Abstract
Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = −16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.
Similar content being viewed by others
References
Akoh CC, Lee GC, Liaw YC, Huang TH, Shaw JF (2004) GDSL family of serine esterases/lipases. Prog Lipid Res 43:534–552. doi:10.1016/j.plipres.2004.09.002
Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(Pt 1):177–183
Auriol M, Filali-Meknassi Y, Adams CD, Tyagi RD (2006) Natural and synthetic hormone removal using the horseradish peroxidase enzyme: temperature and pH effects. Water Res 40:2847–2856. doi:10.1016/j.watres.2006.05.032
Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26:73–81. doi:10.1111/j.1574-6976.2002.tb00599.x
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Caldwell J, Hutt AJ, Fournel-Gigleux S (1988) The metabolic chiral inversion and dispositional enantioselectivity of the 2-arylpropionic acids and their biological consequences. Biochem Pharmacol 37:105–114. doi:10.1016/0006-2952(88)90762-9
Emond S, André I, Jaziri K, Potocki-Véronèse G, Mondon P, Bouayadi K, Kharrat H, Monsan P, Remaud-Simeon M (2008) Combinatorial engineering to enhance thermostability of amylosucrase. Protein Sci 17:967–976. doi:10.1110/ps.083492608
Evans CT, Wisdom RA, Stabler PJ, Carganico G (1996) Ketoprofen resolution by ester hydrolysis using Trichosporon laibacchii. Google Patents
Godoy-Ruiz R, Perez-Jimenez R, Ibarra-Molero B, Sanchez-Ruiz JM (2004) Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations. J Mol Biol 336:313–318. doi:10.1016/j.jmb.2003.12.048
Gu Q-M, Sih CJ (1992) Improving the enantioselectivity of the Candida Cylindracea lipase via chemical modification. Biocatal Biotransform 6:115–126. doi:10.3109/10242429209014887
Guo F, Xu H, Xu H, Yu H (2013) Compensation of the enantioselectivity-activity trade-off in the directed evolution of an esterase from Rhodobacter sphaeroides by site-directed saturation mutagenesis. Appl Microbiol Biotechnol 97:3355–3362. doi:10.1007/s00253-012-4516-z
Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzym Microb Technol 39:235–251. doi:10.1016/j.enzmictec.2005.10.016
Hayball P (1996) Chirality and nonsteroidal anti-inflammatory drugs. Drugs 52:47–58. doi:10.2165/00003495-199600525-00006
Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13:390–397
Ji J, Fan K, Tian X, Zhang X, Zhang Y, Yang K (2012) Iterative combinatorial mutagenesis as an effective strategy for generation of deacetoxycephalosporin C synthase with improved activity toward penicillin G. Appl Environ Microbiol 78:7809–7812. doi:10.1128/aem. 02122-12
Jiang Y, Morley KL, Schrag JD, Kazlauskas RJ (2011) Different active-site loop orientation in serine hydrolases versus acyltransferases. Chembiochem 12:768–776. doi:10.1002/cbic.201000693
Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL Repository and associated resources. Nucleic Acids Res 37:D387–D392. doi:10.1093/nar/gkn750
Kim YW, Choi JH, Kim JW, Park C, Kim JW, Cha B, Lee SB, Oh BH, Moon TW, Park KH (2003) Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Appl Environ Microbiol 69:4866–4874
Kim S-B, Lee W, Ryu Y-W (2008) Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus. J Microbiol 46:100–107. doi:10.1007/s12275-007-0185-5
Koeller KM, Wong C-H (2001) Enzymes for chemical synthesis. Nature 409:232–240
Kotik M, Zhao W, Iacazio G, Archelas A (2013) Directed evolution of metagenome-derived epoxide hydrolase for improved enantioselectivity and enantioconvergence. J Mol Catal B Enzym 91:44–51. doi:10.1016/j.molcatb.2013.02.006
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lee EG, Won HS, Ro H-S, Ryu Y-W, Chung BH (2003) Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP. J Mol Catal B Enzym 26:149–156. doi:10.1016/j.molcatb.2003.05.004
Margolin AL (1993) Enzymes in the synthesis of chiral drugs. Enzym Microb Technol 15:266–280
Mauleon D, Artigas R, Garcia ML, Carganico G (1996) Preclinical and clinical development of dexketoprofen. Drugs 52(Suppl 5):24–45, discussion 45–26
Meiering EM, Serrano L, Fersht AR (1992) Effect of active site residues in barnase on activity and stability. J Mol Biol 225:585–589
Quin MB, Schmidt-Dannert C (2011) Engineering of biocatalysts: from evolution to creation. ACS Catal 1:1017–1021. doi:10.1021/cs200217t
Shiraki K (2001) Conformational stability of a hyperthermophilic protein in various conditions for denaturation. Denki Kagaku oyobi Kogyo Butsuri Kagaku 69:949
Urakami T, Komagata K (1981) Electrophoretic comparison of enzymes in the gram negative methanol-utilizing bacteria. J Gen Appl Microbiol 27:381–403. doi:10.2323/jgam.27.381
Verger R (1997) ‘Interfacial activation’ of lipases: facts and artifacts. Trends Biotechnol 15:32–38. doi:10.1016/S0167-7799(96)10064-0
Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43. doi:10.1128/MMBR. 65.1.1-43.2001
Wang M, Si T, Zhao H (2012) Biocatalyst development by directed evolution. Bioresour Technol 115:117–125. doi:10.1016/j.biortech.2012.01.054
Yiyun C, Tongwen X, Rongqiang F (2005) Polyamidoamine dendrimers used as solubility enhancers of ketoprofen. Eur J Med Chem 40:1390–1393. doi:10.1016/j.ejmech.2005.08.002
Yoon S, Kim S, Park S, Hong E, Kim J, Kim S, Yoo T, Ryu Y (2014) Improving the enantioselectivity of an esterase toward (S)-ketoprofen ethyl ester through protein engineering. J Mol Catal B Enzym 100:25–31. doi:10.1016/j.molcatb.2013.11.008
Acknowledgments
This study was supported by the Basic Science Research Program (2011-0014093) and the Priority Research Centers Program (2012-0006687) through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kim, J., Kim, S., Yoon, S. et al. Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases. Appl Microbiol Biotechnol 99, 6293–6301 (2015). https://doi.org/10.1007/s00253-015-6422-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-015-6422-7