The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase

Sandy Schmidt, Maika Genz, Kathleen Balke, Uwe T. Bornscheuer

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

Original languageEnglish
Pages (from-to)199-211
Number of pages13
JournalJournal of Biotechnology
Volume214
DOIs
Publication statusPublished - 20 Nov 2015
Externally publishedYes

Fingerprint

Mixed Function Oxygenases
Disulfides
Mutation
Enzymes
Acinetobacter
Lactones
Ketones
Freezing
Oxidation
Esters
Biocatalysts
Enantioselectivity
Computational methods
Scaffolds
Labeling
Industrial applications
Temperature
Melting point
cyclohexanone oxygenase
Substrates

Keywords

  • Acinetobacter
  • Bacterial Proteins
  • Cysteine
  • Disulfides
  • Enzyme Stability
  • Escherichia coli
  • Mutation
  • Oxygenases
  • Protein Engineering
  • Recombinant Proteins
  • Serine
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase. / Schmidt, Sandy; Genz, Maika; Balke, Kathleen; Bornscheuer, Uwe T.

In: Journal of Biotechnology, Vol. 214, 20.11.2015, p. 199-211.

Research output: Contribution to journalArticleResearchpeer-review

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title = "The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase",
abstract = "Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60{\%} activity after incubation with 25 mM H2O2 whereas the wild type retained only 16{\%}. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33{\%} residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.",
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T1 - The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase

AU - Schmidt, Sandy

AU - Genz, Maika

AU - Balke, Kathleen

AU - Bornscheuer, Uwe T.

N1 - Copyright © 2015 Elsevier B.V. All rights reserved.

PY - 2015/11/20

Y1 - 2015/11/20

N2 - Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

AB - Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

KW - Acinetobacter

KW - Bacterial Proteins

KW - Cysteine

KW - Disulfides

KW - Enzyme Stability

KW - Escherichia coli

KW - Mutation

KW - Oxygenases

KW - Protein Engineering

KW - Recombinant Proteins

KW - Serine

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.jbiotec.2015.09.026

DO - 10.1016/j.jbiotec.2015.09.026

M3 - Article

VL - 214

SP - 199

EP - 211

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

ER -