3-ketosteroid-Ξ1-dehydrogenase (gene kstD1) from Arthrobacter simplex 156
Enzyme Description
Sequence
MDWAEEYDVLVAGSGAGGMAGTYTAAREGLSVCLVEAGDKFGGTTAYSGGGGAWFPANPVLLRAGTDDTIEDALEYYRAVVGDRTPADLQETYVRGGAGLVAYLEEDDHFSFESYPWPDYFGDAPKARRDGQRHIIPTPLPVPSAPELREVVRGPLDNDRLGTPQPDDLFIGGRALVARFLTALATYPHATLVRETALAELVVEDGVVVGAIVETDGVRRAIRARRGVLLAAGGFEANDELRQKYGVPGVARDTMGPPTNVGAAHQAAIAVGADTDLMGEAWWSPGLTHPDGRSAFALWFTGGIFVDGAGRRFVNESAPYDRLGRAVIDHLTEGGVTPRYWMVYDHKEGSIPPVRATNVSMVDEEQYVAAGLWHTADTLPELAALIGVPADALVATVARFNELVADGYDADFGRGGEAYDRFFSGGEPPLVSIDEGPFHAAAFGISDLGTKGGLRTDTSARVLTADGTPIGGLYAAGNTMAAPSGTTYPGGGNPIGTSMLFSHLAVRHMGTEDAR
Shuhong Mao et al. (2018)
β Engineering of 3-ketosteroid-β1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates
Microbial Cell Factories
Β· doi:10.1186/s12934-018-0981-0 β
Β· Activity - Classical Stability - Incubation
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Database ID
UniProt: P77815 β
Sequence Annotation
Explicit - Provided GenBank Accession Number
Protein Source
Recombinant, host bacterium Escherichia coli BL21 (DE3)
Experimental Data (32 measurements)
32 measurements β page 1 of 2
| Property | Assay | Solvent | Solvent Volume | Aqueous Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 35% | 100.0 | 4.4 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 30% | 100.0 | 14.6 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 25% | 100.0 | 23.7 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 20% | 100.0 | 44.0 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 15% | 100.0 | 59.5 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 10% | 100.0 | 75.6 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 7.5% | 100.0 | 83.6 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Activity - Classical | Activity measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in the presence of organic solvent | Methanol | 5% | 100.0 | 94.2 | % | 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | 7.5 | 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Classical aqueous control (in %) | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethylformamide (DMF) | 10% | 100.0 | 131.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Acetone | 50% | 100.0 | 60.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Acetone | 30% | 100.0 | 122.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Acetone | 20% | 100.0 | 132.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Acetone | 10% | 100.0 | 126.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethylformamide (DMF) | 50% | 100.0 | 116.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethylformamide (DMF) | 30% | 100.0 | 137.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethylformamide (DMF) | 20% | 100.0 | 146.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Methanol | 10% | 100.0 | 124.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethyl Sulfoxide (DMSO) | 50% | 100.0 | 126.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethyl Sulfoxide (DMSO) | 30% | 100.0 | 148.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
| Stability - Incubation | Activity after incubation (2 hours at 4Β°C), measured by absorbance spectrophotometry (reduction of DCPIP to DCPIPH2, due to reduction of PMS into PMSH2 by the enzyme-FADH2, causing a decrease in absorbance, measured at 600 nm) in aqueous phase | Dimethyl Sulfoxide (DMSO) | 20% | 100.0 | 169.0 | % | Incubation: 50 mM Tris-HCl buffer, Assay: 50 mM TrisβHCl buffer, 2% Methanol, 1.5 mM phenazine methosulphate, 40 ΞΌM 6-dichlorophenolindophenol | Incubation: 8, Assay: 7.5 | Incubation: 4Β°C, Assay: 30Β°C | 0.5 mM Androstenedione | Boldenone | FAD | β | Non-incubated control (in %), assay in aqueous phase | Control with 2% methanol for subtrate solubilization |
Visualization : Activity β Classical
One bar per measurement. Colour = solvent, shade = solvent volume.
Visualization : Stability β Incubation
One bar per measurement. Colour = solvent, shade = solvent volume.