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Literature summary for 1.14.14.28 extracted from

  • Dong, Y.; Yan, J.; Du, H.; Chen, M.; Ma, T.; Feng, L.
    Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis (2012), Appl. Microbiol. Biotechnol., 94, 1019-1029.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
-
Geobacillus thermodenitrificans

Protein Variants

Protein Variants Comment Organism
A102D hydroxylation activity of purified LadA mutant on hexadecane is 2.1fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.3fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C16 to C28 Geobacillus thermodenitrificans
A102D/F146C/L320V/N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
A102D/F146C/N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
A102D/L320V mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
A102E hydroxylation activity of purified LadA mutant on hexadecane is 2.2fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.2fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein Geobacillus thermodenitrificans
F146C mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
F146C/L320V/N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
F146C/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.9fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.9fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein Geobacillus thermodenitrificans
F146E/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.0fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.7fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C28 Geobacillus thermodenitrificans
F146N/N376I hydroxylation activity of purified LadA mutant on hexadecane is 3.4fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 3.4fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. The mutant enzyme shows a shift in optimum temperature from 60°C (for the wild-type enzyme) to 75°C. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C28 Geobacillus thermodenitrificans
F146Q/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.3fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.3fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C14 to C24 Geobacillus thermodenitrificans
F146R/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.5fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.8fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. The mutant enzyme is more heat resistant than wild-type protein, with more than half of the initial activity being retained after incubation at 60°C for 12 h, compared to a 60°C incubation of 4 h or less resulting in the loss of half of the initial activity in the wild-type and F146N/N376I mutant. The mutant enzyme shows a shift in optimum temperature from 60°C (for the wild-type enzyme) to 65°C. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C24 Geobacillus thermodenitrificans
L320A hydroxylation activity of purified LadA mutant on hexadecane is 2.2fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.5fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C22 Geobacillus thermodenitrificans
L320V hydroxylation activity of purified LadA mutant on hexadecane is 2.5fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.4fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein Geobacillus thermodenitrificans
N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.4
-
hexadecane pH 7.5, 60°C, mutant enzyme F146E/N376I Geobacillus thermodenitrificans
1.5
-
hexadecane pH 7.5, 60°C, mutant enzyme A102E Geobacillus thermodenitrificans
1.8
-
hexadecane pH 7.5, 60°C, mutant enzyme A102D Geobacillus thermodenitrificans
2
-
hexadecane pH 7.5, 60°C, mutant enzyme F146R/N376I Geobacillus thermodenitrificans
2.7
-
hexadecane pH 7.5, 60°C, mutant enzyme F146Q/N376I Geobacillus thermodenitrificans
2.8
-
hexadecane pH 7.5, 60°C, mutant enzyme F146C/N376I Geobacillus thermodenitrificans
7.1
-
hexadecane pH 7.5, 60°C, mutant enzyme L320A Geobacillus thermodenitrificans
8.9
-
hexadecane pH 7.5, 60°C, mutant enzyme F146N/N376I Geobacillus thermodenitrificans
9.1
-
hexadecane pH 7.5, 60°C, wild-type enzyme Geobacillus thermodenitrificans
11
-
hexadecane pH 7.5, 60°C, mutant enzyme L320V Geobacillus thermodenitrificans

Organism

Organism UniProt Comment Textmining
Geobacillus thermodenitrificans A4IU28
-
-
Geobacillus thermodenitrificans NG80-2 A4IU28
-
-

Purification (Commentary)

Purification (Comment) Organism
wild-type and N-terminal His-tagged fusion proteins Geobacillus thermodenitrificans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
docosan + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-docosanol + FMN + H2O
-
?
docosan + FMNH2 + O2
-
Geobacillus thermodenitrificans NG80-2 1-docosanol + FMN + H2O
-
?
dotriacontan + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-dotriacontanol + FMN + H2O
-
?
dotriacontan + FMNH2 + O2
-
Geobacillus thermodenitrificans NG80-2 1-dotriacontanol + FMN + H2O
-
?
hexacosan + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-hexacosanol + FMN + H2O
-
?
hexacosan + FMNH2 + O2
-
Geobacillus thermodenitrificans NG80-2 1-hexacosanol + FMN + H2O
-
?
hexadecane + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-hexadecanol + FMN + H2O
-
?
hexadecane + FMNH2 + O2
-
Geobacillus thermodenitrificans NG80-2 1-hexadecanol + FMN + H2O
-
?
hexatriacontan + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-hexatriacontanol + FMN + H2O
-
?
hexatriacontan + FMNH2 + O2
-
Geobacillus thermodenitrificans NG80-2 1-hexatriacontanol + FMN + H2O
-
?
octacosan + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-octacosanol + FMN + H2O
-
?
octadecane + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-octadecanol + FMN + H2O
-
?
pentadecane + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-pentadecanol + FMN + H2O
-
?
tetracosan + FMNH2 + O2
-
Geobacillus thermodenitrificans 1-tetracosanol + FMN + H2O
-
?

Subunits

Subunits Comment Organism
homodimer
-
Geobacillus thermodenitrificans

Synonyms

Synonyms Comment Organism
LADA
-
Geobacillus thermodenitrificans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
wild-type enzyme Geobacillus thermodenitrificans
65
-
mutant enzyme F146R/N376I Geobacillus thermodenitrificans
75
-
mutant enzyme F146N/N376I Geobacillus thermodenitrificans

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
40 90 both the wild-type and the mutants are active at temperatures ranging from 40°C to 90°C Geobacillus thermodenitrificans

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
60
-
incubation of 4 h or less results in the loss of half of the initial activity in the wild-type and F146N/N376I mutant. Mutant enzyme F146R/N376I retains more than half of the initial activity after incubation for 12 h Geobacillus thermodenitrificans

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.02
-
hexadecane pH 7.5, 60°C, wild-type enzyme Geobacillus thermodenitrificans
0.043
-
hexadecane pH 7.5, 60°C, mutant enzyme F146E/N376I Geobacillus thermodenitrificans
0.045
-
hexadecane pH 7.5, 60°C, mutant enzyme A102D Geobacillus thermodenitrificans
0.047
-
hexadecane pH 7.5, 60°C, mutant enzyme A102E Geobacillus thermodenitrificans
0.047
-
hexadecane pH 7.5, 60°C, mutant enzyme L320A Geobacillus thermodenitrificans
0.05
-
hexadecane pH 7.5, 60°C, mutant enzyme F146Q/N376I Geobacillus thermodenitrificans
0.053
-
hexadecane pH 7.5, 60°C, mutant enzyme L320V Geobacillus thermodenitrificans
0.055
-
hexadecane pH 7.5, 60°C, mutant enzyme F146R/N376I Geobacillus thermodenitrificans
0.063
-
hexadecane pH 7.5, 60°C, mutant enzyme F146C/N376I Geobacillus thermodenitrificans
0.073
-
hexadecane pH 7.5, 60°C, mutant enzyme F146N/N376I Geobacillus thermodenitrificans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
mutant enzyme F146R/N376I Geobacillus thermodenitrificans
7.5
-
wild-type enzyme Geobacillus thermodenitrificans

pH Range

pH Minimum pH Maximum Comment Organism
6 8.8 both the wild-type and the mutants are active at pH values from 6.0 to 8.8 Geobacillus thermodenitrificans

Cofactor

Cofactor Comment Organism Structure
FMN
-
Geobacillus thermodenitrificans
FMNH2
-
Geobacillus thermodenitrificans

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0025
-
hexadecane pH 7.5, 60°C, wild-type enzyme Geobacillus thermodenitrificans
0.0048
-
hexadecane pH 7.5, 60°C, mutant enzyme L320V Geobacillus thermodenitrificans
0.0067
-
hexadecane pH 7.5, 60°C, mutant enzyme L320A Geobacillus thermodenitrificans
0.0082
-
hexadecane pH 7.5, 60°C, mutant enzyme F146N/N376I Geobacillus thermodenitrificans
0.0185
-
hexadecane pH 7.5, 60°C, mutant enzyme F146Q/N376I Geobacillus thermodenitrificans
0.022
-
hexadecane pH 7.5, 60°C, mutant enzyme F146C/N376I Geobacillus thermodenitrificans
0.0245
-
hexadecane pH 7.5, 60°C, mutant enzyme A102D Geobacillus thermodenitrificans
0.0295
-
hexadecane pH 7.5, 60°C, mutant enzyme F146R/N376I Geobacillus thermodenitrificans
0.031
-
hexadecane pH 7.5, 60°C, mutant enzyme F146E/N376I Geobacillus thermodenitrificans
0.0315
-
hexadecane pH 7.5, 60°C, mutant enzyme A102E Geobacillus thermodenitrificans