Information on EC 3.5.1.77 - N-carbamoyl-D-amino-acid hydrolase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.5.1.77
-
RECOMMENDED NAME
GeneOntology No.
N-carbamoyl-D-amino-acid hydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
an N-carbamoyl-D-amino acid + H2O = a D-amino acid + NH3 + CO2
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
hydrolysis of linear amides
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
N-carbamoyl-D-amino-acid amidohydrolase
This enzyme, along with EC 3.5.1.87 (N-carbamoyl-L-amino-acid hydrolase), EC 5.1.99.5 (hydantoin racemase) and hydantoinase, forms part of the reaction cascade known as the "hydantoinase process", which allows the total conversion of D,L-5-monosubstituted hydantoins into optically pure D- or L-amino acids [2]. It has strict stereospecificity for N-carbamoyl-D-amino acids and does not act upon the corresponding L-amino acids or on the N-formyl amino acids, N-carbamoyl-sarcosine, -citrulline, -allantoin and -ureidopropanoate, which are substrates for other amidohydrolases.
CAS REGISTRY NUMBER
COMMENTARY hide
71768-08-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 1-671
-
-
Manually annotated by BRENDA team
strain NTRRL B11291
-
-
Manually annotated by BRENDA team
Blastobacter sp.
strain A17p-4
-
-
Manually annotated by BRENDA team
strain E222c
-
-
Manually annotated by BRENDA team
strain E222c
-
-
Manually annotated by BRENDA team
strain S-5
SwissProt
Manually annotated by BRENDA team
strain S-5
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
AJ11199
-
-
Manually annotated by BRENDA team
AJ11199
-
-
Manually annotated by BRENDA team
AJ11221
-
-
Manually annotated by BRENDA team
AJ11221
-
-
Manually annotated by BRENDA team
strain KNK003A and strain KNK505
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-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
DL-5-(p-Hydroxyphenyl)hydantoin + H2O
?
show the reaction diagram
Blastobacter sp.
-
-
-
-
-
N-carbamoyl-D-Ala + H2O
D-Ala + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-Asp + H2O
D-Asp + NH3 + CO2
show the reaction diagram
-
81% of the activity with N-carbamoyl-D-p-hydroxyphenylglycine
-
-
-
N-carbamoyl-D-Glu + H2O
D-Glu + NH3 + CO2
show the reaction diagram
-
19% of the activity with N-carbamoyl-D-phenylglycine
-
-
-
N-carbamoyl-D-hydroxyphenylglycine + H2O
D-hydroxyphenylglycine + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-Ile + H2O
D-Ile + NH3 + CO2
show the reaction diagram
-
72% of the activity with N-carbamoyl-D-p-hydroxyphenylglycine
-
-
-
N-carbamoyl-D-Leu + H2O
D-Leu + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-Met + H2O
D-Met + NH3 + CO2
show the reaction diagram
48% of activity with N-carbamoyl-D-p-hydroxyphenylglycine
-
-
?
N-Carbamoyl-D-p-chloro-D-phenylglycine + H2O
D-p-Chloro-D-phenylglycine + NH3 + CO2
show the reaction diagram
-
78% of the activity with N-carbamoyl-D-phenylglycine
-
-
-
N-carbamoyl-D-p-hydroxyphenylglycine + H2O
D-p-hydroxyphenylglycine + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-p-methoxy-D-phenylglycine + H2O
D-p-methoxy-D-phenylglycine + NH3 + CO2
show the reaction diagram
-
62% of the activity with N-carbamoyl-D-phenylglycine
-
-
-
N-carbamoyl-D-Phe + H2O
D-Phe + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-phenylglycine + H2O
D-phenylglycine + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-Ser + H2O
D-Ser + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-Trp + H2O
D-Trp + NH3 + CO2
show the reaction diagram
N-carbamoyl-D-Tyr + H2O
D-Tyr + NH3 + CO2
show the reaction diagram
-
-
-
-
-
N-carbamoyl-D-Val + H2O
D-Val + NH3 + CO2
show the reaction diagram
N-Carbamoyl-DL-2-amino-n-butyric acid + H2O
?
show the reaction diagram
N-Carbamoyl-DL-Met + H2O
?
show the reaction diagram
N-Carbamoyl-DL-norleucine + H2O
?
show the reaction diagram
N-Carbamoyl-DL-norvaline + H2O
?
show the reaction diagram
N-Carbamoyl-DL-Thr + H2O
?
show the reaction diagram
N-Carbamoyl-Gly + H2O
Gly + NH3 + CO2
show the reaction diagram
-
12% of the activity with N-carbamoyl-D-p-hydroxyphenylglycine
-
-
-
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
N-carbamoyl-D-p-hydroxyphenylglycine + H2O
D-p-hydroxyphenylglycine + NH3 + CO2
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Al3+
-
slightly enhances activity to about 110% of the standard activity
Ca2+
2 mM, enhances activity to about 110%
Fe3+
-
slightly enhances activity to about 110% of the standard activity
Pb2+
-
slightly enhances activity to about 110% of the standard activity
Sn2+
-
slightly enhances activity to about 110% of the standard activity
additional information
-
no metal ion requirement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoic acid)
D-Phe
H2O2
32% loss of activity after treatment with 0.1 mM H2O2 for 30 min, 57% loss of activity after treatment with 0.2 mM H2O2 for 30 min
iodoacetamide
-
-
iodoacetate
Mn2+
2 mM, 58% inhibition
N-carbamoyl-D-Phe
Blastobacter sp.
-
-
NaAsO2
Blastobacter sp.
-
-
Sn2+
2 mM, 46.5% inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4 - 11.6
N-carbamoyl-D-Ala
0.36 - 3.6
N-carbamoyl-D-Leu
0.79
N-carbamoyl-D-norleucine
Blastobacter sp.
-
-
0.52 - 19
N-carbamoyl-D-p-hydroxyphenylglycine
0.5 - 19.7
N-carbamoyl-D-Phe
0.88 - 26.9
N-Carbamoyl-D-phenylglycine
2.4 - 23.7
N-carbamoyl-D-Ser
0.41 - 1
N-carbamoyl-D-Val
0.71 - 7.5
N-Carbamoyl-DL-Met
4.8
N-Carbamoyl-DL-norleucine
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03 - 22.47
N-carbamoyl-D-p-hydroxyphenylglycine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.8 - 6.8
N-carbamoyl-D-p-hydroxyphenylglycine
1597
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.34
-
-
9.25
enzyme from Sinorhizobium morelens
21.4
enzyme recombinantly expressed in Escherichia coli
additional information
Blastobacter sp.
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3 - 7.4
-
-
8 - 9
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N-carbamoyl-D-Phe
8
-
activity assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 11
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pH 5.0: about 40% of maximal activity, pH 11: about 25% of maximal activity
5.5 - 10.7
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about 70% of maximal activity at pH 5.5 and pH 10.7
6 - 8.2
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pH 6.0: about 50% of maximal activity, pH 8.2: about 65% loss of maximal activity
6 - 9
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relative enzyme activity 80-30%, pH 7.0 100%
6.2 - 8.7
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pH 6.2: about 45% of maximal activity, pH 8.7: about 35% of maximal activity
6.5 - 7.5
pH 6.5: about 90% of maximal activity, pH 7.5: about 95% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 60
-
about 50% of maximal activity at 25C and 60C
30 - 70
-
30C: about 25% of maximal activity, 30C: about 30% of maximal activity
30 - 60
Blastobacter sp.
-
30C: about 35% of maximal activity, 20C: about 20% of maximal activity
30 - 50
-
30C: about 50% of maximal activity, 50C: about 55% of maximal activity
35 - 80
-
relative enzyme activity 30-25%, 70C 100%
40 - 70
-
40C: about 50% of maximal activity, 70C: about 70% of maximal activity
45 - 65
45C: about 50% of maximal activity, 65C: about 75% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
-
2 * 32000, SDS-PAGE
34000
-
2 * 34000, calculation from nucleotide sequence
34285
-
2 * 34285, calculation from nucleotide sequence
34363
4 * 34363, calculated from sequence
38000
4 * 38000, SDS-PAGE
38100
-
2 * 38100, SDS-PAGE
40564
-
x * 40564, calculation from amino acid content
67000
-
gel filtration
84000
-
gel filtration
111000 - 120000
Blastobacter sp.
-
gel filtration
117000
-
gel filtration
150000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
trimer
Blastobacter sp.
-
3 * 40000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting-drop vapour-diffusion method. Native crystals belong to space group P2(1)2(1)2 with a = 67.84 A, b = 137.83 A, c = 68.39 A. Structure determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices
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hanging drop vapor-diffusion method. Crystal structure of mutant enzymes C172A, C172S, R175A, N173A and the complexes of mutant enzymes R175A and N173A with the substrate N-carbamoyl-D-p-hydroxyphenylglycine
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hanging drop vapour diffusion method using protein solutions containing 15 mg/ml protein with precipitating solution at room temperature. A222C and A302C mutants are initially grown as micro-crystals with the precipitating condition of 1.2 M lithium sulfate and 0.1 M Hepes buffer (pH 7.0). A micro-seeding method is then applied to obtain large single crystals. For P95C/F304C and P178C, no crystals are obtained. Crystals structures of D-NCase mutants, A302C and A222C
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hanging-drop vapor-diffusion method, crystals belong to space group P2(1) with unit cell dimensions a = 70.23 A, b = 67.53 A, c = 137.48 A and beta = 96.12. There are 4 molecules per asymmetric unit
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hanging-drop vapour diffusion method using lithium sulfate as precipitant. It crystallizes in space group P2(1) with unit-cell parameters a = 69.8 A, b = 67.9 A and c = 137.8 A and beta = 96.4. There are 4 molecules per asymmetric unit
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.3
30 min, 30 min, stable
667845
7 - 9
-
30C, 20 min, stable
31891
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
57
-
the activity of CDase-M3 is almost completely lost after heat treatment for 60 min at 57C
63
-
63.1C, 20 min, 50% loss of activity, wild-type enzyme; 63.5C, 20 min, 50% loss of activity, mutant enzyme A222C
64
-
63.6C, 20 min, 50% loss of activity, mutant enzyme P178C
68
-
Tm-value of mutant enzyme P178C: 68.3C, Tm-value of mutant enzyme A222C: 67.8C
69
-
Tm-value of wild-type enzyme: 68.6C
70
-
30 min, 90% loss of wild-type activity, 80% loss of activity of mutant enzyme M184L, 49% loss of activity of mutant enzyme T262A, 46% loss of activity of mutant enzyme M184L/T262A 81% loss of activity of mutant enzyme Q23L, 93% loss of activity of mutant enzyme V40A, 80% loss of activity of mutant enzyme H58Y, 96% loss of activity of mutant enzyme G75S, 21% loss of activity of mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A
71
-
20 min, 50% loss of activity, mutant enzyme A302C
72
-
Tm-value of mutant enzyme P295C/F304C: 72.3C
73
-
30 min, mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A, 50% loss of activity
77
-
Tm-value of mutant enzyme A302C: 77.4C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
high thermal stability and high stability in repeated batch reactions of the immobilized enzyme
-
immobilized enzyme is most stable at pH 7.0
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immobilized enzyme is stabilized by dithiothreitol, L-Cys, cysteamine, and sodium hydrosulfite. After 14times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of the glutaraldehyde with dithiothreitol in the reaction mixture is 12%, 18%, and 63% respectively
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stable in absence rather than in presence of 2-mercaptoethanol
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
high oxidative stability
667845
residual activity after incubation with 0.2 mM hydrogen peroxide for 30 min at 25C is 5% for the wild-type enzyme, 20% for mutant enzyme M184L, 40.9% for mutant enzyme T262A, 54.8% for mutant enzyme M184L/T262A, 63.9% for mutant enzyme Q23L, 20.6% for mutant enzymes V40A and H58Y, 42.5% for mutant enzyme G75S, 79.3% for mutant enzyme Q23L/V40A/H58Y/G75S/M184L/T262A
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657225
residual activity after treatment with 0.5 mM H2O2 for 15 min is 21% for wild-type enzyme, 51% for mutant enzyme M5L, 8% for mutant enzyme M31L, 84% for mutant enzyme M73L, 37% for mutant enzyme M167L/M169L, 57% for mutant enzymeM184L, 62% for mutant enzyme M220L. The mutant enzymes M239L, M244L and M239L/M244L are stable to treatment with 0.5 mM H2O2 for 15 min
-
654422
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, phosphate buffer, progressive loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
denatured recombinant enzyme
-
recombinant enzyme
recombinant mutant A18T/Y30N/K34E
-
using metal affinity resins, immobilization on chitin beads
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
into the vector pET28b, a mutagenesis library is created
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mutant A18T/Y30N/K34E is overexpressed in Escherichia coli and up to 80% of it is soluble
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the plasmids pET20bl and pET-Chi are used for expression of the protein in Escherichia coli cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H57L
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
H57Y
-
mutant enzymes His57Tyr, Pro203Leu or Pro203Ser show an improved thermostability by about 5C compared with those of the wild type enzyme
P203A
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
P203H
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
P203I
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
P203L
-
mutant enzymes His57Tyr, Pro203Leu or Pro203Ser show an improved thermostability by about 5C compared with those of the wild type enzyme
P203N
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
P203S
-
mutant enzymes His57Tyr, Pro203Leu or Pro203Ser show an improved thermostability by about 5C compared with those of the wild type enzyme
P203T
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
V236A
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
V236S
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
V236T
-
mutant Val236Ala shows a 10C increase in thermostability compared to the wild type enzyme. The following mutant enzymes show increased thermostability: His57Leu, Pro203Asn, Pro203Glu, Pro203Ala, Pro203Ile, Pro203His, Pro203Thr, Val236Thr, Val236Ser
H57Y
-
mutant enzymes His57Tyr, Pro203Leu or Pro203Ser show an improved thermostability by about 5C compared with those of the wild type enzyme
-
P203L
-
mutant enzymes His57Tyr, Pro203Leu or Pro203Ser show an improved thermostability by about 5C compared with those of the wild type enzyme
-
P203S
-
mutant enzymes His57Tyr, Pro203Leu or Pro203Ser show an improved thermostability by about 5C compared with those of the wild type enzyme
-
A222C
-
crystal structure is nearly identical to wild-type enzyme, half-life at 50C is 1.1fold higher than that of wild-type enzyme
A302C
-
crystal structure is nearly identical to wild-type enzyme, 123.9% of wild-type activity, temperature-optimum is 10C higher than that of wild-type enzyme, half-life at 50C is 2.5fold higher than that of wild-type enzyme, 4.2fold increase in kcat/Km at 65C, 1.5fold increase at 55C and 1.15fold increase at 37C
G75S
-
96% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 42.5% compared to 5% for the wild-type enzyme
G75S/V237A
-
mutant, thermal stability 55.6%, wild-type 3.0%, oxidative stability 42.8%, wild-type 5.0%
H129A
-
inactive mutant enzyme
H129N
-
inactive mutant enzyme
H129R
-
inactive mutant enzyme
H144A
-
5% of the activity of wild-type enzyme
H215A
-
17% of the activity of wild-type enzyme
H58Y
-
80% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 20.6% compared to 5% for the wild-type enzyme
I286V/F287A
-
mutant, thermal stability 4.5%, wild-type 3.0%, oxidative stability 4.8%, wild-type 5.0%
M167L/M169L
-
residual activity after treatment with 0.5 mM H2O2 for 15 min is 37% for mutant enzyme, compared to 21% for wild-type enzyme
M184L/T262A
-
46% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 54.8% compared to 5% for the wild-type enzyme. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 58.6% of the wild-type ratio
M220L
-
residual activity after treatment with 0.5 mM H2O2 for 15 min is 62% for mutant enzyme, compared to 21% for wild-type enzyme
M239L
-
mutant enzyme is stable after treatment with 0.5 mM H2O2 for 15 min compared to 79% loss of wild-type activity
M239L/M244L
-
mutant enzyme is stable after treatment with 0.5 mM H2O2 for 15 min compared to 79% loss of wild-type activity
M244L
-
mutant enzyme is stable after treatment with 0.5 mM H2O2 for 15 min compared to 79% loss of wild-type activity
M31L
-
residual activity after treatment with 0.5 mM H2O2 for 15 min is 8% for mutant enzyme, compared to 21% for wild-type enzyme
M5L
-
residual activity after treatment with 0.5 mM H2O2 for 15 min is 51% for mutant enzyme, compared to 21% for wild-type enzyme
M73L
-
residual activity after treatment with 0.5 mM H2O2 for 15 min is 84% for mutant enzyme, compared to 21% for wild-type enzyme
N173A
-
KM-value for N-carbamoyl-D-p-hydroxyphenylglycine is 40% of the wild-type value. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 5.5fold lower than wild-type ratio. Tm is 68C compared to 63 for wild-type enzyme
P178C
-
half-life at 50C is nearly identical to wild-type enzyme
P295C/F304C
-
107.3% of wild-type activity, temperature-optimum is 5C higher than that of wild-type enzyme, half-life at 50C is 2fold higher than that of wild-type enzyme. 2.5fold increase in kcat/Km at 65C, 1.1fold increase at 55C and at 37C
Q23L
-
81% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 63.9% compared to 5% for the wild-type enzyme
Q23L/V40A/H58Y/G75S/M184L/T262A
-
mutant enzyme with improved oxidative and thermostability, 79.3% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 72.4% of the wild-type ratio
R175A
-
inactive mutant enzyme
R175K
-
KM-value for N-carbamoyl-D-p-hydroxyphenylglycine is 2.5fold higher than the wild-type value. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 4706fold lower than wild-type ratio. Tm is 65C compared to 63 for wild-type enzyme
R176A
-
inactive mutant enzyme
R176K
-
KM-value for N-carbamoyl-D-p-hydroxyphenylglycine is 4.2fold higher than the wild-type value. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 364fold lower than wild-type ratio. Tm is 65C compared to 63 for wild-type enzyme
V237A/C279S
-
mutant, thermal stability 22.4%, wild-type 3.0%, oxidative stability 31.2%, wild-type 5.0%
V40A/G75S/V237A
-
mutant, thermal stability 68.4%, wild-type 3.0%, oxidative stability 80.3%, wild-type 5.0%
V40A/G75S/V237A/I286V/F287A
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mutant, thermal stability 72.3%, wild-type 3.0%, oxidative stability 83.1%, wild-type 5.0%
M184L
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80% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 20% compared to 5% for the wild-type enzyme
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M184L/T262A
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46% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 54.8% compared to 5% for the wild-type enzyme. The ratio of turnover number to Km-value for N-carbamoyl-D-p-hydroxyphenylglycine is 58.6% of the wild-type ratio
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Q23L
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81% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 63.9% compared to 5% for the wild-type enzyme
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T262A
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1.5fold increased activity, improvement in expression levels, mutation results in a significant improvement in both oxidative stability and thermostability, increases stability of N-carbamoylase in vivo, thereby preventing it from degradation by cellular proteases; 49% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 40.9% compared to 5% for the wild-type enzyme
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V40A
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2fold increased activity, improvement in expression levels, mutation mainly gives rise to the increase in oxidative stability rather than thermostability; 93% loss of activity of mutant enzyme M184L after 30 min at 70C, compared to 90% loss of wild-type enzyme. Residual activity after incubation with 0.2 mM H2O2 for 30 min at 25C is 20.6% compared to 5% for the wild-type enzyme
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C172A
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mutant enzyme Cys172Ala or Cys172Ser is completely inactive. Substitution of any of the other Cys has no effect on enzyme activity
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C172S
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mutant enzyme Cys172Ala or Cys172Ser is completely inactive. Substitution of any of the other Cys has no effect on enzyme activity
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A164T
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mutant, screening for thermostable enzyme
A18T/Y30N/K34E
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kinetic properties and thermodynamic parameters of the mutant enzyme are identical with those of the wild-type enzyme. More than 80% improve in solubility compared to wild-type enzyme
A36E
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mutant, screening for thermostable enzyme
A36V
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mutant, screening for thermostable enzyme
H248Q
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mutant, screening for thermostable enzyme
H248Q/T262A
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the mutant displays a T50 value of 65C and a DELTAT50 enhancement of 8C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
H58Y
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mutant, screening for thermostable enzyme
K34D
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mutant has higher solubility than wild-type enzyme
K34E
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mutant has higher solubility than wild-type enzyme
Q12A
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saturation mutagenesis at position Gln12, thermostability 13.3%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12C
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saturation mutagenesis at position Gln12, thermostability 9.3%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12D
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saturation mutagenesis at position Gln12, thermostability 8.9%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12E
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saturation mutagenesis at position Gln12, thermostability 9.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12F
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saturation mutagenesis at position Gln12, thermostability 14.3%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12G
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saturation mutagenesis at position Gln12, thermostability 20.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12H
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saturation mutagenesis at position Gln12, thermostability 12.8%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12I
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saturation mutagenesis at position Gln12, thermostability 14.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12K
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saturation mutagenesis at position Gln12, thermostability 11.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12L/Q23L/H248Q/T262A/T263S
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the mutant exhibits an increase of 10C in thermostability compared with the parental enzyme M3
Q12L/Q23L/H248Q/T262A/T263S/A273K
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the mutant displays a T50 value of 69C and a DELTAT50 enhancement of 12C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/E266D
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the mutant displays a T50 value of 68C and a DELTAT50 enhancement of 11C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/M31L
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the mutant displays a T50 value of 68C and a DELTAT50 enhancement of 11C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/M31L/N242G
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the mutant displays a T50 value of 71C and a DELTAT50 enhancement of 14C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/M31L/Q207E
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the mutant displays a T50 value of 71C and a DELTAT50 enhancement of 14C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/N242G
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the mutant displays a T50 value of 69C and a DELTAT50 enhancement of 12C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/N242G/A273P
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the mutant displays a T50 value of 71C and a DELTAT50 enhancement of 14C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/N242G/T271I/A273P
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the mutant displays a T50 value of 72C and a DELTAT50 enhancement of 15C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/N93Y
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the mutant displays a T50 value of 68C and a DELTAT50 enhancement of 11C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/Q207E
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the mutant displays a T50 value of 68C and a DELTAT50 enhancement of 11C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/Q207E/N242G
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the mutant displays a T50 value of 71C and a DELTAT50 enhancement of 14C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/T271I
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the mutant displays a T50 value of 69C and a DELTAT50 enhancement of 12C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/H248Q/T262A/T263S/T271I/A273K
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the mutant displays a T50 value of 71C and a DELTAT50 enhancement of 14C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P
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the mutant displays a T50 value of 73C and a DELTAT50 enhancement of 16C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%). The mutant retains over 50% of its initial activity after heat treatment at 57C for at least 100 min
Q12M
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saturation mutagenesis at position Gln12, thermostability 9.2%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12N
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saturation mutagenesis at position Gln12, thermostability 15.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12P
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saturation mutagenesis at position Gln12, thermostability 12.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12R
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saturation mutagenesis at position Gln12, thermostability 9.2%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12S
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saturation mutagenesis at position Gln12, thermostability 12.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12T
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saturation mutagenesis at position Gln12, thermostability 14.8%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12V
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saturation mutagenesis at position Gln12, thermostability 20.3%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12W
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saturation mutagenesis at position Gln12, thermostability 16.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
Q12Y
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saturation mutagenesis at position Gln12, thermostability 11.9%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T262A
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mutant, screening for thermostable enzyme
T263A
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saturation mutagenesis at position Thr263, thermostability 8.6%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263C
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saturation mutagenesis at position Thr263, thermostability 13.2%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263D
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saturation mutagenesis at position Thr263, thermostability 10.3%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263E
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saturation mutagenesis at position Thr263, thermostability 12.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263F
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saturation mutagenesis at position Thr263, thermostability 13.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263G
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saturation mutagenesis at position Thr263, thermostability 15.7%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263H
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saturation mutagenesis at position Thr263, thermostability 15.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263I
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saturation mutagenesis at position Thr263, thermostability 14.7%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263K
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saturation mutagenesis at position Thr263, thermostability 11.8%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263L
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saturation mutagenesis at position Thr263, thermostability 10.4%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263M
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saturation mutagenesis at position Thr263, thermostability 23.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263N
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saturation mutagenesis at position Thr263, thermostability 14.7%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263P
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saturation mutagenesis at position Thr263, thermostability 14.6%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263Q
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saturation mutagenesis at position Thr263, thermostability 10.7%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263R
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saturation mutagenesis at position Thr263, thermostability 9.6%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263S
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mutant, screening for thermostable enzyme; saturation mutagenesis at position Thr263, thermostability 25.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263V
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saturation mutagenesis at position Thr263, thermostability 8.7%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263W
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saturation mutagenesis at position Thr263, thermostability 9.5%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
T263Y
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saturation mutagenesis at position Thr263, thermostability 12.0%, wild-type 12.3%, incubation at various temperatures, residual activity is calculated relative to the activity of the non-heat treated enzyme
V237A
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mutant, screening for thermostable enzyme
H248Q/T262A
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the mutant displays a T50 value of 65C and a DELTAT50 enhancement of 8C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
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Q12L
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the mutant displays a T50 value of 63C and a DELTAT50 enhancement of 6C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
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Q12L/Q23L/H248Q/T262A/T263S
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the mutant exhibits an increase of 10C in thermostability compared with the parental enzyme M3
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Q12L/Q23L/H248Q/T262A/T263S/M31L
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the mutant displays a T50 value of 68C and a DELTAT50 enhancement of 11C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%)
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Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P
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the mutant displays a T50 value of 73C and a DELTAT50 enhancement of 16C compared with CDase-M3 (T50 is defined as the temperature at which heat treatment for 15 min reduces the initial activity by 50%). The mutant retains over 50% of its initial activity after heat treatment at 57C for at least 100 min
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
refolding of purified denatured DCaseH is best yielded at 50% glycerol for 3 days with a 20% renaturation yield. Approximately 38% of the native secondary structure is reformed. The refolding with the addition of glycerol partially recovers both the activity and the native-like secondary structure of the denatured enzyme
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
synthesis
Show AA Sequence (507 entries)
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