998). Quantitative transformation of racemic 2-hydroxy acids into (R)-2-hydroxy acids by enantioselective oxidation with glycolate oxidase and subsequent reduction of 2-keto acids with D-lactate dehydrogenase. Tetrahedron: Asymmetry, 9(2), 351–355.CrossRefGoogle Scholar
- 32.Shen, N. D., Ni, Y., Ma, H. M., Wang, L. J., Li, C. X., Zheng, G. W., Zhang, J., & Xu, J. H. (2012). Efficient synthesis of a chiral precursor for angiotensin-converting enzyme (ACE) inhibitors in high space-time yield by a new reductase without external cofactors. Organic Letters, 14(8), 1982–1985.CrossRefGoogle Scholar