Details
| Original language | English |
|---|---|
| Pages (from-to) | 44-51 |
| Number of pages | 8 |
| Journal | Molecular Catalysis |
| Volume | 468 |
| Early online date | 22 Feb 2019 |
| Publication status | Published - May 2019 |
| Externally published | Yes |
Abstract
Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and product tolerance compared to the wild type. The wild type CHMO and the four variants were applied in a convergent cascade coupled with an alcohol dehydrogenase from Thermoanaerobacter ethanolicus for ECL synthesis. M15 DS and M16 DS variants performed better than the wild type and the parental variants under operational conditions. Subsequently, the cascade was further optimized by means of Design of Experiments, doubling the product yield (21 mM, 27%) with a reduced cofactor amount of 0.5 mM NADP+.
Keywords
- Baeyer-Villiger monooxygenase, Enzymatic cascades, Protein engineering, Stability, ε-Caprolactone
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemical Engineering(all)
- Process Chemistry and Technology
- Chemistry(all)
- Physical and Theoretical Chemistry
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In: Molecular Catalysis, Vol. 468, 05.2019, p. 44-51.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Characterization of new Baeyer-Villiger monooxygenases for lactonizations in redox-neutral cascades
AU - Engel, Jennifer
AU - Mthethwa, Katlego S.
AU - Opperman, Diederik J.
AU - Kara, Selin
N1 - Funding Information: This work was supported by Deutsche Forschungsgemeinschaft (DFG) (grant number KA 4399/1-1).JE acknowledges Deutscher Akademischer Austauschdienst (DAAD) for financial support of the research stay at University of the Free State (South Africa). Funding Information: This work was supported by Deutsche Forschungsgemeinschaft (DFG) (grant number KA 4399/1-1 ).
PY - 2019/5
Y1 - 2019/5
N2 - Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and product tolerance compared to the wild type. The wild type CHMO and the four variants were applied in a convergent cascade coupled with an alcohol dehydrogenase from Thermoanaerobacter ethanolicus for ECL synthesis. M15 DS and M16 DS variants performed better than the wild type and the parental variants under operational conditions. Subsequently, the cascade was further optimized by means of Design of Experiments, doubling the product yield (21 mM, 27%) with a reduced cofactor amount of 0.5 mM NADP+.
AB - Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and product tolerance compared to the wild type. The wild type CHMO and the four variants were applied in a convergent cascade coupled with an alcohol dehydrogenase from Thermoanaerobacter ethanolicus for ECL synthesis. M15 DS and M16 DS variants performed better than the wild type and the parental variants under operational conditions. Subsequently, the cascade was further optimized by means of Design of Experiments, doubling the product yield (21 mM, 27%) with a reduced cofactor amount of 0.5 mM NADP+.
KW - Baeyer-Villiger monooxygenase
KW - Enzymatic cascades
KW - Protein engineering
KW - Stability
KW - ε-Caprolactone
UR - http://www.scopus.com/inward/record.url?scp=85061786408&partnerID=8YFLogxK
U2 - 10.1016/j.mcat.2019.02.006
DO - 10.1016/j.mcat.2019.02.006
M3 - Article
AN - SCOPUS:85061786408
VL - 468
SP - 44
EP - 51
JO - Molecular Catalysis
JF - Molecular Catalysis
SN - 2468-8231
ER -