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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

Abstract

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.

Keywords

Esterase Enzyme catalysis Enantioselectivity Crystal structure X-ray crystallography 

Notes

Acknowledgements

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.

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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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