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

Abstract

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.

Keywords

Activity Domain exchange Esterase Protein engineering Thermostability 

Notes

Acknowledgments

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)

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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

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