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Literature summary for 2.4.1.20 extracted from

  • De Groeve, M.; De Baere, M.; Hoflack, L.; Desmet, T.; Vandamme, E.; Soetaert, W.
    Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase (2009), Protein Eng., 22, 393-399.
    View publication on PubMed
Search for more literature for 2.4.1.20

Cloned(Commentary)

Cloned (Comment) Organism
CP gene, expression of His-tagged wild-type and mutant enzymes in Escherichia coli Cellulomonas uda

Protein Variants

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Cellulomonas uda Q7WTR6 DSM 20108
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by immobilized metal ion affinity chromatography Cellulomonas uda

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms Comment Organism
lactose phosphorylase created through directed evolution Cellulomonas uda

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Cellulomonas uda

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.6
-
 assay at Cellulomonas uda

kcat/KM [mM/s]

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