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Literature summary 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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.1.2.2 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

EC Number Protein Variants Comment Organism
5.1.2.2 additional information directed evolution of mandelate racemase by a high-throughput screening method, overview Pseudomonas putida
5.1.2.2 V22I site-directed mutagenesis, kinetic analysis Pseudomonas putida
5.1.2.2 V22I/V29I site-directed mutagenesis, kinetic analysis Pseudomonas putida
5.1.2.2 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
5.1.2.2 V22I/Y54F site-directed mutagenesis, kinetic analysis Pseudomonas putida
5.1.2.2 V29I site-directed mutagenesis, kinetic analysis Pseudomonas putida
5.1.2.2 V29I/Y54F site-directed mutagenesis, kinetic analysis Pseudomonas putida
5.1.2.2 Y54F site-directed mutagenesis, kinetic analysis Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.1.2.2 9.94
-
(R)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 10.62
-
(S)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 10.9
-
(S)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 11.66
-
(R)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 11.99
-
(R)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 13.74
-
(S)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 14.19
-
(S)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 14.35
-
(S)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 14.52
-
(R)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 14.82
-
(R)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 15.1
-
(R)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 15.51
-
(R)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 15.97
-
(R)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 17.74
-
(S)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 17.94
-
(S)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 19.91
-
(S)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
5.1.2.2 Mg2+ required Pseudomonas putida

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
5.1.2.2 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
5.1.2.2 0.00057
-
wild-type enzyme, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00065
-
wild-type enzyme, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00089
-
mutant Y45F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00103
-
mutant V29I, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00118
-
mutant Y45F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00125
-
mutant V22I, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00133
-
mutant V29I, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00134
-
mutant V29I/Y45F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00143
-
mutant V22I, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00143
-
mutant V22I/Y45F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00149
-
mutant V22I/V29I, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00152
-
mutant V29I/Y45F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00155
-
mutant V22I/V29I, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00174
-
mutant V22I/V29I/Y54F, pH 7.5, 25°C, substrate (R)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00175
-
mutant V22I/Y45F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida
5.1.2.2 0.00194
-
mutant V22I/V29I/Y54F, pH 7.5, 25°C, substrate (S)-2-chloromandelate Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.1.2.2 (R)-2-chloromandelate
-
Pseudomonas putida (S)-2-chloromandelate
-
r
5.1.2.2 (R)-2-chloromandelate
-
Pseudomonas putida ATCC 12633 (S)-2-chloromandelate
-
r
5.1.2.2 (S)-2-chloromandelate binding structure Pseudomonas putida (R)-2-chloromandelate
-
r
5.1.2.2 (S)-2-chloromandelate binding structure Pseudomonas putida ATCC 12633 (R)-2-chloromandelate
-
r
5.1.2.2 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 ?
-
?
5.1.2.2 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

EC Number Synonyms Comment Organism
5.1.2.2 mdlA
-
Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.1.2.2 25
-
assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.1.2.2 0.9
-
(R)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.99
-
(S)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.23
-
(R)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.33
-
(S)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.45
-
(R)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.73
-
(R)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.82
-
(R)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.83
-
(S)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 1.96
-
(S)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 2.35
-
(S)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 2.4
-
(R)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 2.47
-
(S)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 2.51
-
(S)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 2.57
-
(R)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 2.6
-
(R)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 3.49
-
(S)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.1.2.2 7.5
-
assay at Pseudomonas putida

General Information

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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.1.2.2 0.056
-
(S)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.061
-
(R)-2-chloromandelate wild-type enzyme, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.106
-
(R)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.118
-
(R)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.121
-
(R)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.122
-
(S)-2-chloromandelate mutant Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.124
-
(S)-2-chloromandelate mutant V22I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.128
-
(S)-2-chloromandelate mutant V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.15
-
(R)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.159
-
(S)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.172
-
(R)-2-chloromandelate mutant V22I/V29I, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.174
-
(R)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.177
-
(R)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.177
-
(S)-2-chloromandelate mutant V22I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.185
-
(S)-2-chloromandelate mutant V29I/Y45F, pH 7.5, 25°C Pseudomonas putida
5.1.2.2 0.195
-
(S)-2-chloromandelate mutant V22I/V29I/Y54F, pH 7.5, 25°C Pseudomonas putida