Information on EC 4.2.2.7 - heparin lyase

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

EC NUMBER
COMMENTARY hide
4.2.2.7
-
RECOMMENDED NAME
GeneOntology No.
heparin lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Eliminative cleavage of polysaccharides containing (1->4)-linked D-glucuronate or L-iduronate residues and (1->4)-alpha-linked 2-sulfoamino-2-deoxy-6-sulfo-D-glucose residues to give oligosaccharides with terminal 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends
show the reaction diagram
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-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
-
-
-
-
additional information
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
heparin degradation
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-
SYSTEMATIC NAME
IUBMB Comments
Heparin lyase
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CAS REGISTRY NUMBER
COMMENTARY hide
1000607-06-6
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9025-39-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
; phylogenetic tree
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-
Manually annotated by BRENDA team
; phylogenetic tree
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
strain WAL2926
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
novel species isolated from the effluents of a food mill
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
2-O-desulfated heparin
?
show the reaction diagram
acharan sulfate
n 4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyl-(1->4)-N-acetyl-D-glucosamine
show the reaction diagram
de-N-sulfated heparin
?
show the reaction diagram
-
8% activity compared to heparin
-
?
deaminated heparin
?
show the reaction diagram
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54% activity compared to heparin
-
?
heparan sulfate
?
show the reaction diagram
heparan sulfate
disaccharides
show the reaction diagram
heparin
-
show the reaction diagram
heparin
?
show the reaction diagram
heparin
disaccharide + tetrasaccharide + hexasaccharide
show the reaction diagram
-
-
-
?
Heparin
Disaccharide + tetrasaccharide + hexasaccharides
show the reaction diagram
heparin
disaccharides
show the reaction diagram
heparin
n 4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyl-(1->4)-N-acetyl-6-O-sulfo-D-glucosamine
show the reaction diagram
Heparin monosulfate
?
show the reaction diagram
Heparin sulfate
?
show the reaction diagram
-
heparin-like portion
-
-
-
N-acetyl heparin
?
show the reaction diagram
-
78% activity compared to heparin
-
?
N-acetyl-de-O-sulfated heparin
?
show the reaction diagram
-
250% activity compared to heparin
-
?
partially de-N-sulfated forms of heparin
(DELTA4,5-unsaturated hexuronic acid)-(N-unsubstituted glucosamine(6S)) + (DELTA4,5-unsaturated hexuronic acid(2S))-(N-unsubstituted glucosamine) + (DELTA4,5-unsaturated hexuronic acid(2S))-(N-unsubstituted glucosamine(6S))
show the reaction diagram
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heparinase II
-
-
?
additional information
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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
heparan sulfate
?
show the reaction diagram
-
the enzymatic disruption of heparan sulfate chains on cell surface proteoglycans alters bone morphogenetic protein (BMP) activity and Wnt activity so as to enhance the lineage commitment and osteogenic differentiation of human mesenchymal stem cells
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-
?
heparin
?
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
-
up to 300 mM
additional information
optimal condition: 75 mmol/l NaCl
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
carbodiimide
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0.1 mM, 27% inhibition
diethyl dicarbonate
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EDTA
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1 mM EDTA completely inactivates heparinase I in the presence of 2 mM Ca2+. Inhibition is reversible, the reintroduction of calcium ions into the system completely restores the enzymatic activity of heparinase I
Fe3+
-
-
heparin with reduced carboxylate group
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competitive inhibitor
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iodoacetic acid
-
-
Mg2+
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0.1 mM, 17% inhibition
Morus alba extract
-
-
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N-Acetylimidazole
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80% inhibition at 1 mM concentration of inhibitor, indicates the necessity of tyrosine residues in the active site
N-bromosuccinimide
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30% inhibition at 1 mM concentration of inhibitor
N-tosyl-L-lysine-chloromethyl ketone
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0.1 mM, 84% inhibition
Na+-heparin
-
-
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p-Chloromercuriphenyl sulfonic acid
Polyvinyl sulfate
-
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resveratrol
-
-
SO42-
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slight
additional information
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specific desulfation of the alpha-L-iduronic acid, 2-O-sulfate ring or the desulfation of the acid alpha-L-iduronic, 2-O-sulfate ring, followed by epimerization of its iduronic to galacturonic acid abolishes its degradation by heparinase I, as well as leading to the formation of competitive inhibitors
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
-
dithiothreitol
-
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iodoacetic acid
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0.1 mM, 53% increase in activity
Urea
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up to 1 M of urea
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0036 - 0.21
heparan sulfate
0.0023 - 1.2
heparin
0.0046 - 0.2395
heparin, 20-mer
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0.0541 - 5.015
heparin, 6-mer
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0.0182
heparin, 8-mer
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wild-type protein
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0.042
N-acetyl-de-O-sulfated heparin
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pH 7.4, 37ºC
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0.018 - additional information
acharan sulfate
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4 - 29.1
heparin, 20-mer
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14.5 - 78.4
heparin, 6-mer
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73.7
heparin, 8-mer
Bacteroides thetaiotaomicron
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wild-type protein
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16.5 - 6311
heparin, 20-mer
14938
3 - 1448
heparin, 6-mer
14939
4054
heparin, 8-mer
Bacteroides thetaiotaomicron
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wild-type protein
27239
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00008
heparin with reduced carboxylate group
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pH not specified in the publication temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
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heparin (20-mer), deletion mutant 191-213 protein
19.5
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pH 7.2, 40ºC
21.1
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heparin (6-mer), deletion mutant 191-213 protein
27.74
heparan sulfate
40.6
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heparin (20-mer), wild-type protein
44.6
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purified enzyme, pH 7.0, 30°C; purified enzyme, pH 7.5, 30°C
101.4
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heparin (8-mer), wild-type protein
107.8
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heparin (6-mer), wild-type protein
109.9
acharan sulfate
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 7.2
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7.4
substrate heparin
additional information
-
effect of blocking agents on pH optimum, immobilized enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.8 - 5.5
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intermediate activity at 5.5 and 3.8
5 - 9
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5.8 - 7.9
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pH 5.8: about 55% of maximal activity, pH 7.9: about 55% of maximal activity
6 - 8.5
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high activity range
6 - 8.4
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10% of the maximal activity below and above this range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
43
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enzyme assay at
additional information
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Q10: 1.45
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 60
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highly active within the temperature range of 25-35°C, but activity is lost rapidly above 35°C
25 - 40
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30 - 56
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30°C: about 40% of maximal activity, 56°C: about 650% of maximal actvity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.35
calculated for mature protein
8.5
-
isoelectric focusing
9
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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optimization of enzyme production, simple carbon sources, including sucrose, lactose, maltose, and complex carbon sources such as starch and cellulose are able to support growth of the organism, but enzyme can be produced only in the presence of maltose, sorbitol, mannitol, arabinose, and mannose. Phosphate inhibits enzyme production and cell growth, while heparin inhibits cell growth but is required for enzyme induction, overview. Heparinase production in optimized medium is highest, i.e., 75.4 units, when the pH of the medium is kept at pH 6.5, inoculum size of 0.5-1.0% of spore suspension, and for an incubation temperature of 30°C. Growth profile, heparin utilization, and heparinase production in optimized medium, overview
Manually annotated by BRENDA team
additional information
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propagated in bovine lung microvascular endothelial cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482)
Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23380
-
x * 24000, SDS-PAGE, x * 23380, mass spectrometry
24000
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x * 24000, SDS-PAGE; x * 24000, SDS-PAGE, x * 23380, mass spectrometry
45000
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SDS-PAGE, Flavobacterium heparinum
48000
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x * 48000, SDS-PAGE
50000
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gel permeation chromatography, Flavobacterium heparinum
63000
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SDS-PAGE, Bacteroides heparinolyticus
70000
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gel filtration
70100
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1 * 70100, SDS-PAGE
75670
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mass spectrometry
85000
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2 * 85000, calculated: 84545
85400
x * 85400, calculated
111000
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x * 111000, SDS-PAGE
111200
x * 111200, from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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2 * 85000, calculated: 84545
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop method
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hanging drop vapor diffusion method
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hanging-drop vapour-diffusion method, space group: P2(1)2(1)2(1), unit-cell parameters: a = 70.0 A, b = 119.3 A, c = 200.7 A, alpha = beta = gamma = 90°. Two molecules in the asymmetric unit
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structure of the enzyme in ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.3 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 11
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0°C, 30 min, stable. Inactivated out of this range
681056
7
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stability maximum
5972, 5982
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15
-
30 min, stable
28
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purified native enzyme, catalytic activity after about 72 h shows a loss of 40.4% in activity
35
-
30 min, rapid inactivation at 35°C or above
40
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rapid loss of activity above, more than 25% loss of activity after 20 min
45
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completely inactivated after 5 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
CaCl2 is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4°C or freezing-thawing
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enzyme is stabilized by dithiothreitol
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Stability maximum: 0.15 M NaCl
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, purified native enzyme, 72 h, below 12% loss of activity
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1 month at -15°C
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4°C, in the presence of CaCl2 15% loss of activity after 5 days
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4°C, purified native enzyme, 72 h, below 22% loss of activity
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4°C, without any additives, 15 days, retains its activity, more stable at 4°C than at -20°C, activity decreases after a few cycles of freezing and thawing
At 4°C, some loss of activity after 5 weeks
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Frozen or lyophilized at -20°C, protein concentration: 0.2-0.4 mg/ml, more stable in presence of 7.5% glycerol or 5% sucrose
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unstable if stored at 4ºC, stability is increased with bovine serum albumin addition
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; immobilization
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; large scale
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ammonium sulfate precipitation followed by DEAE-Sephacel and Sephacryl S-200 column chromatography
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by several chromatographic steps, including QAE-cellulose, DEAE-cellulose, Sephadex and hydroxyapatite
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enhancement of enzyme production by 92% is achieved by manipulation of the fermentation conditions (pH: 6.5, inoculum size of 0.5-1.0%, incubation temperature 30°C) and medium ingredients (replacement of dextrose by mannitol, soy peptone by chitin and ammonium sulfate by ammonium nitrate and supplementation of pyridoxine hydrochloride and glutaric acid)
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enzyme expressed in Escherichia coli as a fusion protein with GST at the N-terminus, one-step affinity purification
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immobilization
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immobilized metal ion affinity chromatography, ion exchange chromatography, gel filtration
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ion exchange chromatography
ion-exchange chromatography (SP-Sepharose), hydroxylapatite chromatography, mutant proteins by immobilized metal-chelate chromatography, His-tag is removed, further purification by ion-exchange chromatography
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large scale
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native enzyme 40.5fold by anion and cation exchange chromatography, and gel filtration; native enzyme 40.5fold by anion exchange and hydrophobic interaction chromatography, and gel filtration
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purification of a recombinant enzyme by ammonium sulfate precipitation followed by DEAE-Sephacel and Sephacryl S-200 column chromatography
three steps osmotic shock procedure, followed by chromatography on SP-Sepharose and Source 30S resin
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of recombinant Escherichia coli for over-production of soluble heparinase I by fusion to maltose-binding protein at high yield
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expressed in Escherichia coli BL21(DE3)
expressed in Pedobacter heparinus
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expression as fusion protein, fused to the C-terminus of soluble partners
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expression in Escherichia coli
expression in Escherichia coli as a fusion protein with GST at the N-terminus
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expression of heparinase I in Escherichia coli
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expression of the recombinant wild type and several mutants in Escherichia coli
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His-tagged version expressed in Escherichia coli BL21(DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
heparin induces the enzyme in cells, enzyme production is also upleveled by addition of N-acetyl D-glucosamine, glutaric acid, mannitol, arabinose, pyridoxine, diammonium biphosphate, ammonium nitrate, and especially by chitin, slight enhancement by L-leucine, L-glutamine, and L-histidine, overview
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inducible heparinase
phosphate suppresses the enzyme production in cells, enzyme production is also reduced by potassium nitrate, maltose, mannose, and especially by sucrose, overview
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA 156-221
-
no activity
DELTA 191-213
-
about 5% of wild-type activity
H151A
-
no activity
K185A
-
about 100% of wild-type activity
K252A
-
about 40% of wild-type activity
K353A
-
no activity
Q149A
-
no activity
Q223A
-
about 40% of wild-type activity
Q22A
-
about 20% of wild-type activity
R156A
-
about 90% of wild-type activity
Y357A
-
no activity
H151A
-
no activity
-
K185A
-
about 100% of wild-type activity
-
K353A
-
no activity
-
Q22A
-
about 20% of wild-type activity
-
R156A
-
about 90% of wild-type activity
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D210A
-
mutation does not affect the product profile compared to the wild type anzyme
D212A
-
mutation does not affect the product profile compared to the wild type anzyme
E207A
-
mutation does not affect the product profile compared to the wild type anzyme
E381A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
G213A
-
mutation does not affect the product profile compared to the wild type anzyme
G378A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
H202/H406A
-
His-tagged version expressed in Escherichia coli BL21(DE3)
H202A
-
no activity in a real time assay, His-tagged version expressed in Escherichia coli BL21(DE3)
H202A/Y257A
-
His-tagged version expressed in Escherichia coli BL21(DE3)
H406A
-
no activity in a real time assay, His-tagged version expressed in Escherichia coli BL21(DE3)
N375A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
N405A
-
site-directed mutagenesis, compared to the wild-type enzyme, the mutant shows broadened substrate specificity and cleaves the resistant linkage proximate to the 3-O-sulfoglucosamine residue
N405A/H406A
-
site-directed mutagenesis
N405G
-
site-directed mutagenesis, compared to the wild-type enzyme, the mutant shows broadened substrate specificity and cleaves the resistant linkage proximate to the 3-O-sulfoglucosamine residue
N405G/H406A
-
site-directed mutagenesis
S377A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
T216A
-
mutation does not affect the product profile compared to the wild type anzyme
T373A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
T382A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
Y257A
-
no activity in a real time assay, His-tagged version expressed in Escherichia coli BL21(DE3)
Y257A/H406A
-
His-tagged version expressed in Escherichia coli BL21(DE3)
Y257F
-
no activity in a real time assay, His-tagged version expressed in Escherichia coli BL21(DE3)
Y379A
-
produces lower amounts of disaccharides and tetrasaccharides and a greater fraction of larger digestion fragments
additional information
-
construction of several double and triple mutants of the calcium-coordinating residues
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis
-
expression as fusion protein, fused to the C-terminus of soluble partners translation initiation factor 2 domain I, glutathione S-transferase, maltose-binding protein, small ubiquitin modifying protein and N-utilization substance A, and purification of hybrid proteins. Except for NusA, the fusion partners dramatically improve the soluble expression of recombinant HepA, with translation initiation factor 2 domain I-HepA and small ubiquitin modifying protein-HepA creating almost completely soluble HepA where 98% and 94% of expressed HepA fusions are soluble, respectively