Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3) | Clostridium beijerinckii |
Protein Variants | Comment | Organism |
---|---|---|
additional information | computational design of highly stable and soluble alcohol dehydrogenase mutants for NADPH regeneration, several mutants are created and evaluated, overview. A NADP+-specific ADH from Clostridium beijerinckii is engineered for cofactor recycling using an automated algorithm. The mutant is selected for large-scale production and industrial usage | Clostridium beijerinckii |
S24P/G182A/G196A/H222D/S250E/S254R | the mutant CbADH-6M exhibits a favorable soluble and highly active expression with an activity of 46.3 U/ml, which is 16times higher than the wild-type (2.9 U/ml) and a more stable protein conformation with an enhanced thermal stability. The activity of CbADH-6M is upgraded to 2401.8 U/ml by high cell density fermentation strategy using recombinant Escherichia coli. Improving CbADH solubility by chaperone buffering. The efficiency for NADPH regeneration of the mutant enzyme is testified in the synthesis of some fine chiral aromatic alcohols coupling with another ADH from Lactobacillus kefir (LkADH). Method optimization, overview. Although the Asp225-His222 salt bridge in the original wild-type CbADH protein disappears due to the amino acid substitution H222D, the replacement of H222D, S250E and S254R leads to the formation of four new salt bridges including Arg254-Glu250, Arg254-Asp225, Arg254-Glu280 and Arg254-Asp222, which constitute a salt bridge network centered on Arg254 | Clostridium beijerinckii |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
soluble | - |
Clostridium beijerinckii | - |
- |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | required | Clostridium beijerinckii |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Clostridium beijerinckii | - |
- |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
activity of the crude recombinant mutant CbADH-6M is 46.3 U/ml, that of the crude recombinant wild-type enzyme is 2.9 U/ml | Clostridium beijerinckii |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
1-(3,4,5-trifluorophenyl)ethan-1-one + NADPH + H+ | - |
Clostridium beijerinckii | (1S)-1-(3,4,5-trifluorophenyl)ethan-1-ol + NADP+ | 90.8% conversion, 99% ee | r | |
1-(3-chlorophenyl)ethan-1-one + NADPH + H+ | - |
Clostridium beijerinckii | (1S)-1-(3-chlorophenyl)ethan-1-ol + NADP+ | 99.5% conversion, 99% ee | r | |
1-(4-fluorophenyl)ethan-1-one + NADPH + H+ | - |
Clostridium beijerinckii | (1S)-1-(4-fluorophenyl)ethan-1-ol + NADP+ | 99.5% conversion, 99% ee | r | |
1-(4-methylphenyl)ethan-1-one + NADPH + H+ | - |
Clostridium beijerinckii | (1S)-1-(4-methylphenyl)ethan-1-ol + NADP+ | 99.5% conversion, 99% ee | r | |
1-phenylethan-1-one + NADPH + H+ | - |
Clostridium beijerinckii | (1S)-1-phenylethan-1-ol + NADP+ | 99.5% conversion, 99% ee | r | |
additional information | the enatiomeric excess with all substrates is over 99% | Clostridium beijerinckii | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
dimer | - |
Clostridium beijerinckii |
Synonyms | Comment | Organism |
---|---|---|
CbADH | - |
Clostridium beijerinckii |
NADPH-dependent ADH | - |
Clostridium beijerinckii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
35 | - |
assay at | Clostridium beijerinckii |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
63.7 | - |
half-life of the wild-type enzyme is 60 min | Clostridium beijerinckii |
66.8 | - |
half-life of the enzyme mutant CbADH-6M is 60 min | Clostridium beijerinckii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Clostridium beijerinckii |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Clostridium beijerinckii | |
NADPH | - |
Clostridium beijerinckii |