Applied Microbiology and Biotechnology

, Volume 98, Issue 4, pp 1719–1726 | Cite as

Improved thermostability of a Bacillus subtilis esterase by domain exchange

  • Markus G. Gall
  • Alberto Nobili
  • Ioannis V. Pavlidis
  • Uwe T. Bornscheuer
Biotechnologically relevant enzymes and proteins


A moderately thermostable esterase from Geobacillus stearothermophilus (BsteE) and its homolog from Bacillus subtilis (BsubE) show a high structural similarity with more than 95 % homology and 74 % amino acid identity. Interestingly, their thermal stability differs significantly by 30 °C in their melting temperature. In order to identify the positions that are responsible for this difference, most of the flexible amino acids assumed to confer instability were found to be in the cap region. For this reason, a 30 amino acid long cap domain fragment containing ten differing positions derived from BsteE was incorporated into the homologous gene encoding for the more labile BsubE by spliced overlap-extension PCR. The melting temperature of the two wild-type esterases and the mutant was evaluated by circular dichroism spectroscopy, while the kinetic parameters and the stability were determined with a photometric assay. The cap domain mutant maintained its activity, with a catalytic efficiency more similar to BsteE, while it exhibited an increase of the melting temperature by 4 °C compared to BsubE. Additional point mutations based on the differences of the parent enzymes gave a further increase of the thermostability up to 11 °C compared to BsubE; however, a significant reduction in activity was observed.


Activity Domain exchange Esterase Protein engineering Thermostability 



This work was supported by the European Union within the Marie-Curie Action, project "European Network on Directed Evolution of Functional Proteins" [grant number 215560]. We thank Stefan Saß from our group for his help in protein purification.

Supplementary material

253_2013_5053_MOESM1_ESM.docx (82 kb)
ESM 1 (DOCX 82 kb)


  1. Alsop E, Silver M, Livesay DR (2003) Optimized electrostatic surfaces parallel increased thermostability: a structural bioinformatic analysis. Protein Eng 16:871–874. doi: 10.1093/protein/gzg131 PubMedCrossRefGoogle Scholar
  2. Bommarius AS, Broering JM, Chaparro-Riggers JF, Polizzi KM (2006) High-throughput screening for enhanced protein stability. Curr Opin Biotechnol 17:606–610. doi: 10.1016/j.copbio.2006.10.001 PubMedCrossRefGoogle Scholar
  3. Deléage G, Geourjon C (1993) An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Comput Appl Biosci 9:197–199PubMedGoogle Scholar
  4. Dill KA, Ozkan SB, Weikl TR, Chodera JD, Voelz VA (2007) The protein folding problem: when will it be solved? Curr Opin Struct Biol 17:342–346. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  5. Eijsink VGH, Bjørk A, Gåseidnes S, Sirevåg R, Synstad B, van den Burg B, Vriend G (2004) Rational engineering of enzyme stability. J Biotechnol 113:105–120. doi: 10.1016/j.jbiotec.2004.03.026 PubMedCrossRefGoogle Scholar
  6. Gumulya Y, Reetz MT (2011) Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary pathways. ChemBioChem 12:2502–2510. doi: 10.1002/cbic.201100412 PubMedCrossRefGoogle Scholar
  7. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580PubMedCrossRefGoogle Scholar
  8. Henke E, Bornscheuer UT (2002) Esterases from Bacillus subtilis and B. stearothermophilus share high sequence homology but differ substantially in their properties. Appl Microbiol Biotechnol 60:320–326. doi: 10.1007/s00253-002-1126-1 PubMedCrossRefGoogle Scholar
  9. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59PubMedCrossRefGoogle Scholar
  10. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension sequences. Gene 77:61–68PubMedCrossRefGoogle Scholar
  11. Jäckel C, Bloom JD, Kast P, Arnold FH, Hilvert D (2010) Consensus protein design without phylogenetic bias. J Mol Biol 399:541–546. doi: 10.1016/j.jmb.2010.04.039 PubMedCentralPubMedCrossRefGoogle Scholar
  12. Jaenicke R, Böhm G (1998) The stability of proteins in extreme environments. Curr Opin Struct Biol 8:738–748PubMedCrossRefGoogle Scholar
  13. Jochens H, Aerts D, Bornscheuer UT (2010) Thermostabilization of an esterase by alignment-guided focussed directed evolution. Protein Eng Des Sel 23:903–909. doi: 10.1093/protein/gzq071 PubMedCrossRefGoogle Scholar
  14. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637. doi: 10.1002/bip.360221211 PubMedCrossRefGoogle Scholar
  15. Kourist R, Jochens H, Bartsch S, Kuipers R, Padhi SK, Gall M, Böttcher D, Joosten H-J, Bornscheuer UT (2010) The α/β-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering. ChemBioChem 11:1635–1643. doi: 10.1002/cbic.201000213 PubMedCrossRefGoogle Scholar
  16. Lehmann M, Pasamontes L, Lassen SF, Wyss M (2000) The consensus concept for thermostability engineering of proteins. Biochim Biophys Acta 1543:408–415PubMedCrossRefGoogle Scholar
  17. Li WF, Zhou XX, Lu P (2005) Structural features of thermozymes. Biotechnol Adv 23:271–281. doi: 10.1016/j.biotechadv.2005.01.002 PubMedCrossRefGoogle Scholar
  18. Liu P, Wang Y-F, Ewis HE, Abdelal AT, Lu C-D, Harrison RW, Weber IT (2004) Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30. J Mol Biol 342:551–561. doi: 10.1016/j.jmb.2004.06.069 PubMedCrossRefGoogle Scholar
  19. Mandrich L, Merone L, Pezzullo M, Cipolla L, Nicotra F, Rossi M, Manco G (2005) Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family. J Mol Biol 345:501–512. doi: 10.1016/j.jmb.2004.10.035 PubMedCrossRefGoogle Scholar
  20. Palm GJ, Fernández-Álvaro E, Bogdanović X, Bartsch S, Sczodrok J, Singh RK, Böttcher D, Atomi H, Bornscheuer UT, Hinrichs W (2011) The crystal structure of an esterase from the hyperthermophilic microorganism Pyrobaculum calidifontis VA1 explains its enantioselectivity. Appl Microbiol Biotechnol 91:1061–1072. doi: 10.1007/s00253-011-3337-9 PubMedCrossRefGoogle Scholar
  21. Pavlidis IV, Gournis D, Papadopoulos GK, Stamatis H (2009) Lipases in water-in-ionic liquid microemulsions: structural and activity studies. J Mol Catal B Enzym 60:50–56. doi: 10.1016/j.molcatb.2009.03.007 CrossRefGoogle Scholar
  22. Radestock S, Gohlke H (2008) Exploiting the link between protein rigidity and thermostability for data-driven protein engineering. Eng Life Sci 8:507–522. doi: 10.1002/elsc.200800043 CrossRefGoogle Scholar
  23. Radestock S, Gohlke H (2011) Protein rigidity and thermophilic adaptation. Proteins Struct Funct Bioinforma 79:1089–1108. doi: 10.1002/prot.22946 CrossRefGoogle Scholar
  24. Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2:891–903. doi: 10.1038/nprot.2007.72 PubMedCrossRefGoogle Scholar
  25. Reetz MT, Carballeira JD, Vogel A (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew Chem Int Ed 45:7745–7751. doi: 10.1002/anie.200602795 CrossRefGoogle Scholar
  26. Schwab T, Sterner R (2011) Stabilization of a metabolic enzyme by library selection in Thermus thermophilus. ChemBioChem 12:1581–1588. doi: 10.1002/cbic.201000770 PubMedCrossRefGoogle Scholar
  27. Sreerama N, Woody RW (1993) A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal Biochem 209:32–44. doi: 10.1006/abio.1993.1079 PubMedCrossRefGoogle Scholar
  28. Suzuki Y (1989) A general principle of increasing protein thermostability. Proc Jpn Acad 65:146–148CrossRefGoogle Scholar
  29. Vieille C, Zeikus JG (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43. doi: 10.1128/MMBR.65.1.1 PubMedCentralPubMedCrossRefGoogle Scholar
  30. Watanabe K, Chishiro K, Kitamura K, Suzuki Y (1991) Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006. J Biol Chem 266:24287–24294PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Markus G. Gall
    • 1
  • Alberto Nobili
    • 1
  • Ioannis V. Pavlidis
    • 1
  • Uwe T. Bornscheuer
    • 1
  1. 1.Department of Biotechnology and Enzyme Catalysis, Institute of BiochemistryGreifswald UniversityGreifswaldGermany

Personalised recommendations