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

Bisagni, Serena LU ; Abolhalaj, Milad LU ; de Brevern, Alexandre G.; Rebehmed, Joseph; Hatti-Kaul, Rajni LU and Mamo, Gashaw LU (2017) In ChemistrySelect 2(24). p.7169-7177
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... (More)

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.

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organization
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type
Contribution to journal
publication status
published
subject
keywords
Baeyer-Villiger monooxygenases, cyclohexanone, Dietzia species, saturation mutagenesis, structural models
in
ChemistrySelect
volume
2
issue
24
pages
9 pages
publisher
Wiley-Blackwell Publishing Ltd
external identifiers
  • scopus:85041841657
DOI
10.1002/slct.201701212
language
English
LU publication?
yes
id
bf8c234d-646a-4577-8748-47c133e06005
date added to LUP
2018-02-22 13:06:43
date last changed
2019-03-19 03:49:47
@article{bf8c234d-646a-4577-8748-47c133e06005,
  abstract     = {<p>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.</p>},
  author       = {Bisagni, Serena and Abolhalaj, Milad and de Brevern, Alexandre G. and Rebehmed, Joseph and Hatti-Kaul, Rajni and Mamo, Gashaw},
  keyword      = {Baeyer-Villiger monooxygenases,cyclohexanone,Dietzia species,saturation mutagenesis,structural models},
  language     = {eng},
  number       = {24},
  pages        = {7169--7177},
  publisher    = {Wiley-Blackwell Publishing Ltd},
  series       = {ChemistrySelect},
  title        = {Enhancing the Activity of a Dietzia sp. D5 Baeyer-Villiger Monooxygenase towards Cyclohexanone by Saturation Mutagenesis},
  url          = {http://dx.doi.org/10.1002/slct.201701212},
  volume       = {2},
  year         = {2017},
}