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Literature summary for 5.1.2.2 extracted from

  • Yang, C.; Ye, L.; Gu, J.; Yang, X.; Li, A.; Yu, H.
    Directed evolution of mandelate racemase by a novel high-throughput screening method (2017), Appl. Microbiol. Biotechnol., 101, 1063-1072 .
    View publication on PubMed
Search for more literature for 5.1.2.2

Cloned(Commentary)

Cloned (Comment) Organism
gene mdlA, genetic library construction, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
additional information directed evolution of mandelate racemase by a high-throughput screening method, overview Pseudomonas putida
V22I site-directed mutagenesis, kinetic analysis Pseudomonas putida
V22I/V29I site-directed mutagenesis, kinetic analysis Pseudomonas putida
V22I/V29I/Y54F site-directed mutagenesis, the mutant shows 3.5fold greater relative activity as compared to the wild-type enzyme. The enhanced catalytic efficiency mainly arises from the elevated kcat, kinetic analysis Pseudomonas putida
V22I/Y54F site-directed mutagenesis, kinetic analysis Pseudomonas putida
V29I site-directed mutagenesis, kinetic analysis Pseudomonas putida
V29I/Y54F site-directed mutagenesis, kinetic analysis Pseudomonas putida
Y54F site-directed mutagenesis, kinetic analysis Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
9.94
-
 (R)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
10.62
-
 (S)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
10.9
-
 (S)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
11.66
-
 (R)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
11.99
-
 (R)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
13.74
-
 (S)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
14.19
-
 (S)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
14.35
-
 (S)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
14.52
-
 (R)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
14.82
-
 (R)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
15.1
-
 (R)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
15.51
-
 (R)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
15.97
-
 (R)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
17.74
-
 (S)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
17.94
-
 (S)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
19.91
-
 (S)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Pseudomonas putida

Organism

Organism UniProt Comment Textmining
Pseudomonas putida P11444 i.e. Arthrobacter siderocapsulatus
-
Pseudomonas putida ATCC 12633 P11444 i.e. Arthrobacter siderocapsulatus
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Pseudomonas putida

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.00057
-
 wild-type enzyme, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00065
-
 wild-type enzyme, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00089
-
 mutant Y45F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00103
-
 mutant V29I, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00118
-
 mutant Y45F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00125
-
 mutant V22I, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00133
-
 mutant V29I, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00134
-
 mutant V29I/Y45F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00143
-
 mutant V22I, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00143
-
 mutant V22I/Y45F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00149
-
 mutant V22I/V29I, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00152
-
 mutant V29I/Y45F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00155
-
 mutant V22I/V29I, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00174
-
 mutant V22I/V29I/Y54F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
0.00175
-
 mutant V22I/Y45F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
0.00194
-
 mutant V22I/V29I/Y54F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-2-chloromandelate
-
 Pseudomonas putida (S)-2-chloromandelate
-
 r
(R)-2-chloromandelate
-
 Pseudomonas putida ATCC 12633 (S)-2-chloromandelate
-
 r
(S)-2-chloromandelate binding structure Pseudomonas putida (R)-2-chloromandelate
-
 r
(S)-2-chloromandelate binding structure Pseudomonas putida ATCC 12633 (R)-2-chloromandelate
-
 r
additional information enzyme activity detection in the high-throughput screening method of enzymes involving the racemization of (S)-2-chloromandelate, method evaluation, overview Pseudomonas putida ?
-
 ?
additional information enzyme activity detection in the high-throughput screening method of enzymes involving the racemization of (S)-2-chloromandelate, method evaluation, overview Pseudomonas putida ATCC 12633 ?
-
 ?

Synonyms

Synonyms Comment Organism
mdlA
-
 Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.9
-
 (R)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
0.99
-
 (S)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
1.23
-
 (R)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
1.33
-
 (S)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
1.45
-
 (R)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
1.73
-
 (R)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
1.82
-
 (R)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
1.83
-
 (S)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
1.96
-
 (S)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
2.35
-
 (S)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
2.4
-
 (R)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
2.47
-
 (S)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
2.51
-
 (S)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
2.57
-
 (R)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
2.6
-
 (R)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
3.49
-
 (S)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Pseudomonas putida

General Information

General Information Comment Organism
additional information enzyme molecular dynamics simulation and molecular docking Pseudomonas putida

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.056
-
 (S)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
0.061
-
 (R)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
0.106
-
 (R)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
0.118
-
 (R)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
0.121
-
 (R)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
0.122
-
 (S)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
0.124
-
 (S)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
0.128
-
 (S)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
0.15
-
 (R)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
0.159
-
 (S)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
0.172
-
 (R)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
0.174
-
 (R)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
0.177
-
 (R)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
0.177
-
 (S)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
0.185
-
 (S)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
0.195
-
 (S)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida