Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli strain SG13009 | Pseudomonas fluorescens |
Protein Variants | Comment | Organism |
---|---|---|
A28S | mutant shows decarboxylase activity towards 2-keto acids | Pseudomonas fluorescens |
A480I | reduced lyase activity | Pseudomonas fluorescens |
F484I | reduced lyase activity | Pseudomonas fluorescens |
H286A | reduced lyase activity | Pseudomonas fluorescens |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | inhibitory above 7.5 mM | Pseudomonas fluorescens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.045 | - |
Benzoin | mutant enzyme A480I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.047 | - |
Benzoin | mutant enzyme A28S, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.048 | - |
Benzoin | wild type enzyme, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.055 | - |
Benzoin | mutant enzyme H286A, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.067 | - |
Benzoin | mutant enzyme F484I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.3 | - |
2-hydroxypropiophenone | wild type enzyme, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.6 | - |
2-hydroxypropiophenone | mutant enzyme A28S, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
0.9 | - |
2-hydroxypropiophenone | mutant enzyme H286A, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
1.2 | - |
2-hydroxypropiophenone | mutant enzyme F484I, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
5 | - |
2-hydroxypropiophenone | mutant enzyme A480I, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
10 | - |
benzaldehyde | in 50 mM potassium phosphate at 30°C and pH 7.5 | Pseudomonas fluorescens |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | dependent, 1-5 mM | Pseudomonas fluorescens |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
54000 | - |
4 * 54000, gel filtration | Pseudomonas fluorescens |
59800 | - |
His-tagged BAL subunit, SDS-PAGE and MALDI-TOF-MS | Pseudomonas fluorescens |
216000 | - |
gel filtration | Pseudomonas fluorescens |
Organic Solvent | Comment | Organism |
---|---|---|
DMSO | the addition of 20% (v/v) DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL | Pseudomonas fluorescens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas fluorescens | - |
strain biovar I | - |
Purification (Comment) | Organism |
---|---|
immobilized metal ion chromatography, after which the enzyme shows only 35-45% of its activity, however, activity is completely regained after incubation in the presence of Mg2+ and thiamine diphosphate | Pseudomonas fluorescens |
Storage Stability | Organism |
---|---|
30°C, in cofactor-free potassium phosphate buffer at pH 7.0, 3 h, 65% loss of activity | Pseudomonas fluorescens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxypropiophenone | - |
Pseudomonas fluorescens | ? | - |
? | |
benzaldehyde + (R)-2,2-dimethyl-1,3-dioxolan-4-carbaldehyde | - |
Pseudomonas fluorescens | acyloin + benzoin | - |
? | |
benzoin | - |
Pseudomonas fluorescens | benzaldehyde + benzaldehyde | - |
r | |
additional information | unable to cleave acetoin | Pseudomonas fluorescens | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
tetramer | 4 * 54000, gel filtration | Pseudomonas fluorescens |
Synonyms | Comment | Organism |
---|---|---|
benzaldehyde lyase | the wild type enzyme does not show any decarboxylase activity towards 2-keto acids | Pseudomonas fluorescens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
35 | - |
maximal activity for the benzoin lyase reaction | Pseudomonas fluorescens |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
a concentration of 1 mM MgSO4 and 0.01 mM thiamine diphosphate is sufficient to keep BAL stable at 25°C | Pseudomonas fluorescens |
37 | - |
reaction temperature shall not exceed 37°C, in the presence of 5 mM MgSO4 and 0.1 mM thiamine diphosphate the enzyme's activity is reduced by only 15% over 3 h at 37°C, however, a concentration of 1 mM MgSO4 and 0.01 mM thiamine diphosphate leads to more than 40% inactivation within 3 h at 37°C | Pseudomonas fluorescens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.6 | - |
2-hydroxypropiophenone | mutant enzyme A28S, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
2.8 | - |
2-hydroxypropiophenone | mutant enzyme H286A, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
3.4 | - |
2-hydroxypropiophenone | wild type enzyme, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
4 | - |
2-hydroxypropiophenone | mutant enzyme F484I, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
4.9 | - |
2-hydroxypropiophenone | mutant enzyme A480I, at 30°C and pH 7.5 | Pseudomonas fluorescens | |
6.6 | - |
Benzoin | mutant enzyme A480I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
9.5 | - |
Benzoin | mutant enzyme A28S, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
22 | - |
Benzoin | mutant enzyme F484I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
56.4 | - |
Benzoin | mutant enzyme H286A, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens | |
66.9 | - |
Benzoin | wild type enzyme, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 | Pseudomonas fluorescens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
pH-optimum of both the lyase and the ligase reaction, in potassium phosphate, Tris, and imidazole buffers | Pseudomonas fluorescens |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
6 | 8 | in potassium phosphate, Tris, and imidazole buffers, pH values below 6 lead to complete inactivation after about one day, while the enzyme shows some activity when kept above pH 8 | Pseudomonas fluorescens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | dependent, the optimal concentration is 0.1-5 mM, a significant loss of activity is observed with a concentration below 0.01 mM | Pseudomonas fluorescens |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Pseudomonas fluorescens | isoelectric focusing | - |
4.6 |