Cloned (Comment) | Organism |
---|---|
CP gene, expression of His-tagged wild-type and mutant enzymes in Escherichia coli | Cellulomonas uda |
Protein Variants | Comment | Organism |
---|---|---|
A397V | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows reduced activity with lactose compared to the wild-type enzyme | Cellulomonas uda |
A397V/T508A/A512T/D557N/N667T/G681S | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows 3fold increased activity with lactose and 6fold decreased activity with cellobiose compared to the wild-type enzyme | Cellulomonas uda |
A512T | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows similar activity with lactose compared to the wild-type enzyme | Cellulomonas uda |
D557N | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows similar activity with lactose compared to the wild-type enzyme | Cellulomonas uda |
G681S | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows reduced activity with lactose compared to the wild-type enzyme | Cellulomonas uda |
additional information | random, site-saturation, and site-directed mutagenesis for directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase activity, useful for the production of alpha-D-galactose 1-phosphate, overview. Location of mutations in the 3D structure, overview | Cellulomonas uda |
T508A/N667T | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows increased activity with lactose compared to the wild-type enzyme | Cellulomonas uda |
T508I/N667A | random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows 7.5fold increased activity with lactose compared to the wild-type enzyme | Cellulomonas uda |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.7 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant T508I/N667A | Cellulomonas uda | |
2.9 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant T508A/N667T | Cellulomonas uda | |
2.9 | - |
cellobiose | pH 6.6, 37°C, recombinant wild-type enzyme | Cellulomonas uda | |
3.3 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S | Cellulomonas uda | |
228 | - |
lactose | pH 6.6, 37°C, recombinant wild-type enzyme | Cellulomonas uda | |
230 | - |
lactose | pH 6.6, 37°C, recombinant mutant T508I/N667A | Cellulomonas uda | |
254 | - |
lactose | pH 6.6, 37°C, recombinant mutant T508A/N667T | Cellulomonas uda | |
330 | - |
lactose | pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S | Cellulomonas uda |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
cellobiose + phosphate | Cellulomonas uda | - |
alpha-D-glucose 1-phosphate + D-glucose | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Cellulomonas uda | Q7WTR6 | DSM 20108 | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by immobilized metal ion affinity chromatography | Cellulomonas uda |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.033 | - |
purifed recombinant wild-type enzyme, substrate lactose | Cellulomonas uda |
0.1 | - |
purifed recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S, substrate lactose | Cellulomonas uda |
0.171 | - |
purifed recombinant mutant T508I/N667A, substrate lactose | Cellulomonas uda |
0.249 | - |
purifed recombinant mutant T508A/N667T, substrate lactose | Cellulomonas uda |
1.35 | - |
purifed recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S, substrate cellobiose | Cellulomonas uda |
1.71 | - |
purifed recombinant mutant T508I/N667A, substrate cellobiose | Cellulomonas uda |
2.61 | - |
purifed recombinant mutant T508A/N667T, substrate cellobiose | Cellulomonas uda |
7.78 | - |
purifed recombinant wild-type enzyme, substrate cellobiose | Cellulomonas uda |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cellobiose + phosphate | - |
Cellulomonas uda | alpha-D-glucose 1-phosphate + D-glucose | - |
r | |
lactose + phosphate | - |
Cellulomonas uda | alpha-D-galactose 1-phosphate + D-glucose | - |
r |
Synonyms | Comment | Organism |
---|---|---|
lactose phosphorylase | created through directed evolution | Cellulomonas uda |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Cellulomonas uda |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.109 | - |
lactose | pH 6.6, 37°C, recombinant wild-type enzyme | Cellulomonas uda | |
0.35 | - |
lactose | pH 6.6, 37°C, recombinant mutant T508I/N667A | Cellulomonas uda | |
0.53 | - |
lactose | pH 6.6, 37°C, recombinant mutant T508A/N667T | Cellulomonas uda | |
1.11 | - |
lactose | pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S | Cellulomonas uda | |
2.01 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant T508I/N667A | Cellulomonas uda | |
2.61 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant T508A/N667T | Cellulomonas uda | |
3.28 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S | Cellulomonas uda | |
13.2 | - |
cellobiose | pH 6.6, 37°C, recombinant wild-type enzyme | Cellulomonas uda |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.6 | - |
assay at | Cellulomonas uda |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00048 | - |
lactose | pH 6.6, 37°C, recombinant wild-type enzyme | Cellulomonas uda | |
0.0015 | - |
lactose | pH 6.6, 37°C, recombinant mutant T508I/N667A | Cellulomonas uda | |
0.0021 | - |
lactose | pH 6.6, 37°C, recombinant mutant T508A/N667T | Cellulomonas uda | |
0.0034 | - |
lactose | pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S | Cellulomonas uda | |
0.9 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant T508A/N667T | Cellulomonas uda | |
0.99 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S | Cellulomonas uda | |
1.21 | - |
cellobiose | pH 6.6, 37°C, recombinant mutant T508I/N667A | Cellulomonas uda | |
4.56 | - |
cellobiose | pH 6.6, 37°C, recombinant wild-type enzyme | Cellulomonas uda |