Enhancing the Activity of a Dietzia sp. D5 Baeyer-Villiger Monooxygenase towards Cyclohexanone by Saturation Mutagenesis

Research output: Contribution to journalArticle

Abstract

A recombinant Baeyer-Villiger monooxygenase, BVMO4 from Dietzia sp. D5 has been previously reported. The enzyme exhibited good thermostability and was active with a wide range of cyclic ketone substrates but catalysed poorly the conversion of cyclohexanone to caprolactone. The present work focuses on protein engineering of BVMO4 to improve the conversion of cyclohexanone. Thus, a structural model of BVMO4 was generated and used in combination with literature information on substrate specificity of BVMOs to identify ‘hotspots’ whose mutation would influence substrate specificity. Site saturation mutagenesis was performed on 12 selected sites and 528 mutants were screened with expected coverage of about 98 %. About one-fourth of the mutants screened exhibited more than 50 % increase in cyclohexanone oxidation activity. Compared to the wild type BVMO, the best mutants, Y499I, Y499F and Y499 L have shown about 12-fold increase for caprolactone production.

Details

Authors
Organisations
External organisations
  • Johnson Matthey
  • Institut Gustave Roussy
  • Paris Diderot University
  • Institut National de la Transfusion Sanguine (INTS)
  • Lebanese American University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biocatalysis and Enzyme Technology

Keywords

  • Baeyer-Villiger monooxygenases, cyclohexanone, Dietzia species, saturation mutagenesis, structural models
Original languageEnglish
Pages (from-to)7169-7177
Number of pages9
JournalChemistrySelect
Volume2
Issue number24
Publication statusPublished - 2017
Publication categoryResearch
Peer-reviewedYes