Application | Comment | Organism |
---|---|---|
synthesis | two co-expressed enantiocomplementary carbonyl reductases, BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis) and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) are used for asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) or (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity and coupled with cofactor regeneration by GDH. Enantiomerically pure (R)-1-phenyl-1,2-ethanediol ((R)-PED) can be used as a building block for the preparation of (R)-norfluoxetine, (R)-fluoxetine, and beta-lactam antibiotics | Gluconobacter oxydans |
Cloned (Comment) | Organism |
---|---|
gene GoSCR, recombinant expression of His-tagged enzyme GoSCR in Escherichia coli strain BL21 (DE3), coexpression with 2,3-butanediol dehydrogenase (BDHA) from Bacillus subtilis for asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) or (S)-1-phenyl-1,2-ethanediol ((S)-PED) | Gluconobacter oxydans |
Protein Variants | Comment | Organism |
---|---|---|
additional information | in vitro bioreduction of 2-hydroxyacetophenone (2-HAP) is catalyzed by GoSCR coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration. The two coexpressed enantiocomplementary carbonyl reductases, BDHA (2, 3-butanediol dehydrogenase from Bacillus subtilis) and GoSCR are used for asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) or (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, method optimization, overview. Products (R)-PED and (S)-PED are obtained with 99% yield, over 99% enantiomeric excess and 18.0 g/l/h volumetric productivity. The reaction is carried out in 5 ml sodium phosphate buffer (pH 7.0, 100 mM) at 30°C, containing 10 U/ml BDHA (cell free extract of Escherichia coli (BDHA)), 15 U/ml GoSCR (cell free extract of Escherichia coli (GoSCR)), 10 U/ml GDH (cell free extract of Escherichia coli (GDH)), 50-200 mM 2-HAP (with 10% DMSO as cosolvent), 60-250 mM D-glucose. Strong tolerance of BDHA and GoSCR against high substrate concentration | Gluconobacter oxydans |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Gluconobacter oxydans | |
0.8 | - |
2-hydroxyacetophenone | pH 7.0, 25°C, recombinant His-tagged enzyme | Gluconobacter oxydans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxyacetophenone + NADH + H+ | Gluconobacter oxydans | - |
(S)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | Gluconobacter oxydans 621H | - |
(S)-1-phenyl-1,2-ethanediol + NAD+ | - |
? |
Organic Solvent | Comment | Organism |
---|---|---|
additional information | DMSO is selected as the co-solvent, it does not affect the activity at up 30% v/v, but is inhibitory above | Gluconobacter oxydans |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Gluconobacter oxydans | Q5FNX9 | - |
- |
Gluconobacter oxydans 621H | Q5FNX9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged GoSCR from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and dialysis | Gluconobacter oxydans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxyacetophenone + NADH + H+ | - |
Gluconobacter oxydans | (S)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | 99% enantiomeric excess | Gluconobacter oxydans | (S)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | - |
Gluconobacter oxydans 621H | (S)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | 99% enantiomeric excess | Gluconobacter oxydans 621H | (S)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
additional information | an enantiocomplementary carbonyl reductase, polyol dehydrogenase (GoSCR) from Gluconobacter oxydans is discovered to convert 2-hydroxyacetophenone (2-HAP) to (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity. No activity with NADPH | Gluconobacter oxydans | ? | - |
- |
|
additional information | an enantiocomplementary carbonyl reductase, polyol dehydrogenase (GoSCR) from Gluconobacter oxydans is discovered to convert 2-hydroxyacetophenone (2-HAP) to (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity. No activity with NADPH | Gluconobacter oxydans 621H | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
GoSCR | - |
Gluconobacter oxydans |
More | cf. EC 1.1.1.76 | Gluconobacter oxydans |
polyol dehydrogenase | - |
Gluconobacter oxydans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
45 | - |
reduction of 2-HAP | Gluconobacter oxydans |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
purified recombinant His-tagged enzyme, inactivation within 2 h | Gluconobacter oxydans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
reduction of 2-HAP | Gluconobacter oxydans |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
7 | - |
purified recombinant His-tagged enzyme, more than 80% residual activity after 18 h at pH 7.0. The activity decreases significantly in acidic circumstances | Gluconobacter oxydans |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | no activity with NADPH | Gluconobacter oxydans | |
NADH | dependent on | Gluconobacter oxydans |