Application | Comment | Organism |
---|---|---|
synthesis | two coexpressed 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 | Bacillus subtilis subsp. subtilis |
Cloned (Comment) | Organism |
---|---|
gene bdhA, recombinant expression of His-tagged enzyme BDHA in Escherichia coli strain BL21 (DE3), co-expression with polyol dehydrogenase (GoSCR) from Gluconobacter oxydans 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) | Bacillus subtilis subsp. subtilis |
Protein Variants | Comment | Organism |
---|---|---|
additional information | in vitro bioreduction of 2-hydroxyacetophenone (2-HAP) is catalyzed by BDHA coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration. The two coexpressed enantiocomplementary carbonyl reductases, BDHA 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, 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 co-solvent), 60-250 mM D-glucose. Strong tolerance of BDHA and GoSCR against high substrate concentration | Bacillus subtilis subsp. subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Bacillus subtilis subsp. subtilis | |
1 | - |
2-hydroxyacetophenone | pH 7.0, 25°C, recombinant His-tagged enzyme | Bacillus subtilis subsp. subtilis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxyacetophenone + NADH + H+ | Bacillus subtilis subsp. subtilis | - |
(R)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | Bacillus subtilis subsp. subtilis 168 | - |
(R)-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 | Bacillus subtilis subsp. subtilis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis subsp. subtilis | O34788 | - |
- |
Bacillus subtilis subsp. subtilis 168 | O34788 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged BDHA from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and dialysis | Bacillus subtilis subsp. subtilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxyacetophenone + NADH + H+ | - |
Bacillus subtilis subsp. subtilis | (R)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | over 99% enantiomeric excess | Bacillus subtilis subsp. subtilis | (R)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | - |
Bacillus subtilis subsp. subtilis 168 | (R)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
2-hydroxyacetophenone + NADH + H+ | over 99% enantiomeric excess | Bacillus subtilis subsp. subtilis 168 | (R)-1-phenyl-1,2-ethanediol + NAD+ | - |
? | |
additional information | an enantiocomplementary carbonyl reductase, 2,3-butanediol dehydrogenase (BDHA) from Bacillus subtilis is discovered to convert 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) with excellent stereochemical selectivity. No activity with NADPH | Bacillus subtilis subsp. subtilis | ? | - |
- |
|
additional information | an enantiocomplementary carbonyl reductase, 2,3-butanediol dehydrogenase (BDHA) from Bacillus subtilis is discovered to convert 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) with excellent stereochemical selectivity. No activity with NADPH | Bacillus subtilis subsp. subtilis 168 | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
2,3-butanediol dehydrogenase | - |
Bacillus subtilis subsp. subtilis |
BdhA | - |
Bacillus subtilis subsp. subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
reduction of 2-HAP | Bacillus subtilis subsp. subtilis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
purified recombinant His-tagged enzyme, inactivation within 2 h | Bacillus subtilis subsp. subtilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
reduction of 2-HAP | Bacillus subtilis subsp. subtilis |
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 | Bacillus subtilis subsp. subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | no activity with NADPH | Bacillus subtilis subsp. subtilis | |
NADH | dependent on | Bacillus subtilis subsp. subtilis |