Applied Microbiology and Biotechnology

, Volume 101, Issue 6, pp 2333–2342 | Cite as

Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester

  • Jinyeong Kim
  • Seung-hyeon Seok
  • Eunsoo Hong
  • Tae Hyeon Yoo
  • Min-duk SeoEmail author
  • Yeonwoo RyuEmail author
Biotechnologically relevant enzymes and proteins


Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.


Esterase Enzyme catalysis Enantioselectivity Crystal structure X-ray crystallography 



This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (No. NRF-2011-0014093).

Compliance with ethical standards

This study was funded by National Research Foundation of Korea (NRF-2011-0014093). All authors declare that we have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Cryst D 66(2):213–221. doi: 10.1107/S0907444909052925 CrossRefGoogle Scholar
  2. Adams PD, Grosse-Kunstleve RW, Hung L-W, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Cryst D 58(11):1948–1954. doi: 10.1107/S0907444902016657 CrossRefGoogle Scholar
  3. Alber T, Dao-pin S, Wilson K, Wozniak JA, Cook SP, Matthews BW (1987) Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme. Nature 330(6143):41–46CrossRefPubMedGoogle Scholar
  4. Alvarez-Macarie E, Augier-Magro V, Baratti J (1999) Characterization of a thermostable esterase activity from the moderate Thermophile Bacillus licheniformis. Biosci Biotechnol Biochem 63(11):1865–1870. doi: 10.1271/bbb.63.1865 CrossRefPubMedGoogle Scholar
  5. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(1):177–183CrossRefPubMedPubMedCentralGoogle Scholar
  6. Ateşlier ZBB, Metin K (2006) Production and partial characterization of a novel thermostable esterase from a thermophilic Bacillus sp. Enzym Microb Technol 38(5):628–635. doi: 10.1016/j.enzmictec.2005.07.015 CrossRefGoogle Scholar
  7. Bjerrum OJ, Bhakdi S (1983) Detergent Immunoelectrophoresis of membrane proteins—general principles. Scand J Immunol 17:289–301. doi: 10.1111/j.1365-3083.1983.tb04032.x CrossRefGoogle Scholar
  8. Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH (2005) Evolving strategies for enzyme engineering. Curr Opin Struct Biol 15(4):447–452. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  9. Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26(1):73–81. doi: 10.1111/j.1574-6976.2002.tb00599.x CrossRefPubMedGoogle Scholar
  10. Bornscheuer UT, Pohl M (2001) Improved biocatalysts by directed evolution and rational protein design. Curr Opin Chem Biol 5(2):137–143. doi: 10.1016/S1367-5931(00)00182-4 CrossRefPubMedGoogle Scholar
  11. 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(1–2):248–254. doi: 10.1016/0003-2697(76)90527-3 CrossRefPubMedGoogle Scholar
  12. Chayen NE, Shaw Stewart PD, Maeder DL, Blow DM (1990) An automated system for micro-batch protein crystallization and screening. J Appl Crystallogr 23(4):297–302. doi: 10.1107/S0021889890003260 CrossRefGoogle Scholar
  13. Choi G-S, Kim J-Y, Kim J-H, Ryu Y-W, Kim G-J (2003) Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester. Protein Expr Purif 29(1):85–93. doi: 10.1016/S1046-5928(03)00009-3 CrossRefPubMedGoogle Scholar
  14. Cosme J, Johnson EF (2000) Engineering Microsomal Cytochrome P450 2C5 to Be a soluble, Monomeric enzyme: mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding. J Biol Chem 275(4):2545–2553. doi: 10.1074/jbc.275.4.2545 CrossRefPubMedGoogle Scholar
  15. D’Amico S, Marx J-C, Gerday C, Feller G (2003) Activity-stability relationships in Extremophilic enzymes. J Biol Chem 278(10):7891–7896. doi: 10.1074/jbc.M212508200 CrossRefPubMedGoogle Scholar
  16. Eijsink VGH, Bjørk A, Gåseidnes S, Sirevåg R, Synstad B, Burg B, Vriend G (2004) Rational engineering of enzyme stability. J Biotechnol 113(1–3):105–120. doi: 10.1016/j.jbiotec.2004.03.026 CrossRefPubMedGoogle Scholar
  17. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Cryst D 66(4):486–501. doi: 10.1107/S0907444910007493 CrossRefGoogle Scholar
  18. Fernández L, Beerthuyzen MM, Brown J, Siezen RJ, Coolbear T, Holland R, Kuipers OP (2000) Cloning, characterization, controlled Overexpression, and inactivation of the major Tributyrin esterase Gene of Lactococcus lactis. Appl Environ Microbiol 66(4):1360–1368. doi: 10.1128/aem.66.4.1360-1368.2000 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Fukuchi S, Nishikawa K (2001) Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria. J Mol Biol 309(4):835–843. doi: 10.1006/jmbi.2001.4718 CrossRefPubMedGoogle Scholar
  20. 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(2):313–318. doi: 10.1016/j.jmb.2003.12.048 CrossRefPubMedGoogle Scholar
  21. Grosdidier A, Zoete V, Michielin O (2011) SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res 39(suppl 2):W270–W277. doi: 10.1093/nar/gkr366 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzym Microb Technol 39(2):235–251. doi: 10.1016/j.enzmictec.2005.10.016 CrossRefGoogle Scholar
  23. Hayball P (2012) Chirality and Nonsteroidal anti-inflammatory drugs. Drugs 52(5):47–58. doi: 10.2165/00003495-199600525-00006 Google Scholar
  24. Jaeger K-E, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53(1):315–351. doi: 10.1146/annurev.micro.53.1.315 CrossRefPubMedGoogle Scholar
  25. Kim J, Deng L, Hong E, Ryu Y (2015a) Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197. Protein Expr Purif 116:90–97. doi: 10.1016/j.pep.2015.08.009 CrossRefPubMedGoogle Scholar
  26. Kim J, Kim S, Yoon S, Hong E, Ryu Y (2015b) 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(15):6293–6301. doi: 10.1007/s00253-015-6422-7 CrossRefPubMedGoogle Scholar
  27. Kim S, Joo S, Yoon HC, Ryu Y, Kim KK, Kim TD (2007) Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase. Acta Cryst F 63(7):579–581. doi: 10.1107/S1744309107026152 CrossRefGoogle Scholar
  28. Kim S-B, Lee W, Ryu Y-W (2008) Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus. J Microbiol 46(1):100–107. doi: 10.1007/s12275-007-0185-5 CrossRefPubMedGoogle Scholar
  29. Kim Y-J, Choi G-S, Kim S-B, Yoon G-S, Kim Y-S, Ryu Y-W (2006) Screening and characterization of a novel esterase from a metagenomic library. Protein Expr Purif 45(2):315–323. doi: 10.1016/j.pep.2005.06.008 CrossRefPubMedGoogle Scholar
  30. Koudelakova T, Chaloupkova R, Brezovsky J, Prokop Z, Sebestova E, Hesseler M, Khabiri M, Plevaka M, Kulik D, Kuta Smatanova I, Rezacova P, Ettrich R, Bornscheuer UT, Damborsky J (2013) Engineering enzyme stability and resistance to an organic Cosolvent by modification of residues in the access tunnel. Angew Chem Int Ed 52(7):1959–1963. doi: 10.1002/anie.201206708 CrossRefGoogle Scholar
  31. Kramer Ryan M, Shende Varad R, Motl N, Pace CN, Scholtz JM (2012) Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility. Biophys J 102(8):1907–1915. doi: 10.1016/j.bpj.2012.01.060 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Krebsfänger N, Schierholz K, Bornscheuer UT (1998) Enantioselectivity of a recombinant esterase from Pseudomonas fluorescens towards alcohols and carboxylic acids. J Biotechnol 60(1–2):105–111. doi: 10.1016/S0168-1656(97)00192-2 CrossRefPubMedGoogle Scholar
  33. Kumar R, Singh R, Kaur J (2013) Characterization and molecular modelling of an engineered organic solvent tolerant, thermostable lipase with enhanced enzyme activity. J Mol Catal B Enzym 97:243–251. doi: 10.1016/j.molcatb.2013.09.001 CrossRefGoogle Scholar
  34. Liu AMF, Somers NA, Kazlauskas RJ, Brush TS, Zocher F, Enzelberger MM, Bornscheuer UT, Horsman GP, Mezzetti A, Schmidt-Dannert C, Schmid RD (2001) Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes. Tetrahedron Asymmetry 12(4):545–556. doi: 10.1016/S0957-4166(01)00072-6 CrossRefGoogle Scholar
  35. Liu Y-Y, Xu J-H, Hu Y (2000) Enhancing effect of Tween-80 on lipase performance in enantioselective hydrolysis of ketoprofen ester. J Mol Catal B Enzym 10(5):523–529. doi: 10.1016/S1381-1177(00)00093-X CrossRefGoogle Scholar
  36. Ma J, Wu L, Guo F, Gu J, Tang X, Jiang L, Liu J, Zhou J, Yu H (2012) Enhanced enantioselectivity of a carboxyl esterase from Rhodobacter sphaeroides by directed evolution. Appl Microbiol Biotechnol 97(11):4897–4906. doi: 10.1007/s00253-012-4396-2 CrossRefPubMedGoogle Scholar
  37. Mauleón D, Artigas R, García ML, Carganico G (1996) Preclinical and clinical development of Dexketoprofen. Drugs 52(5):24–46. doi: 10.2165/00003495-199600525-00005 CrossRefPubMedGoogle Scholar
  38. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40(4):658–674. doi: 10.1107/S0021889807021206 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Meiering EM, Serrano L, Fersht AR (1992) Effect of active site residues in barnase on activity and stability. J Mol Biol 225(3):585–589. doi: 10.1016/0022-2836(92)90387-Y CrossRefPubMedGoogle Scholar
  40. Mohr P, Rösslein L, Tamma C (1989) Kinetic resolution of racemic β,γ-epoxy esters with pig liver esterase (PLE, e.C. Tetrahedron Lett 30(19):2513–2516. doi: 10.1016/S0040-4039(01)80438-X CrossRefGoogle Scholar
  41. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol 276:307–326. doi: 10.1016/S0076-6879(97)76066-X CrossRefGoogle Scholar
  42. Patel RN (2001) ChemInform abstract: Stereoselective biocatalysis for synthesis of some chiral pharmaceutical intermediates. ChemInform 32(21). doi: 10.1002/chin.200121256
  43. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  44. Sathishkumar M, Jayabalan R, Mun SP, Yun SE (2010) Role of bicontinuous microemulsion in the rapid enzymatic hydrolysis of (R,S)-ketoprofen ethyl ester in a micro-reactor. Bioresour Technol 101(20):7834–7840. doi: 10.1016/j.biortech.2010.05.032 CrossRefPubMedGoogle Scholar
  45. Shoichet BK, Baase WA, Kuroki R, Matthews BW (1995) A relationship between protein stability and protein function. Proc Natl Acad Sci 92(2):452–456. doi: 10.1073/pnas.92.2.452 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Tao W, Shengxue F, Duobin M, Xuan Y, Congcong D, Xihua W (2013) Characterization of a new thermophilic and acid tolerant esterase from Thermotoga maritima capable of hydrolytic resolution of racemic ketoprofen ethyl ester. J Mol Catal B Enzym 85–86:23–30. doi: 10.1016/j.molcatb.2012.08.006 CrossRefGoogle Scholar
  47. Tokuriki N, Tawfik DS (2009) Stability effects of mutations and protein evolvability. Curr Opin Struct Biol 19(5):596–604. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  48. Toledo MV, José C, Collins SE, Ferreira ML, Briand LE (2015) Towards a green enantiomeric esterification of R/S-ketoprofen: a theoretical and experimental investigation. J Mol Catal B Enzym 118:52–61. doi: 10.1016/j.molcatb.2015.05.003 CrossRefGoogle Scholar
  49. Ulmer K (1983) Protein engineering. Science 219(4585):666–671. doi: 10.1126/science.6572017 CrossRefPubMedGoogle Scholar
  50. Varley PG, Pain RH (1991) Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus. J Mol Biol 220(2):531–538. doi: 10.1016/0022-2836(91)90028-5 CrossRefPubMedGoogle Scholar
  51. Wei Y, Contreras JA, Sheffield P, Osterlund T, Derewenda U, Kneusel RE, Matern U, Holm C, Derewenda ZS (1999) Crystal structure of brefeldin a esterase, a bacterial homolog of the mammalian hormone-sensitive lipase. Nat Struct Mol Biol 6(4):340–345CrossRefGoogle Scholar
  52. Yeung DT, Josse D, Nicholson JD, Khanal A, McAndrew CW, Bahnson BJ, Lenz DE, Cerasoli DM (2004) Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model. Biochim Biophys Acta 1702(1):67–77. doi: 10.1016/j.bbapap.2004.08.002 CrossRefPubMedGoogle Scholar
  53. Yoon S, Kim S, Park S, Hong E, Kim J, Kim S, Yoo TH, 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 CrossRefGoogle Scholar
  54. Yoon S, Kim S, Ryu Y, Kim TD (2007) Identification and characterization of a novel (S)-ketoprofen-specific esterase. Int J Biol Macromol 41(1):1–7. doi: 10.1016/j.ijbiomac.2006.11.010 CrossRefPubMedGoogle Scholar
  55. Zhang J, Guan R, Tan Z, Yu Y, Hou Z, Qi Z, Wang S (2005) Purification and properties of lipases/esterases from a Bacillus strain for enantioselective resolution of (S)-ketoprofen. Artif Cell Blood Sub 33(4):435–445. doi: 10.1080/10731190500290105 CrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Molecular Science and TechnologyAjou UniversitySuwonSouth Korea
  2. 2.College of PharmacyAjou UniversitySuwonSouth Korea

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