Ene-reductase (gene oyeRo2a) from Rhodococcus opacus 1CP

Enzyme Description

Species

Extremophile

EC Number

1.6.99.1 ↗

Sequence

Length: 366 amino acids

MSVLFEPITFRGVTVPNRVWMAPMCQYSADVTGRDVGVPGDWHRTHLVTRAIGGAGLILTEATAVSPEGRISPADLGIWNDTQTEAFAEINRRIREFGAVPGIQLGHAGRKGSAHVPWRGGGSLDGDDRLSWQTVAPSAIGFGDHTPPAAATTADIRKVVADFAAAAERASRAGFKVVEIHAAHGYLLHQFLSPVSNHRTDEYGGSFAGRIRLLLEVVDAVRGVWPAELPVFVRVSATDWLSEEPGLDADSWTPDQTVSLVQALADLGVDLVDVSSGGVASARIPIGPGYQVPFARRIQNETTVPAAAVGLITEPEQAERIVESGEAVAVFLGRELLRDPYWPRKAALVLNAQVTPQIPAQYARAY

Anika Riedel et al. (2015) — Functional characterization and stability improvement of a 'thermophilic-like ' ene-reductase from Rhodococcus opacus 1CP

Frontiers in Microbiology · doi:10.3389/fmicb.2015.01073 ↗ · Activity - Classical Stability - Incubation

22 measurements ▼

Database ID

UniParc: UPI0007209AF6 ↗

Sequence Annotation

Explicit - Provided

Protein Source

Recombinant, host bacterium Escherichia coli BL21 (DE3)

Experimental Data (22 measurements)

22 measurements — page 2 of 2

Property	Assay	Solvent	Solvent Volume	Aqueous Reference	Measured Value	Units	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
Stability - Incubation	Activity after incubation (15 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase	Ethanol	20%	100	6	%	Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer	Incubation: 7.1, Assay: 7.1	Incubation: 25°C, Assay: 25°C	1 mM Maleimide	Succinimide	Assay: 140 μM NADPH	—	Non-incubated control (in %), assay in aqueous phase \| Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent
Stability - Incubation	Activity after incubation (20 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase	Ethanol	20%	100	0	%	Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer	Incubation: 7.1, Assay: 7.1	Incubation: 25°C, Assay: 25°C	1 mM Maleimide	Succinimide	Assay: 140 μM NADPH	—	Non-incubated control (in %), assay in aqueous phase \| Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

Activity - Classical

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

Ethanol

30%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Dimethyl Sulfoxide (DMSO)

40%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Dimethyl Sulfoxide (DMSO)

30%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Dimethyl Sulfoxide (DMSO)

20%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Dimethyl Sulfoxide (DMSO)

10%

100

101

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Ethanol

10%

100

106

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Ethanol

25%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Ethanol

20%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Ethanol

15%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Ethanol

10%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Ethanol

100

101

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Dimethylformamide (DMF)

10%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Dimethyl Sulfoxide (DMSO)

10%

100

101

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Acetonitrile

10%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Acetone

10%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Activity - Classical

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

Methanol

10%

100

25 mM phosphate buffer

7.1

25°C

1 mM Maleimide

Succinimide

140 μM NADPH

—

Classical aqueous control (in %)

Stability - Incubation

Activity after incubation (1 minute at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase

Ethanol

20%

100

Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer

Incubation: 7.1, Assay: 7.1

Incubation: 25°C, Assay: 25°C

1 mM Maleimide

Succinimide

Assay: 140 μM NADPH

—

Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

Stability - Incubation

Activity after incubation (3 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase

Ethanol

20%

100

Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer

Incubation: 7.1, Assay: 7.1

Incubation: 25°C, Assay: 25°C

1 mM Maleimide

Succinimide

Assay: 140 μM NADPH

—

Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

Stability - Incubation

Activity after incubation (5 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase

Ethanol

20%

100

Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer

Incubation: 7.1, Assay: 7.1

Incubation: 25°C, Assay: 25°C

1 mM Maleimide

Succinimide

Assay: 140 μM NADPH

—

Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

Stability - Incubation

Activity after incubation (10 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase

Ethanol

20%

100

Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer

Incubation: 7.1, Assay: 7.1

Incubation: 25°C, Assay: 25°C

1 mM Maleimide

Succinimide

Assay: 140 μM NADPH

—

Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

Stability - Incubation

Activity after incubation (15 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase

Ethanol

20%

100

Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer

Incubation: 7.1, Assay: 7.1

Incubation: 25°C, Assay: 25°C

1 mM Maleimide

Succinimide

Assay: 140 μM NADPH

—

Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

Stability - Incubation

Activity after incubation (20 minutes at 25°C), measured by absorbance spectrophotometry (NADPH absorbance measurement, 340 nm) in aqueous phase

Ethanol

20%

100

Incubation: 25 mM phosphate buffer, Assay: 25 mM phosphate buffer

Incubation: 7.1, Assay: 7.1

Incubation: 25°C, Assay: 25°C

1 mM Maleimide

Succinimide

Assay: 140 μM NADPH

—

Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether the assay was in aqueous phase or in the presence of organic solvent

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Visualization : Activity — Classical

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Visualization : Stability — Incubation

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

Structure

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Ene-reductase (gene oyeRo2a) from Rhodococcus opacus 1CP

Enzyme Description

Sequence

Experimental Data (22 measurements)

Visualization : Activity — Classical

Visualization : Stability — Incubation

Structure

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