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Applied Microbiology and Biotechnology

, Volume 102, Issue 12, pp 5121–5131 | Cite as

Biocatalytic versatility of engineered and wild-type tyrosinase from R. solanacearum for the synthesis of 4-halocatechols

  • Reeta Davis
  • Susan Molloy
  • Blathnaid Quigley
  • Jasmina Nikodinovic-Runic
  • Francisco Solano
  • Kevin E. O’Connor
Biotechnological products and process engineering
  • 151 Downloads

Abstract

We evaluated the kinetic characteristics of wild type (WT) and three engineered variants (RVC10, RV145, and C10_N322S) of tyrosinase from Ralstonia solanacearum and their potential as biocatalysts to produce halogenated catechols. RV145 exhibited a 3.6- to 14.5-fold improvement in catalytic efficiency (kcat/Km) with both reductions in Km and increases in kcat compared to WT, making it the best R. solanacearum tyrosinase variant towards halogenated phenols. RVC10 also exhibited increases in catalytic efficiency with all the tested phenols. A single-mutation variant (C10_N322S) exhibited the greatest improvement in kcat but lowest improvement in catalytic efficiency due to an increase in Km compared to WT. Consistent with kinetic characteristics, biotransformation experiments showed that RV145 was a superior biocatalyst in comparison to WT. To prevent through conversion of the catechol to quinone, ascorbic acid (AA) was added to the biotransformation medium in 1:2 (substrate:AA) ratio resulting in a catechol yield of > 90%. Flask experiments with 10 mM 4-iodophenol and 10 μg/mL of the RV145 enzyme yielded 9.5 mM 4-iodocatechol in the presence of 20 mM AA in 30 min. Similarly, 10 mM 4-fluorophenol was completely consumed by 20 μg/mL of RV145 enzyme and yielded 9.2 mM 4-fluorocatechol in the presence of 20 mM AA in 80 min. The biotransformation of 20 mM 4-fluorphenol was incomplete (93%) and the yield of 4-flurocatechol was 87.5%. The 4-halophenol conversion rates and product yields obtained in this study are the highest reported using tyrosinase or any other enzyme.

Keywords

Tyrosinase Enzyme engineering Enzyme catalysis 4-Halophenol 4-Halocatechol 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Reeta Davis
    • 1
  • Susan Molloy
    • 1
  • Blathnaid Quigley
    • 1
  • Jasmina Nikodinovic-Runic
    • 1
    • 2
  • Francisco Solano
    • 3
  • Kevin E. O’Connor
    • 1
  1. 1.School of Biomolecular and Biomedical ScienceUniversity College DublinDublin 4Ireland
  2. 2.Institute for Molecular Genetics and Genetic EngineeringUniversity of BelgradeBelgradeSerbia
  3. 3.Department of Biochemistry and Molecular Biology B and ImmunologyUniversity of MurciaMurciaSpain

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