Cyclohexanone monooxygenase from Acinetobacter calcoaceticus

Enzyme Description

Species

Extremophile

EC Number

1.14.13.22 ↗

Sequence

Length: 543 amino acids

MSQKMDFDAIVIGGGFGGLYAVKKLRDELELKVQAFDKATDVAGTWYWNRYPGALTDTETHLYCYSWDKELLQSLEIKKKYVQGPDVRKYLQQVAEKHDLKKSYQFNTAVQSAHYNEADALWEVTTEYGDKYTARFLITALGLLSAPNLPNIKGINQFKGELHHTSRWPDDVSFEGKRVGVIGTGSTGVQVITAVAPLAKHLTVFQRSAQYSVPIGNDPLSEEDVKKIKDNYDKSLGWCMNSALAFALNESTVPAMSVSAEERKAVFEKAWQTGGGFRFMFETFGDIATNMEANIEAQNFIKGKIAEIVKDPAIAQKLMPQDLYAKRPLCDSGYYNTFNRDNVRLEDVKANPIVEITENGVKLENGDFVELDMLICATGFDAVDGNYVRMDIQGKNGLAMKDYWKEGPSSYMGVTVNNYPNMFMVLGPNGPFTNLPPSIESQVEWISDTIQYTVENNVESIEATKEAEEQWTQTCANIAEMTLFPKAQSWIFGANIPGKKNTVYFYLGGLKEYRTCASNCKNHAYEGFDIQLQRSDIKQPANA

Francesco Secundo et al. (2010) — Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus : A comparative study

Biotechnology and Bioengineering · doi:10.1002/bit.22963 ↗ · Activity - Classical Stability - C50 Activity + Stability - Incubation

27 measurements ▼

Database ID

UniProt: P12015 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Recombinant, host bacterium Escherichia coli BL21

Experimental Data (27 measurements)

27 measurements — page 2 of 2

Property	Assay	Solvent	Solvent Volume	Aqueous Reference	Measured Value	Units	Extraction method	Data type	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
Activity + Stability - Incubation	Activity after incubation (24 hours at 25°C) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent	Methanol	5 % v/v	1.83	0.0	U/mg	Digital	Continuous	Incubation: 50 mM Tris-HCl, Assay: 50 mM Tris-HCl	Incubation: 8.6, Assay: 8.6	Incubation: 25°C, Assay: 25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Water-incubated control in water (in U/mg), assay in organic solvent
Activity + Stability - Incubation	Activity after incubation (24 hours at 25°C) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent	Methanol	1 % v/v	1.83	1.8	U/mg	Digital	Continuous	Incubation: 50 mM Tris-HCl, Assay: 50 mM Tris-HCl	Incubation: 8.6, Assay: 8.6	Incubation: 25°C, Assay: 25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Water-incubated control in water (in U/mg), assay in organic solvent
Stability - C50	Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)	Methanol	—	—	7.0	% (v/v)	Direct	Continuous	50 mM Tris-HCl	8.6	25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Not applicable control (in % (v/v))
Stability - C50	Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)	Ethanol	—	—	3.0	% (v/v)	Direct	Continuous	50 mM Tris-HCl	8.6	25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Not applicable control (in % (v/v))
Stability - C50	Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)	1,4-Dioxane	—	—	5.0	% (v/v)	Direct	Continuous	50 mM Tris-HCl	8.6	25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Not applicable control (in % (v/v))
Stability - C50	Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)	Acetonitrile	—	—	6.0	% (v/v)	Direct	Continuous	50 mM Tris-HCl	8.6	25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Not applicable control (in % (v/v))
Stability - C50	Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)	2,2,2-Trifluoroethanol (TFE)	—	—	1.0	% (v/v)	Direct	Continuous	50 mM Tris-HCl	8.6	25°C	Molecular oxygen (O2), Thioanisole	H2O , methyl phenyl sulfoxide	0.1 mM NADPH	—	Not applicable control (in % (v/v))

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Acetonitrile

5 % v/v

3.0

1.84

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

1,4-Dioxane

20 % v/v

3.0

0.09

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

1,4-Dioxane

10 % v/v

3.0

0.65

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

1,4-Dioxane

5 % v/v

3.0

1.36

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

2,2,2-Trifluoroethanol (TFE)

20 % v/v

3.0

0.0

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

2,2,2-Trifluoroethanol (TFE)

10 % v/v

3.0

0.05

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

2,2,2-Trifluoroethanol (TFE)

5 % v/v

3.0

0.11

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

2,2,2-Trifluoroethanol (TFE)

1 % v/v

3.0

0.3

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Ethanol

20 % v/v

3.0

0.24

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Ethanol

10 % v/v

3.0

0.48

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Ethanol

1 % v/v

3.0

2.26

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Acetonitrile

1 % v/v

3.0

2.54

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Acetonitrile

10 % v/v

3.0

0.33

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Acetonitrile

20 % v/v

3.0

0.06

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

1 % v/v

3.0

3.4

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

2 % v/v

3.0

3.66

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

5 % v/v

3.0

1.84

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

10 % v/v

3.0

1.1

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

20 % v/v

3.0

0.6

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity - Classical

Activity (in U/mg) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Ethanol

5 % v/v

3.0

0.91

U/mg

Digital

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Classical aqueous control (in U/mg)

Activity + Stability - Incubation

Activity after incubation (24 hours at 25°C) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

5 % v/v

1.83

0.0

U/mg

Digital

Continuous

Incubation: 50 mM Tris-HCl, Assay: 50 mM Tris-HCl

Incubation: 8.6, Assay: 8.6

Incubation: 25°C, Assay: 25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Water-incubated control in water (in U/mg), assay in organic solvent

Activity + Stability - Incubation

Activity after incubation (24 hours at 25°C) measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in the presence of organic solvent

Methanol

1 % v/v

1.83

1.8

U/mg

Digital

Continuous

Incubation: 50 mM Tris-HCl, Assay: 50 mM Tris-HCl

Incubation: 8.6, Assay: 8.6

Incubation: 25°C, Assay: 25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Water-incubated control in water (in U/mg), assay in organic solvent

Stability - C50

Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)

Methanol

—

7.0

% (v/v)

Direct

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Not applicable control (in % (v/v))

Stability - C50

Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)

Ethanol

—

3.0

% (v/v)

Direct

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Not applicable control (in % (v/v))

Stability - C50

Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)

1,4-Dioxane

—

5.0

% (v/v)

Direct

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Not applicable control (in % (v/v))

Stability - C50

Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)

Acetonitrile

—

6.0

% (v/v)

Direct

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Not applicable control (in % (v/v))

Stability - C50

Concentration (in % v/v) for which 50% of enzyme activity is lost, as measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm)

2,2,2-Trifluoroethanol (TFE)

—

1.0

% (v/v)

Direct

Continuous

50 mM Tris-HCl

8.6

25°C

Molecular oxygen (O2), Thioanisole

H2O, methyl phenyl sulfoxide

0.1 mM NADPH

—

Not applicable control (in % (v/v))

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Visualization : Activity — Classical

Francesco Secundo et al. (2010)

One bar per measurement. Colour = solvent, shade = solvent volume.

Visualization : Activity + Stability — Incubation

Francesco Secundo et al. (2010)

One bar per measurement. Colour = solvent, shade = solvent volume.

Visualization : Stability — C₅₀

Francesco Secundo et al. (2010)

One bar per measurement. Colour = solvent, shade = temperature. Hover for details.

Elvira Romero et al. (2016) — Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

Angewandte Chemie International Edition · doi:10.1002/anie.201608951 ↗ · Activity + Stability - Conversion

5 measurements ▼

Database ID

UniProt: P12015 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Recombinant, host bacterium Escherichia coli NEB 10-β

Experimental Data (5 measurements)

5 measurements

Property	Assay	Solvent	Solvent Volume	Aqueous Reference	Measured Value	Units	Extraction method	Data type	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
Activity + Stability - Conversion	Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography	Methanol	30 % v/v	75	3	%	Direct	Continuous	50 mM Tris-HCl buffer, 20 mM phosphite	7	17°C	11 mM 2-Butanone	Ethyl Acetate	150 µM NADPH	200 rpm	Classical aqueous control (in %)
Activity + Stability - Conversion	Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography	Ethanol	30 % v/v	75	1	%	Direct	Continuous	50 mM Tris-HCl buffer, 20 mM phosphite	7	17°C	11 mM 2-Butanone	Ethyl Acetate	150 µM NADPH	200 rpm	Classical aqueous control (in %)
Activity + Stability - Conversion	Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography	1,4-Dioxane	15 % v/v	75	7	%	Direct	Continuous	50 mM Tris-HCl buffer, 20 mM phosphite	7	17°C	11 mM 2-Butanone	Ethyl Acetate	150 µM NADPH	200 rpm	Classical aqueous control (in %)
Activity + Stability - Conversion	Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography	1,3-Dioxane	15 % v/v	75	1	%	Direct	Continuous	50 mM Tris-HCl buffer, 20 mM phosphite	7	17°C	11 mM 2-Butanone	Ethyl Acetate	150 µM NADPH	200 rpm	Classical aqueous control (in %)
Activity + Stability - Conversion	Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography	2-Methyl-1,3-Dioxolane	15 % v/v	75	26	%	Direct	Continuous	50 mM Tris-HCl buffer, 20 mM phosphite	7	17°C	11 mM 2-Butanone	Ethyl Acetate	150 µM NADPH	200 rpm	Classical aqueous control (in %)

Activity + Stability - Conversion

Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography

Methanol

30 % v/v

Direct

Continuous

50 mM Tris-HCl buffer, 20 mM phosphite

17°C

11 mM 2-Butanone

Ethyl Acetate

150 µM NADPH

200 rpm

Classical aqueous control (in %)

Activity + Stability - Conversion

Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography

Ethanol

30 % v/v

Direct

Continuous

50 mM Tris-HCl buffer, 20 mM phosphite

17°C

11 mM 2-Butanone

Ethyl Acetate

150 µM NADPH

200 rpm

Classical aqueous control (in %)

Activity + Stability - Conversion

Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography

1,4-Dioxane

15 % v/v

Direct

Continuous

50 mM Tris-HCl buffer, 20 mM phosphite

17°C

11 mM 2-Butanone

Ethyl Acetate

150 µM NADPH

200 rpm

Classical aqueous control (in %)

Activity + Stability - Conversion

Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography

1,3-Dioxane

15 % v/v

Direct

Continuous

50 mM Tris-HCl buffer, 20 mM phosphite

17°C

11 mM 2-Butanone

Ethyl Acetate

150 µM NADPH

200 rpm

Classical aqueous control (in %)

Activity + Stability - Conversion

Conversion after incubation (48 hours at 17°C) in the presence of organic solvent, measured by gas chromatography

2-Methyl-1,3-Dioxolane

15 % v/v

Direct

Continuous

50 mM Tris-HCl buffer, 20 mM phosphite

17°C

11 mM 2-Butanone

Ethyl Acetate

150 µM NADPH

200 rpm

Classical aqueous control (in %)

Visualization : Activity + Stability — Conversion

Elvira Romero et al. (2016)

One bar per measurement. Colour = solvent, shade = solvent volume.

Structure

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Cyclohexanone monooxygenase from Acinetobacter calcoaceticus

Enzyme Description

Sequence

Experimental Data (27 measurements)

Visualization : Activity — Classical

Visualization : Activity + Stability — Incubation

Visualization : Stability — C50

Experimental Data (5 measurements)

Visualization : Activity + Stability — Conversion

Structure

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Visualization : Stability — C₅₀