Information on EC 3.1.1.76 - poly(3-hydroxyoctanoate) depolymerase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.1.1.76
-
RECOMMENDED NAME
GeneOntology No.
poly(3-hydroxyoctanoate) depolymerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolyses the polyester poly{oxycarbonyl[(R)-2-pentylethylene]} to oligomers
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
hydrolysis of C-O bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
poly{oxycarbonyl[(R)-2-pentylethylene]} hydrolase
The main product after prolonged incubation is the dimer [3]. Besides hydrolysing polymers of 3-hydroxyoctanoic acid, the enzyme also hydrolyses other polymers derived from medium-chain-length (C6-C12) hydroxyalkanoic acids and copolymers of mixtures of these. It also hydrolyses p-nitrophenyl esters of fatty acids. Polymers of short-chain-length hydroxyalkanoic acids such as poly[(R)-3-hydroxybutanoic acid] and poly[(R)-3-hydroxypentanoic acid] are not hydrolysed.
CAS REGISTRY NUMBER
COMMENTARY hide
140208-16-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
enzyme type I and II
-
-
Manually annotated by BRENDA team
enzyme type I and II
-
-
Manually annotated by BRENDA team
orf Bd3709
-
-
Manually annotated by BRENDA team
P37C
-
-
Manually annotated by BRENDA team
P37C
-
-
Manually annotated by BRENDA team
gene phaZ, strain ATCC 17741
-
-
Manually annotated by BRENDA team
isolated from fuel-oil contaminated soil
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-
Manually annotated by BRENDA team
gene phaZ, precursor; gene phaZ
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Pseudomonas indica K2 / DSM 16298
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
gene phaZ
SwissProt
Manually annotated by BRENDA team
RY-1
-
-
Manually annotated by BRENDA team
RY-1
-
-
Manually annotated by BRENDA team
1317
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-
Manually annotated by BRENDA team
enzyme type I and II according to the lipase box in the catalytic domain
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-
Manually annotated by BRENDA team
enzyme type I and II according to the lipase box in the catalytic domain
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-
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strain DSM 15344
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Manually annotated by BRENDA team
isolated from soil, gene phaZSomSO2
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Manually annotated by BRENDA team
isolated from soil, gene phaZSomSO2
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Manually annotated by BRENDA team
isolated from sludge, gene phaZSroSL3
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Manually annotated by BRENDA team
isolated from sludge, gene phaZSroSL3
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
isolated from a soil sample taken at the Campus of the University of the Basque Country, Bizkaia, Spain
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Manually annotated by BRENDA team
gene phaZSveSO1
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Manually annotated by BRENDA team
the enzyme is encoded in parts of gene TTHA1605
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-
Manually annotated by BRENDA team
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
the enzyme is encoded in parts of gene TTHA1605
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,2,3-tributyryl-sn-glycerol + H2O
?
show the reaction diagram
4-nitrophenyl acetate + H2O
4-nitrophenol + acetate
show the reaction diagram
4-nitrophenyl decanoate + H2O
?
show the reaction diagram
4-nitrophenyl dodecanoate + H2O
4-nitrophenol + dodecanoate
show the reaction diagram
4-nitrophenyl dodecanoate + H2O
?
show the reaction diagram
4-nitrophenyl hexadecanoate + H2O
?
show the reaction diagram
4-nitrophenyl hexanoate + H2O
4-nitrophenol + hexanoate
show the reaction diagram
4-nitrophenyl octanoate + H2O
4-nitrophenol + octanoate
show the reaction diagram
4-nitrophenyl octanoate + H2O
?
show the reaction diagram
4-nitrophenyl octanoate + H2O
octanoate + 4-nitrophenol
show the reaction diagram
4-nitrophenyl valerate + H2O
?
show the reaction diagram
-
-
-
-
?
fatty acid p-nitrophenyl esters + H2O
?
show the reaction diagram
p-nitrophenyl butyrate + H2O
4-nitrophenol + butyrate
show the reaction diagram
-
1.36% of the activity with p-nitrophenydecanoate
-
-
?
p-nitrophenyl decanoate + H2O
4-nitrophenol + decanoate
show the reaction diagram
PHB-co-PHV (60%) + H2O
?
show the reaction diagram
-
74% of activity with poly(3-hydroxyoctanoate)
-
-
?
poly(3-hydroxyalkanoate) + H2O
3-hydroxalkanoate
show the reaction diagram
poly(3-hydroxyalkanoate) + H2O
oligo(3-hydroxalkanoate)
show the reaction diagram
poly(3-hydroxyalkanoate) + H2O
oligo(3-hydroxyalkanoate) + 3-hydroxyalkanoate
show the reaction diagram
poly(3-hydroxybutyrate) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(3-hydroxybutyrate) + H2O
oligo(3-hydroxybutyrate)
show the reaction diagram
poly(3-hydroxybutyrate) + H2O
oligo(3-hydroxybutyrate) + D-(-)-3-hydroxybutyrate dimer
show the reaction diagram
-
-
-
-
?
poly(3-hydroxybutyrate-co-3-hydroxypropionate) + H2O
?
show the reaction diagram
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) + H2O
?
show the reaction diagram
poly(3-hydroxybutyrate-co-3-mercaptopropionate) + H2O
?
show the reaction diagram
-
co-polymeric substrate, the activity decreases with increasing amount of 3-mercaptopropionate
-
-
?
poly(3-hydroxydecanoate-co-3-hydroxhyoctanoate) + H2O
?
show the reaction diagram
poly(3-hydroxyheptanoate) + H2O
?
show the reaction diagram
-
28.7% of activity with poly(3-hydroxyoctanoate)
-
-
?
poly(3-hydroxyheptanoate) + H2O
di(3-hydroxyheptanoate)
show the reaction diagram
poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate) + H2O
?
show the reaction diagram
poly(3-hydroxynonanoate) + H2O
?
show the reaction diagram
-
13.4% of activity with poly(3-hydroxyoctanoate)
-
-
?
poly(3-hydroxyoctanoate) + H2O
?
show the reaction diagram
poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) + H2O
(R)-3-hydroxyoctanoate
show the reaction diagram
poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) + H2O
(R)-3-hydroxyoctanoate + ?
show the reaction diagram
poly(3-hydroxyoctanoic acid) + H2O
?
show the reaction diagram
poly(3-hydroxyphenylheptanoate) + H2O
?
show the reaction diagram
poly(3-hydroxyphenyloctanoate) + H2O
?
show the reaction diagram
poly(3-hydroxyphenylvalerate) + H2O
?
show the reaction diagram
-
4.1% of activity with poly(3-hydroxyoctanoate)
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-
?
poly(3-hydroxypropionate) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(3-hydroxypropionate) + H2O
oligo(3-hydroxypropionate)
show the reaction diagram
poly(epsilon-caprolactone) + H2O
?
show the reaction diagram
polyhydroxyalkanoate + H2O
3-hydroxyhexanoic acid + 3-hydroxyoctanoic acid
show the reaction diagram
polyhydroxyalkanoate latex + H2O
?
show the reaction diagram
polyhydroxyoctanoate-co-hexanoate + H2O
?
show the reaction diagram
poly[(R)-3-hydroxynonanoate]n + H2O
?
show the reaction diagram
poly[(R)-3-hydroxyoctanoate]n + H2O
poly[(R)-3-hydroxyoctanoate]n-x + poly[(R)-3-hydroxyoctanoate]x
show the reaction diagram
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
poly(3-hydroxyalkanoate) + H2O
3-hydroxalkanoate
show the reaction diagram
poly(3-hydroxyalkanoate) + H2O
oligo(3-hydroxalkanoate)
show the reaction diagram
poly(3-hydroxybutyrate) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(3-hydroxybutyrate) + H2O
oligo(3-hydroxybutyrate)
show the reaction diagram
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) + H2O
?
show the reaction diagram
poly(3-hydroxydecanoate-co-3-hydroxhyoctanoate) + H2O
?
show the reaction diagram
poly(3-hydroxyoctanoate) + H2O
?
show the reaction diagram
poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) + H2O
(R)-3-hydroxyoctanoate
show the reaction diagram
poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) + H2O
(R)-3-hydroxyoctanoate + ?
show the reaction diagram
poly(3-hydroxyoctanoic acid) + H2O
?
show the reaction diagram
poly(3-hydroxypropionate) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(epsilon-caprolactone) + H2O
?
show the reaction diagram
polyhydroxyalkanoate latex + H2O
?
show the reaction diagram
poly[(R)-3-hydroxyoctanoate]n + H2O
poly[(R)-3-hydroxyoctanoate]n-x + poly[(R)-3-hydroxyoctanoate]x
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
stimulates at 1 mM
K+
-
stimulates at 1 mM
Na+
-
stimulates at 1 mM
NaCl
-
a high ionic strength of 0.5 M NaCl in the reaction mixture is required for optimal activity
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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complete inhibition
4-hydroxymercuribenzoate
Acetic anhydride
bis(1-methylethyl) fluorophosphate
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carbodiimide
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slight inhibition at 10 mM
Cetyltrimethylammonium bromide
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88% inhibition at 0.05 vol%
deoxycholate
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84% inhibition at 0.05 vol%
diisopropyl fluorophosphate
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83% inhibition at 5 mM
diisopropylfluorophosphate
dithiothreitol
DTNB
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complete inhibition
ethanol
iodoacetamide
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slight inhibition at 10 mM
iodoacetate
mercaptoethanol
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N-bromosuccimide
N-bromosuccinimide
n-Propanol
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strong inhibition at 10% v/v
NaN3
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slight inhibition at 10 mM
NEM
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10 mM, 29% inhibition
phenylmethylsulfonyl fluoride
Sodium azide
sodium cholate
sodium deoxycholate
-
35% inhibition at 0.5 mM
Triton X-100
Tween 20
Tween 80
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-propanol
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2fold activation at 5% (v/v)
EDTA
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10% activation at 10 mM
SDS
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3fold activation at 0.01% (w/v)
sodium cholate
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30% activation at 0.02 mM, 18% at 0.2 mM
Triton X-100
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activates 1.7fold at 0.005% (w/v), inhibits by 72% at 0.03% (w/v)
Tween 80
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activates1.8fold at 0.005% (w/v), inhibits 90% at 0.01% (w/v)
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.122
4-nitrophenyl octanoate
-
pH 9.0, 37°C
2.8
poly(3-hydroxybutyrate)
-
pH 7.9, 30°C
additional information
additional information
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12.1
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purified enzyme, pH 9.5, 30°C, substrate poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate)
17.7
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purified enzyme
19.4
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substrate 4-nitrophenyl octanoate, pH 9.5, 40°C
26
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purified enzyme
55 - 560
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purified recombinant His-tagged enzme, pH 8.0, 37°C, different assay methods
76
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substrate poly(3-hydroxyoctanoic acid), pH 9.5, 40°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9 - 9.5
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maximum activity in the alkaline pH-range
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 11
-
activity range, almost inactive at pH 12.0
6 - 11
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the purified enzyme is most active at pH range of pH 7.0-9.0, while the maximal activity is observed at pH 8.5. At pH 6.0 and pH 11 the enzyme shows 34% and 7.5% of maximal activity, respectively
7 - 10.5
pH 7.0: about 25% of maximal activity, pH 10.5: about 50% of maximal activity
7.5 - 9
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70% at pH 7.5 and at pH 9.0
7.5 - 11
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over 60% of maximal activity within this range
8 - 11
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over 60% of maximal activity within this range
8 - 10.5
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60% of maximal activity within this range
8.5 - 10.5
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pH 8.5: about 40% of maximal activity, pH 10.5: about 90% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
-
with substrate 4-nitrophenyl octanoate
additional information
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 50
-
activity range, highly active at 4-45°C, 29% of maximal activityy at 50°C
10 - 40
maximal activity at 40°C, 50% activity of maximal activity at 10°C
25 - 50
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25°C: about 65% of maximal activity, 50°C: about 70% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.7
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isoelectric focusing
5.2
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isoelectric focusing
6
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isoelectric focusing
6.45
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peak 1, isoelectric focusing
6.95
-
peak 2, isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26000
-
x * 26000, SDS-PAGE
26500
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native PAGE
26600
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calculated from nucleotide sequence
27000
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x * 27000, SDS-PAGE
27160
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MALDI-TOF/mass spectrometry
27164
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1 * 28000, SDS-PAGE, 1 * 27164, MALDI-TOF/mass spectrometry
27600
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gel filtration
30323
x * 30323, amino acid sequence calculation
31283
x * 31283, sequence calculation
48000
-
gel filtration of native enzyme
49300
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x * 49300, recombinant enzyme, SDS-PAGE
115000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
wide-angle X-ray diffraction, structure determination and analysis
crystal structure determination and analysis at 1.7 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10
-
fairly stable over this range
663666
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
purified enzyme, retains full initial activity after 24 h
37
very unstable above
50
-
over 90% remaining activity after 24 h
60
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1 h, almost complete inactivation
70
-
69% remaining activity after 24 h
additional information
-
thermal inactivation of the enzyme followed first-order kinetics in all cases, a value of 48.7 kcal/mol is calculated for the activation energy of enzyme thermal inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20 to -80°C, purified enzyme, 85% activity remaining after 6 months
-
-20°C to 70°C, purified enzyme, stable for more than 6 months
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-20°C, purified enzyme, 1 year, 70% remaining activity
-
the purified enzyme retains at least 75% of its original activity after 5 months of storage at -20 and -80°C, respectively
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
12.6fold to homogeneity by DEAE ion exchange chromatography and gel filtration
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development of a rapid and easy immobilization method of the enzyme, the extracellular enzyme is tightly adsorbed onto a commercially available polypropylene support with high yield and specificity. The immobilized enzyme catalyzes almost the complete hydrolysis of poly(3-hydroxyoctanoic acid) polymer to (R)-3-hydroxyoctanoic acid monomers
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extracellualr enzyme 4.2fold to homogeneity
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extracellular enzyme by gel filtration and adsorption chromatography
native extracellular enzyme by hydrophobic interaction chromatography, gel filtration, and ion exchange chromatography
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purified from culture medium
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recombinant extracellular enzyme by hydrophobic interaction chromatography, recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)(pETBd1) by nickel affinity chromatography
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recombinant solubilized and refolded His-tagged enzyme from Escherichia coli strain M15 by nickel affinity chromatography. The refolded enzyme cannot be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase is homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate cannot be hydrolyzed by the immobilized biocatalyst, whereas the attached enzyme is active in the hydrolysis of 4-nitrophenyl alkanoate esters, which differed in their alkyl chain length
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the enzyme is purified from cell medium 10.4fold by acetone precipitation, hydrophobic interaction chromatography using Octyl-Sepharose CL-4B, and gel filtration to homogeneity
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to homogeneity by hydrophobic interaction chromatography and gel filtration
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to homogeneity by ultrafiltration, affinity-based separation, and gel filtration
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to homogeneity by ultrafiltration, heat tretament at 70°C for 30 min, and affinity-based separation
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a stationary sigma factor (rpoS)negative mutant is constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 (phaC1) and polyhydroxyalkanoate depolymerase (phaZ). The rpoS gene of Pseudomaonas putida KT2440 and its promoter region are amplified with primers and cloned into pBBR1MCS-5 to obtain pBBRrpoS. This plasmid is transferred to Pseudomonas putida SDM75 and SDMPC1.
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gene phaZ, DNA and amino acid sequence determination and analysis, expression and subcloning in Escherichia coli strain DH5alpha; gene phaZ, DNA and amino acid sequence determination and analysis, expression and subcloning in Escherichia coli strain DH5alpha
gene phaZ, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3) in inclusion bodies
gene phaZ, functional expression in Escherichia coli strain JM109
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gene phaZSomSO2, DNA and amino acid sequence determination and analysis
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gene phaZSroSL3, DNA and amino acid sequence determination and analysis
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gene phaZSveSO1, DNA and amino acid sequence determination and analysis
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in Escherichia coli
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orf Bd3709, recombinant extracellular expression in different hypersecretor Tol-pal mutant strains of Escherichia coli and Pseudomonas putida, e.g. in Escherichia coli strain DH10B(pIZBd1) periplasm or Pseudomonas putida strain KT2442, expression of C-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)(pETBd1)
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overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative longchain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in Pseudomonas putida KT2442
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overexpression in Escherichia coli
overexpression of the His-tagged enzyme in Escherichia coli strain M15 in inclusion bodies
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recombinant expression of the extracellular enzyme in different hosts, e.g. Escherichia coli strains MC4100 (pUC18) and MC4100 (pLJ1) , and Pseudomonas putida strains KT2442, KT42FadB (pIZ1016), or KT42FadB(pIZPZ), method optimization and evaluation, the highest enzyme activity is obtained using Pseudomonas putida strain KT2442 fadB mutant
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the activity of PhaZ increases 1.5fold during growth on nitrogen limited medium and octanoate
-
the enzyme is induced by growth on Tween 20, overview
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
denaturing of the enzyme by Triton buffer, refolding of the enzyme from inclusion bodies after recombinant expression in Escherichia coli in Tris-HCl, pH 8.0, containing GSSG and GSH at 25°C
recombinant His-tagged enzyme from Escherichia coli strain M15 is solubilized in 8 M urea, immobilized on Ni2+-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis