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Literature summary for 3.5.1.14 extracted from

  • Parker, B.M.; Taylor, I.N.; Woodley, J.M.; Ward, J.M.; Dalby, P.A.
    Directed evolution of a thermostable l-aminoacylase biocatalyst (2011), J. Biotechnol., 155, 396-405.
    View publication on PubMed
Search for more literature for 3.5.1.14

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli Thermococcus litoralis

Protein Variants

Protein Variants Comment Organism
D212Y lower temperature stability than wild type protein Thermococcus litoralis
E336A total knockout Thermococcus litoralis
E336I total knockout Thermococcus litoralis
E336S partial activity Thermococcus litoralis
F251K impaired activity towards N-benzoyl-L-valine but not towards N-benzoyl-L-phenylalanine Thermococcus litoralis
F251R impaired activity towards N-benzoyl-L-valine but not towards N-benzoyl-L-phenylalanine Thermococcus litoralis
F251Y 32% increase in activity towards N-benzoyl-L-valine, activity towards N-benzoyl-L-phenylalanine unaffected, tetrameric form retained after 1 h at 55°C like wild type protein Thermococcus litoralis
G246E/I274K decreased activity on N-benzoyl-L-phenylalanine at higher temperature Thermococcus litoralis
H169N lower activity than mutant protein H169R Thermococcus litoralis
H169Q lower activity than mutant protein H169R Thermococcus litoralis
H169R low activity Thermococcus litoralis
K226R/P234H/F251Y/I254N 37% increase in activity towards N-benzoyl-L-phenylalanine, lower temperature stability than wild type protein Thermococcus litoralis
L187F may be involved in altering the relative flexibility or conformation Thermococcus litoralis
P237S tetrameric form retained after 1 h at 55°C like wild type protein Thermococcus litoralis
S100T/M106K 51% increase in activity towards N-benzoyl-L-valine, activity towards N-benzoyl-L-phenylalanine unaffected Thermococcus litoralis
S100T/M106K/F251Y no further improvement in activity (non-additive interaction between the mutated sites) Thermococcus litoralis
S217T/S244I/T250I tetrameric form retained after 1 h at 55°C like wild type protein, decreased activity on N-benzoyl-L-phenylalanine at higher temperature Thermococcus litoralis
T299H about 50% activity Thermococcus litoralis
T299K about 50% activity Thermococcus litoralis
T306V may be involved in altering the relative flexibility or conformation Thermococcus litoralis
V242D 32% increase in activity towards N-benzoyl-L-phenylalanine, lower temperature stability than wild type protein Thermococcus litoralis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4
-
 N-benzoyl-L-phenylalanine S100T/M106K mutant protein, pH 8.0, 55°C Thermococcus litoralis
5
-
 N-benzoyl-L-valine F251K mutant protein, pH 8.0, 55°C Thermococcus litoralis
6.7
-
 N-benzoyl-L-valine wild type protein, pH 8.0, 55°C Thermococcus litoralis
8.5
-
 N-benzoyl-L-phenylalanine wild type protein, pH 8.0, 55°C Thermococcus litoralis
9.7
-
 N-benzoyl-L-phenylalanine F251K mutant protein, pH 8.0, 55°C Thermococcus litoralis
10.7
-
 N-benzoyl-L-valine S100T/M106K mutant protein, pH 8.0, 55°C Thermococcus litoralis

Organism

Organism UniProt Comment Textmining
Thermococcus litoralis
-
-
-

Purification (Commentary)

Purification (Comment) Organism
anion exchange chromatography, hydrophobic interaction chromatography Thermococcus litoralis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0006
-
 S100T/M106K mutant protein, substrate N-acetylpipecolic acid, pH 8.0, 55°C Thermococcus litoralis
0.003
-
 F251K mutant protein, substrate N-acetylpipecolic acid, pH 8.0, 55°C Thermococcus litoralis
0.003
-
 S100T/M106K mutant protein, substrate N-benzoyl-L-tert-leucine, pH 8.0, 55°C Thermococcus litoralis
0.005
-
 F251K mutant protein, substrate N-benzoyl-L-isoleucine, pH 8.0, 55°C Thermococcus litoralis
0.008
-
 F251K mutant protein, substrate N-benzoyl-L-tert-leucine, pH 8.0, 55°C Thermococcus litoralis
0.024
-
 wild type protein, substrate N-acetylpipecolic acid, pH 8.0, 55°C Thermococcus litoralis
0.026
-
 S100T/M106K mutant protein, substrate N-benzoyl-L-isoleucine, pH 8.0, 55°C Thermococcus litoralis
0.075
-
 wild type protein, substrate N-benzoyl-L-tert-leucine, pH 8.0, 55°C Thermococcus litoralis
0.112
-
 F251K mutant protein, substrate N-benzoyl-L-valine, pH 8.0, 55°C Thermococcus litoralis
0.19
-
 wild type protein, substrate N-benzoyl-L-isoleucine, pH 8.0, 55°C Thermococcus litoralis
1.256
-
 F251K mutant protein, substrate N-acetyl-L-phenylalanine, pH 8.0, 55°C Thermococcus litoralis
1.405
-
 S100T/M106K mutant protein, substrate N-acetyl-L-phenylalanine, pH 8.0, 55°C Thermococcus litoralis
1.55
-
 S100T/M106K mutant protein, substrate N-benzoyl-L-phenylalanine, pH 8.0, 55°C Thermococcus litoralis
1.679
-
 wild type protein, substrate N-benzoyl-L-valine, pH 8.0, 55°C Thermococcus litoralis
2.15
-
 F251K mutant protein, substrate N-benzoyl-L-phenylalanine, pH 8.0, 55°C Thermococcus litoralis
3.32
-
 S100T/M106K mutant protein, substrate N-benzoyl-L-valine, pH 8.0, 55°C Thermococcus litoralis
5.555
-
 wild type protein, substrate N-acetyl-L-phenylalanine, pH 8.0, 55°C Thermococcus litoralis
6.8
-
 wild type protein, substrate N-benzoyl-L-phenylalanine, pH 8.0, 55°C Thermococcus litoralis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-acetyl-L-phenylalanine + H2O
-
 Thermococcus litoralis acetate + L-phenylalanine
-
 ?
N-acetylpipecolic acid + H2O
-
 Thermococcus litoralis acetate + L-piperidine-2-carboxylate
-
 ?
N-benzoyl-L-isoleucine + H2O
-
 Thermococcus litoralis benzoate + L-isoleucine
-
 ?
N-benzoyl-L-phenylalanine + H2O
-
 Thermococcus litoralis benzoate + L-phenylalanine
-
 ?
N-benzoyl-L-tert-leucine + H2O
-
 Thermococcus litoralis benzoate + L-tert-isoleucine
-
 ?
N-benzoyl-L-valine + H2O
-
 Thermococcus litoralis benzoate + L-valine
-
 ?

Subunits

Subunits Comment Organism
homotetramer SDS-PAGE before and after boiling with beta-mercaptoethanol Thermococcus litoralis

Synonyms

Synonyms Comment Organism
L-aminoacylase
-
 Thermococcus litoralis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
98.1
-
 N-benzoyl-L-valine F251K mutant protein, pH 8.0, 55°C Thermococcus litoralis
1760
-
 N-benzoyl-L-valine wild type protein, pH 8.0, 55°C Thermococcus litoralis
3620
-
 N-benzoyl-L-phenylalanine S100T/M106K mutant protein, pH 8.0, 55°C Thermococcus litoralis
5900
-
 N-benzoyl-L-phenylalanine F251K mutant protein, pH 8.0, 55°C Thermococcus litoralis
9380
-
 N-benzoyl-L-valine S100T/M106K mutant protein, pH 8.0, 55°C Thermococcus litoralis
21300
-
 N-benzoyl-L-phenylalanine wild type protein, pH 8.0, 55°C Thermococcus litoralis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
19.6
-
 N-benzoyl-L-valine F251K mutant protein, pH 8.0, 55°C Thermococcus litoralis
263
-
 N-benzoyl-L-valine wild type protein, pH 8.0, 55°C Thermococcus litoralis
608
-
 N-benzoyl-L-phenylalanine F251K mutant protein, pH 8.0, 55°C Thermococcus litoralis
874
-
 N-benzoyl-L-valine S100T/M106K mutant protein, pH 8.0, 55°C Thermococcus litoralis
904
-
 N-benzoyl-L-phenylalanine S100T/M106K mutant protein, pH 8.0, 55°C Thermococcus litoralis
2500
-
 N-benzoyl-L-phenylalanine wild type protein, pH 8.0, 55°C Thermococcus litoralis