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Information on EC 1.13.11.88 - isoeugenol monooxygenase
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1.13.11.88 isoeugenol monooxygenaseIUBMB Comments
Contains Fe(II). The enzyme, charcterised from the bacteria Pseudomonas putida and Pseudomonas nitroreducens, catalyses the epoxidation of the double bond in the side chain of isoeugenol, followed by a second oxygenation and cleavage of the side chain in the form of acetaldehyde.
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Word Map
- 1.13.11.88
- vanillin
- nitroreducens
- putida
-
phenylpropanoids
-
fragrance
- synthesis
-
bl21de3
- trans-anethole
- acetaldehyde
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pet21a
-
undesired
The enzyme appears in viruses and cellular organisms
Synonyms
isoeugenol monooxygenase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
iem
iem
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
isoeugenol + O2 = vanillin + acetaldehyde
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SYSTEMATIC NAME
IUBMB Comments
isoeugenol:oxygen 7,8-oxidoreductase (bond-cleaving)
Contains Fe(II). The enzyme, charcterised from the bacteria Pseudomonas putida and Pseudomonas nitroreducens, catalyses the epoxidation of the double bond in the side chain of isoeugenol, followed by a second oxygenation and cleavage of the side chain in the form of acetaldehyde.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-methoxy-4-vinylphenol + O2
vanillin + formaldehyde
about 1% of the activity with isoeugenol
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-
?
4-vinylguaiacol + O2
vanillin + formaldehyde
32% conversion
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-
?
isoeugenol + O2
vanillin + acetaldehyde
the conversion is significantly increased at high substrate concentration (50 and 100 mM)
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-
?
isoeugenol + O2
vanillin + acetaldehyde
the conversion is significantly increased at high substrate concentration (50 and 100 mM)
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-
?
isoeugenol + O2
vanillin + acetaldehyde
92% conversion
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-
?
additional information
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oxidative cleavage of isoeugenol by Iem is catalyzed via a monooxygenation reaction, and incorporation of the oxygen atom from O2 into vanillin is preferred over incorporation from water. Iem exhibits no activity toward any other of the phenylpropanoid and styrene compounds tested
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-
?
additional information
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oxidative cleavage of isoeugenol by Iem is catalyzed via a monooxygenation reaction, and incorporation of the oxygen atom from O2 into vanillin is preferred over incorporation from water. Iem exhibits no activity toward any other of the phenylpropanoid and styrene compounds tested
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?
additional information
?
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enzyme catalyzes the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Enzyme catalyzes the incorporation of an oxygen atom from either molecular oxygen or water into vanillin
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-
?
additional information
?
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no substrates: eugenol, coniferyl alcohol, coniferyl aldehyde, ferulic acid, 3,4-dihydroxycinnamic acid, 3,4-dimethoxycinnamic acid, 4-methoxycinnamic acid, 4-hydroxycinnamic acid, 3-pyridinepropionic acid, alpha-methoxycinnamic acid, styrene, trans-beta-methylstyrene, trans-stilbene, trans-stilbene oxide, acrylic acid and trans-2-hexenoic acid
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?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
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enzyme catalyzes the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Enzyme catalyzes the incorporation of an oxygen atom from either molecular oxygen or water into vanillin
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-
?
additional information
?
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no substrates: eugenol, coniferyl alcohol, coniferyl aldehyde, ferulic acid, 3,4-dihydroxycinnamic acid, 3,4-dimethoxycinnamic acid, 4-methoxycinnamic acid, 4-hydroxycinnamic acid, 3-pyridinepropionic acid, alpha-methoxycinnamic acid, styrene, trans-beta-methylstyrene, trans-stilbene, trans-stilbene oxide, acrylic acid and trans-2-hexenoic acid
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-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
additional information
no significant changes of enzyme activity with Ca2+, Co2+, Mg2+, Li+, Mn2+ and Na+
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
66.54% residual activity in the presence of 5 mM 1,10-phenanthroline
EDTA
64.77% residual activity in the presence of 5 mM EDTA
ethyl acetate
in the presence 10% (v/v) ethyl acetate, the enzyme shows 19.35% residual activity with isoeugenol and 35.06% residual activity with 4-vinylguaiacol as substrate
Fe2+
70.55% residual activity in the presence of 5 mM Fe2+
n-Butyl alcohol
in the presence of 10% (v/v) n-butyl alcohol, the enzyme shows 38.19% residual activity with isoeugenol and 21.44% residual activity with 4-vinylguaiacol as substrate
n-caprylic alcohol
in the presence of 10% (v/v) n-caprylic acid, the enzyme shows 74.73% residual activity with isoeugenol as substrate
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n-octane
in the presence of 10% (v/v) n-octane, the enzyme shows 55.34% residual activity with isoeugenol and 70.45% residual activity with 4-vinylguaiacol as substrate
petroleum ether
in the presence of 10% (v/v) petroleum ether, the enzyme shows 46.05% residual activity with isoeugenol as substrate
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Toluene
in the presence of 10% (v/v) toluene, the enzyme shows 39.56% residual activity with isoeugenol and 29.36% residual activity with 4-vinylguaiacol as substrate
Triton X-100
66.54% residual activity in the presence of 5 mM Triton X-100
Vanillin
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there is an incremental decrease in enzyme activity with the increasing vanillin concentration
Cu2+
severe inhibition (3.17% residual activity) at 5 mM Cu2+
Fe3+
62.51% residual activity in the presence of 5 mM Fe3+
Ni2+
74.61% residual activity in the presence of 5 mM Ni2+
Zn2+
77.07% residual activity in the presence of 5 mM Zn2+
additional information
not inhibitory or activating at 1 mM: Mg(II), Mn(II), Mo(II), NADH, FAD, or ascorbic acid, EDTA, 1,10-phenanthroline, and Tiron
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additional information
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not inhibitory or activating at 1 mM: Mg(II), Mn(II), Mo(II), NADH, FAD, or ascorbic acid, EDTA, 1,10-phenanthroline, and Tiron
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additional information
not inhibitory: NAD+, NADH, NADP+, NADPH, FMN and FAD
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additional information
not significantly inhibited by SDS and 2,2-bipyridine
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Trichloromethane
the enzyme activity is substantially improved at presence of 10% (v/v) of trichloromethane (176.16% activity with isoeugenol and 208.47% activity with 4-vinylguaiacol)
additional information
not inhibitory or activating at 1 mM: Mg(II), Mn(II), Mo(II), NADH, FAD, or ascorbic acid, EDTA, 1,10-phenanthroline, and Tiron
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additional information
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not inhibitory or activating at 1 mM: Mg(II), Mn(II), Mo(II), NADH, FAD, or ascorbic acid, EDTA, 1,10-phenanthroline, and Tiron
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.145
isoeugenol
wild-type, 30°C, pH not specified in the publication
0.15
isoeugenol
mutant F281Q, 30°C, pH not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4.21
isoeugenol
wild-type, 30°C, pH not specified in the publication
7.58
isoeugenol
mutant F281Q, 30°C, pH not specified in the publication
10.1
isoeugenol
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mutant enzyme K83R/K95R/L273F, at pH 8.0 and 30°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
over 80% of relative activity is detected under a pH range from 7.0 to 9.0
8 - 9.5
-
the wild type enzyme remains 70% relative activity in a pH range from 8.0 to 9.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 40
the enzyme presents over 80% of relative activity under a temperature range from 20 to 40°C, but almost totally inactivation as the temperature is above 45°C
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
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strain is able to produce high amounts of vanillin when grown in the presence of isoeugenol, and is also capable of growing on isoeugenol as the sole carbon source. In the presence of isoeugenol, a growing culture produces 0.61 g/l vanillin (molar yield of 12.4%), whereas cell free extracts result in 0.9 g/l vanillin (molar yield of 14%)
physiological function
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strain is able to produce high amounts of vanillin when grown in the presence of isoeugenol, and is also capable of growing on isoeugenol as the sole carbon source. In the presence of isoeugenol, a growing culture produces 0.61 g/l vanillin (molar yield of 12.4%), whereas cell free extracts result in 0.9 g/l vanillin (molar yield of 14%)
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). A0A2I5TAF0_SERS3
Serratia sp. (strain ATCC 39006)
496
0
55581
TrEMBL
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
homology modeling of structure and docking of substrate isoeugenol to the active site
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
G398A
-
the mutation results in a 1.3fold increase in specific activity
H167A
mutation of conserved His residue, involved in binding of Fe2+. Loss of catalytic activity
H205A
mutation of conserved His residue, 6.8% residual catalytic activity
H218A
mutation of conserved His residue, involved in binding of Fe2+. Loss of catalytic activity
H282A
mutation of conserved His residue, involved in binding of Fe2+. Loss of catalytic activity
I352R
-
the mutant with 99.7% activity shows better thermostability than the wild type enzyme (70.4% residual activity after 15 min at 35°C)
K83R
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the mutant with 103.9% activity shows better thermostability than the wild type enzyme (84.2% residual activity after 15 min at 35°C)
K83R/I352R
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the mutant with 99.2% activity shows better thermostability than the wild type enzyme (87.8% residual activity after 15 min at 35°C)
K83R/K95R
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the mutant with 104.8% activity shows better thermostability than the wild type enzyme (92.1% residual activity after 15 min at 35°C)
K83R/K95R/G398A
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the mutant with 130.1% activity shows better thermostability than the wild type enzyme (94.1% residual activity after 15 min at 35°C)
K83R/K95R/I352R
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the mutant with 114.3% activity shows better thermostability than the wild type enzyme (90.1% residual activity after 15 min at 35°C)
K83R/K95R/L273F
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compared with the wild type enzyme, the thermal inactivation half-lives (t1/2) of the mutant at 25°C, 30°C, and 35°C increase 2.9fold, 11.9fold, and 24.7fold, respectively. Simultaneously, it also exhibits a 4.8fold increase in kcat, leading to a 1.2fold increase in catalytic efficiency. The tolerance of the mutant to metal ions is also improved
K95R
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the mutant with 106.6% activity shows better thermostability than the wild type enzyme (68% residual activity after 15 min at 35°C)
L273F
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the mutant exhibits 1.17fold increase in specific activity compared to the wild type enzyme
G398A
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the mutation results in a 1.3fold increase in specific activity
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I352R
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the mutant with 99.7% activity shows better thermostability than the wild type enzyme (70.4% residual activity after 15 min at 35°C)
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K83R
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the mutant with 103.9% activity shows better thermostability than the wild type enzyme (84.2% residual activity after 15 min at 35°C)
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K95R
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the mutant with 106.6% activity shows better thermostability than the wild type enzyme (68% residual activity after 15 min at 35°C)
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L273F
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the mutant exhibits 1.17fold increase in specific activity compared to the wild type enzyme
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5 - 11
the enzyme shows a high stability at pH between 5.0 and 11.0, over 80% of initial activity is maintained after 2 h of incubation at universal buffers
764371
9
the enzyme is relatively stable at pH 9.0, with a residual activity over 90% after 12 h
763933
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15 - 20
conversion after 12 h increases from 67.0 to 84.1% with the temperature increase from 15 to 20°C, and it decreases beyond 20°C
20 - 40
at 20°C and 30°C, more than 90% of initial activity residue is detected after 150 min incubation. However, its activity is decreased when incubated at temperatures higher than 40°C, about 50% of its initial activity is detected at 40°C after 30 min of incubation and almost inactivated at 50°C
25 - 35
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the wild type enzyme shows 53.2% residual activity after 15 min at 35°C. The enzyme has half-lives of 967.2, 49.8 and 7.8 min at 25, 30, and 35°C, respectively
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the relative activity of the immobilized enzyme (with 100 mM glutaraldehyde) maintains over 60% after 7 recycles
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
DMSO
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, 20mM Tris/HCl, 10% glycerol, and 1mM dithiothreitol, pH 8.0, stable for several months
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA agarose column chromatography, and Superdex 200 gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
the fusion of amphiphilic short peptide 18A (EWLKAFYEKVLEKLKELF) and isoeugenol monooxygenase is efficiently expressed in Escherichia coli BL21(DE3) cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
transcription of Iem is dependent on the amounts of isoeugenol and the positive regulatory protein IemR. Isoeugenol is the best inducer of Iem, followed by trans-anethole, which induces Iem to 58% of the transcription level observed for isoeugenol. Overproduction of regulatory protein IemR in Escherichia coli increases the transcription of Iem up to 96fold, even in the absence of isoeugenol
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
synthesis
Escherichia coli cells expressing isoeugenol monooxygenase produce 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. No accumulation of undesired by-products, such as vanillic acid or acetaldehyde, is observed
synthesis
the vanillin producing activity is induced by presence of isoeugenol. Under the optimized reaction conditions, Pseudomonas putida cells produce 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide
synthesis
upon expression of mutant F281Q in Escherichia coli and employing sol-gel chitosan membrane for removing the produced vanillin from the biotransformation system, under the optimal conditions (pH 8.5, 30°C, 180 rpm, 0.5 g wet cells, 0.1 g chitosan membrane), vanillin concentrations reach 4.5 g/l after about 40 h
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE