Abstract
The enantioselective biocatalytic hydrolysis of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (rac-cyanodiester, rac-CNDE) to obtain a key (S)-intermediate is crucial for the synthesis of Pregabalin. The gene estZF172 from Pseudomonas CGMCC No. 4184, which encoded a novel esterase EstZF172 with excellent stereoselective catalysis capacity of (R)-CNDE (E = 95), was obtained from genomic library construction through activity screening. EstZF172 was identified to be a new member of the bacterial esterase/lipase Family VIII by sequence alignment, phylogenetic tree analysis and homology model analysis, with preference toward shortchain p-nitrophenyl esters. The esterase was functionally expressed in E. coli BL21(DE3), exhibiting a 120-fold improvement in catalytic activity (4027.5 U/L) over the wild strain (33.43 U/L) toward CNDE. For the chiral hydrolysis of rac-CNDE catalyzed by recombinant cells, the optimum operating temperature and pH were determined to be 35°C and 8.5, respectively, based on the biochemical characterization of the purified EstZF172. Finally, the yield and ee of (S)-CNDE reached 47.7% and > 99.5% after reaction for 7 h with a substrate loading of 127.5 g/L (500 mM). The results suggested that EstZF172 is a potential biocatalyst for the synthesis of an important chiral intermediate of Pregabalin.
Similar content being viewed by others
References
Taylor, C. P., T. Angelotti, and E. Fauman (2007) Pharmacology and mechanism of action of pregabalin: The calcium channel a2-d (alpha2-delta) subunit as a target for antiepileptic drug discovery. Epilepsy Res. 73: 137–150.
Pfizer Inc. and Subsidiary Companies (2013) Financial Report. http:// www.pfizer.com/files/investors/presentations/FinancialReport2013.pdf.
Caner, H., E. Groner, L. Levy, and I. Agranat (2004) Trends in the development of chiral drugs. Drug Discov. Today 9: 105–110.
Evans, D. A., M. D. Ennis, and D. J. Mathre (1982) Asymmetric alkylation reactions of chiral imide enolates. A practical approach to the enantioselective synthesis of a-substituted carboxylic acid derivatives. J. Am. Chem. Soc. 104: 1737–1739.
Yuen, P., G. D. Kanter, C. P. Taylor, and M. G. Vartanian (1994) Enantioselective synthesis of PD144723: A potent stereospecific anticonvulsant. Bioorg. Med. Chem. Lett. 4: 823–826.
Hoekstra, M. S., D. M. Sobieray, M. A. Schwindt, T. A. Mulhern, T. M. Grote, B. K. Huckabee, V. S. Hendrickson, L. C. Franklin, E. J. Granger, and G. L. Karrick (1997) Chemical development of CI-1008, an enantiomerically Pure Anticonvulsant. Org. Process Res. Dev. 1: 26–38.
Huckabee, B. K. and D. M. Sobieray (1996) Methods of making (S)-3-(aminomethyl)-5-methylhexanoic acid. WO Patent 038,405.
Duan, L., Y. Ma, S. Shi, X. Yang, G. Zhang, and Y. Zhu (2006) Preparation method of pregabalin and its intermediate and the said intermediate. WO Patent 136,087.
Burns, M. P., J. K. Weaver, and J. W. Wong (2005) Stereoselective bioconversion of aliphatic dinitriles into cyano carboxylic acids. WO Patent 100,580.
Hedvati, L. and A. Fishman (2008) Use of enzymatic resolution for the preparation of intermediates of pregabalin. US Patent 0,026,433.
Martinez, C. A., S. Hu, Y. Dumond, J. Tao, P. Kelleher, and L. Tully (2008) Development of a chemoenzymatic manufacturing process for pregabalin. Org. Process Res. Dev. 12: 392–398.
Pollard, D. J. and J. M. Woodley (2007) Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol. 25: 66–73.
Wenda, S, S. Illner, A. Mell, and U. Kragl (2011) Industrial biotechnology–the future of green chemistry? Green Chem. 13: 3007–3047.
Woodley, J. M. (2008) New opportunities for biocatalysis: Making pharmaceutical processes greener. Trends Biotechnol. 26: 321–327.
Xie, Z., J. Feng, E. Garcia, M. Bernett, D. Yazbeck, and J. Tao (2006) Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid. J. Mol. Catal. B: Enz. 41: 75–80.
Hedvati, L., G. Sterimbaum, Y. Raizi, and R. Aminov (2010) Stereoselective enzymatic synthesis of (S) or (R)-iso-butyl-glutaric ester. US Patent 0,087,525.
Felluga, F., G. Pitacco, E. Valentin, and C. D. Venneri (2008) A facile chemoenzymatic approach to chiral non-racemic ß-alkyl-γ-amino acids and 2-alkylsuccinic acids. A concise synthesis of (S)-(+)-Pregabalin. Tetrahedron: Asymm. 19: 945–955.
Zheng, R. C., A. P. Li, Z. M. Wu, J. Y. Zheng, and Y. G. Zheng (2012) Enzymatic production of (S)-3-cyano-5-methylhexanoic acid ethyl ester with high substrate loading by immobilized Pseudomonas cepacia lipase. Tetrahedron: Asym. 23: 1517–1521.
Dunn, P. J. (2012) The importance of green chemistry in process research and development. Chem. Soc. Rev. 41: 1452–1461.
Tao, J. and J. H. Xu (2009) Biocatalysis in development of green pharmaceutical processes. Curr. Opin. Chem. Biol. 13: 43–50.
Zheng, R. C., T. Z. Wang, D. J. Fu, A. P. Li, X. J. Li, and Y. G. Zheng (2013) Biocatalytic synthesis of chiral intermediate of pregabalin with high substrate loading by a newly isolated Morgarella morganii ZJB-09203. Appl. Microbiol. Biotechnol. 97: 4839–4847.
Li, X. J., R. C. Zheng, H. Y. Ma, and Y. G. Zheng (2014) Engineering of Thermomyces lanuginosus lipase Lip: Creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin. Appl. Microbiol. Biotechnol. 98: 2473–2483.
Li, X. J., R. C. Zheng, Z. M. Wu, X. Ding, and Y. G. Zheng (2014) Thermophilic esterase from Thermomyces lanuginosus: Molecular cloning, functional expression and biochemical characterization. Protein Express. Purif. 101: 1–7.
Wu, W., L. Yang, G. Xu, and J. Wu (2011) Strain screening and culture condition optimization for the enantioselectively hydrolysis of (3R)-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester. China Biotechnol. 31: 86–93.
Sambrock, J. and D. W. Russel (2001) Molecular cloning: A laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
Oterholm, A. and Z. J. Ordal (1966) Improved method for method for detection of microbial lipolysis. J Dairy Sci. 49: 1281–1284.
Wagner, U. G., F. DiMaio, S. Kolkenbrock, and S. Fetzner (2014) Crystal structure analysis of EstA from Arthrobacter sp. Rue61a–an insight into catalytic promiscuity. FEBS Lett. 588: 1154–1160.
Laskowski, R. A., M. W. MacArthur, D. S. Moss, and J. M. Thornton (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26: 283–291.
Shine, J. and L. Dalgarno (1974) The 3’-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. U.S.A. 71: 1342–1346.
Chen, S. X. and B. Z. Shi (2007) Screening of strain producing a novel esterase with high enantioselectivity and molecular cloning of the enzyme gene. Acta Microbiol. Sin. 47: 452–455.
McKay, D. B., M. P. Jennings, E. A. Godfrey, I. C. MacRae, P. J. Rogers, and I. R. Beacham (1992) Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad. J. Gen. Microbiol. 138: 701–708.
Kim Y. S., H. B. Lee, K. D. Choi, S. Park, and O. J. Yoo (1994) Cloning of Pseudomonas fluorescens carboxylesterase gene and characterization of its product expressed in Escherichia coli. Biosci. Biotechnol. Biochem. 58: 111–116.
Berger, R., M. Hoffmann, and U. Keller (1998) Molecular analysis of a gene encoding a cell-bound esterase from Streptomyces chrysomallus. J. Bacteriol. 180: 6396–6399.
Singh, R., A. Saxena, and H. Singh (2009) Identification of group specific motifs in ß-lactamase family of proteins. J. Biomed. Sci. 16: 109–115.
Goldberg, S. D., W. Iannuccilli, T. Nguyen, J. Ju, and V. W. Cornish (2003) Identification of residues critical for catalysis in a class C ß-lactamase by combinatiorial scanning mutagenesis. Protein Sci. 12: 1633–1645.
Brenner, S. (1988) The molecular evolution of genes and proteins: A tale of two serines. Nature 334: 528–530.
Schütte, M. and S. Fetzner (2007) EstA from Arthrobacter nitroguajacolicus Rü61a, a thermo-and solvent-tolerant carboxylesterase related to class C ß-lactamases. Curr. Microbiol. 54: 230–236.
Oefner, C., A. D’Arcy, J.J. Daly, K. Gubernator, R. L. Charnas, I. Heinze, C. Hubschwerlen, and F. K. Winkler (1990) Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. Nature 343: 284–288.
Lobkovsky, E., E. M. Billings, P. C. Moews, J. Rahil, R. F. Pratt, and J. R. Knox (1994) Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: Mechanistic interpretation of a ß-lactamase transition-state analog. Biochem. 33: 6762–6772.
Dubus, A., P. Ledent, J. Lamotte-Brasseur, and J. M. Frère (1996) The roles of residues Tyr150, Glu272, and His314 in class C ß-lactamases. Proteins Struct. Funct. Genet. 25: 473–485.
Tsukamoto, K., K. Tachibana, N. Yamazaki, Y. Ishii, K. Ujiie, N. Nishida, and T. Sawai (1990) Role of lysine-67 in the active site of class C ß-lactamase from Citrobacter freundii GN346. Eur. J. Biochem. 88: 15–22.
Arpigny, J. L. and K. E. Jaeger (1999) Bacterial lipolytic enzymes: Classification and properties. Biochem. J. 343: 177–183.
Hausmann, S. and K. E. Jaeger (2010) Lipolytic enzymes from bacteria. pp. 1009–1126. In: K. N. Timmis (ed.). Handbook of Hydrocarbon and Lipid Microbiology. Springer Berlin Heidelberg, Berlin, Germany.
Yu, E.Y., M. A. Kwon, M. Lee, J. Y. Oh, J. E. Choi, J. Y. Lee, B. K. Song, D. H. Hahm, and J. K. Song (2011) Isolation and characterization of cold-active family VIII eaterases from an arctic soil metagenome. Appl. Microbiol. Biotechnol. 90: 573–581.
Biver, S. and M. Vandenbol (2013) Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library. J. Ind. Microbiol. Biotechnol. 40: 191–200.
Sharon, C., S. Furugoh, T. Yamakido, H. I. Ogawa, and Y. Kato (1998) Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis. J. Ind. Microbiol. Biotechnol. 20: 304–307.
Petersen, E. I., G. Valinger, B. Sölkner, G. Stubenrauch, and H. Schwab (2001) A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to ß-lactamases and DD-peptidases. J. Biotechnol. 89: 11–25.
Jiang, X., X. Xu, Y. Huo, Y. Wu, X. Zhu, X. Zhang, and M. Wu (2012) Identification and characterization of novel esterases from a deep-sea sediment metagenome. Arch. Microbiol. 194: 207–214.
Dong, H. P., Y. J. Wang, and Y. G. Zheng (2010) Enantioselective hydrolysis of diethyl 3-hydroxyglutarate to ethyl (S)-3-dydroxy-glutarate by immobilized Candida antarctica lipase B. J. Mol. Catal. B: Enz. 66: 90–94.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Xu, F., Chen, S., Xu, G. et al. Discovery and expression of a Pseudomonas sp. esterase as a novel biocatalyst for the efficient biosynthesis of a chiral intermediate of pregabalin. Biotechnol Bioproc E 20, 473–487 (2015). https://doi.org/10.1007/s12257-015-0069-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-015-0069-1