Information on EC 4.2.2.7 - heparin lyase

MetaCyc

heparin degradation

Heparin lyase

-

1000607-06-6

-

9025-39-2

-

2-O-desulfated heparin

?

acharan sulfate

n 4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyl-(1->4)-N-acetyl-D-glucosamine

de-N-sulfated heparin

?

-

Substrates: 8% activity compared to heparin
Products: -

?

deaminated heparin

?

-

Substrates: 54% activity compared to heparin
Products: -

?

fondaparinux

?

heparan sulfate

?

9 entries

heparan sulfate

heparan sulfate disaccharides with 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends

heparin

?

14 entries

heparin

heparin disaccharide + ?

heparin

n 4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyl-(1->4)-N-acetyl-6-O-sulfo-D-glucosamine + ?

heparin

unsaturated heparin disaccharide + ?

heparin

unsaturated heparin disaccharide + unsaturated heparin tetrasaccharide + unsaturated heparin hexasaccharide

5 entries

heparin

unsaturated heparin disaccharide + unsaturated heparin tetrasaccharide + unsaturated heparin pentasaccharide + unsaturated heparin hexasaccharide

heparin

unsaturated heparin disaccharides

heparin + H2O

?

Heparin monosulfate

?

heparin pentasaccharide

heparin oligosaccharides

-

653976

Substrates: oligosaccharide products give bands between those for the heparin disaccharide and tetrasaccharide standards
Products: -

?

Heparin sulfate

?

Flavobacteriia

-

5978

Substrates: heparin-like portion
Products: -

?

heparin, 20-mer

unsaturated heparin disaccharides

heparin, 6-mer

unsaturated heparin disaccharides

heparin, 8-mer

unsaturated heparin disaccharides

N-acetyl heparin

?

-

Substrates: 78% activity compared to heparin
Products: -

?

N-acetyl-de-O-sulfated heparin

?

-

Substrates: 250% activity compared to heparin
Products: -

?

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))

-

665613

Substrates: heparinase II
Products: -

?

additional information

?

-

33 entries

heparan sulfate

?

Homo sapiens

-

682868

Substrates: 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
Products: -

?

heparin

?

Prevotella heparinolytica

additional information

?

-

Ba2+

Ca2+

Co2+

Cu2+

Fe2+

-

activates

5973

K+

-

up to 300 mM

Mg2+

Mn2+

NaCl

Ni2+

Zn2+

additional information

optimal condition: 75 mmol/l NaCl

Ba2+

-

carbodiimide

-

0.1 mM, 27% inhibition

Cd2+

Co2+

Cu2+

diethyl dicarbonate

-

EDTA

Fe3+

-

heparin with reduced carboxylate group

-

competitive inhibitor

-

Hg2+

iodoacetic acid

-

Mg2+

Mn2+

1 mM, about 30% inhibition

747009

Morus alba extract

-

681722

-

N-Acetylimidazole

-

80% inhibition at 1 mM concentration of inhibitor, indicates the necessity of tyrosine residues in the active site

N-bromosuccinimide

-

30% inhibition at 1 mM concentration of inhibitor

N-tosyl-L-lysine-chloromethyl ketone

-

0.1 mM, 84% inhibition

Na+-heparin

-

-

Ni2+

p-chloromercuriphenyl sulfonic acid

Pb2+

Polyvinyl sulfate

-

resveratrol

-

SO42-

-

slight

5984

Zn2+

additional information

-

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

-

2-mercaptoethanol

-

dithiothreitol

-

EDTA

iodoacetic acid

-

0.1 mM, 53% increase in activity

Urea

-

up to 1 M of urea

additional information

-

0.0036 - 0.21

heparan sulfate

0.0023 - 1.2

heparin

32 entries

0.018

heparin pentasaccharide

-

cleavage of a synthetic pentasaccharide containing an antithrombin III binding site into a trisaccharide and a disaccharide, pH 7.0, 30ºC

653976

0.0046 - 0.2395

heparin, 20-mer

-

0.0541 - 5.015

heparin, 6-mer

-

0.0182

heparin, 8-mer

-

wild-type protein

-

0.042

N-acetyl-de-O-sulfated heparin

-

pH 7.4, 37ºC

-

additional information

acharan sulfate

-

8.9 - 27

heparin

7 entries

4 - 29.1

heparin, 20-mer

-

14.5 - 78.4

heparin, 6-mer

-

73.7

heparin, 8-mer

-

wild-type protein

-

0.038 - 125.33

heparin

16.5 - 6311

heparin, 20-mer

-

3 - 1448

heparin, 6-mer

-

4054

heparin, 8-mer

-

wild-type protein

-

additional information

heparin

Km value 4.6 mg/ml, pH 6.0, 50°C

0.00008

heparin with reduced carboxylate group

-

pH not specified in the publication temperature not specified in the publication

-

101.4

-

heparin (8-mer), wild-type protein

107.8

-

heparin (6-mer), wild-type protein

109.9

acharan sulfate

19.5

-

pH 7.2, 40ºC

21.1

-

heparin (6-mer), deletion mutant 191-213 protein

27.74

heparan sulfate

40.6

-

heparin (20-mer), wild-type protein

44.6

5.8

-

heparin (20-mer), deletion mutant 191-213 protein

6.38

heparin

7.8

-

738.3

-

pH 7.5, 35°C

additional information

4.4

-

5.8

-

6

-

6.5

6.7

-

6.7 - 7.3

-

6.8 - 7.2

-

7

7.2

-

45ºC

7.4

substrate heparin

7.5

additional information

-

effect of blocking agents on pH optimum, immobilized enzyme

5981

3.8 - 5.5

Rattus norvegicus

-

intermediate activity at 5.5 and 3.8

5975

4 - 6.5

B3U3D9

almost no activity for wild-type

747009

5

-

almost no activity below

5 - 8

-

5 - 9

-

5.8 - 7.9

-

pH 5.8: about 55% of maximal activity, pH 7.9: about 55% of maximal activity

677819

6 - 8.4

-

10% of the maximal activity below and above this range

6 - 8.5

-

high activity range

9

complete loss of activity

additional information

-

activities in citrate and Tris-HCl buffer are 17% and 37.21% higher than that in Bis-Tris buffer, respectively

30

31

-

32

-

37

40

41

-

43

-

enzyme assay at

45

-

pH 7.2

50

additional information

-

Q10: 1.45

15 - 60

-

highly active within the temperature range of 25-35°C, but activity is lost rapidly above 35°C

20 - 37

-

25 - 40

-

30 - 56

-

30°C: about 40% of maximal activity, 56°C: about 650% of maximal activity

40

90% of maximum activity

65

complete loss of activity

7.35

calculated for mature protein

8.5

Aspergillus flavus MTCC-8654

-

isoelectric focusing

8.9

-

9

Aspergillus oryzae

-

-

-

-

Bacteroides eggerthii

Bacteroides eggerthii 1_2_48FAA

-

747533

E5X103

UniProt

Bacteroides stercoris HJ-15

-

677819, 704591

-

-

-

-

-

-

-

-

-

Niallia circulans HpT298

Pedobacter heparinus ATCC 13125

7 entries

-

Prevotella heparinolytica

-

-

5973

-

Pseudomonas aeruginosa

-

747419, 747935

A0A0H2ZDM9

UniProt

Pseudomonas aeruginosa UCBPP-PA14

-

747419, 747935

A0A0H2ZDM9

UniProt

-

746759, 773001

Pseudopedobacter saltans DSM 12145

UniProt

-

UniProt

-

-

novel species isolated from the effluents of a food mill

-

cell culture

-

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

commercial preparation

-

additional information

-

propagated in bovine lung microvascular endothelial cells

-

-

Highest Expressing Human Cell Lines

Filter by:

Cell Line Links

Gene Links

physiological function

additional information

111000

-

x * 111000, SDS-PAGE

650632

111200

Niallia circulans

Q8KKH9

x * 111200, from amino acid sequence

650636

23380

-

x * 24000, SDS-PAGE, x * 23380, mass spectrometry

24000

43000

45000

-

SDS-PAGE, Flavobacterium heparinum

5982

48000

-

x * 48000, SDS-PAGE

50000

Prevotella heparinolytica

-

gel permeation chromatography, Flavobacterium heparinum

5982

63000

-

SDS-PAGE, Bacteroides heparinolyticus

5973

70000

-

gel filtration

70100

-

1 * 70100, SDS-PAGE

75670

-

mass spectrometry

85000

-

2 * 85000, calculated: 84545

85400

x * 85400, calculated

?

16 entries

homodimer

-

2 * 85000, calculated: 84545

monomer

proteolytic modification

substrate docking, residues Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 are mainly involved in the substrate binding, and 11 hydrogen bonds are formed between heparin and the enzyme

-

sitting drop method

-

hanging drop vapor diffusion method

-

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

-

663480

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

-

665714

homology modeling, the putative catalytic triad involves the residues Asn216, His400 and Tyr270

773001

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

R83A

-

no activity

Y357A

-

no activity

H151A

-

no activity

-

Q149A

-

no activity

-

Q22A

-

about 20% of wild-type activity

-

R156A

-

about 90% of wild-type activity

-

R83A

-

no activity

-

A259D

Q05819

40.43% increase in the specific enzyme activity and a 108.33% increase in the yield

769944

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)

I133T/P316T

B3U3D9

mutant displays about 7fold increase in catalytic efficiency

747009

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

S169D

Q05819

50.18% increase in the specific enzyme activity and a 91.67% increase in the yield

769944

S169D/A259D

Q05819

122.05% increase in the specific enzyme activity and a 75% increase in the yield

769944

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

6 - 9

stable for 1 h

7

-

stability maximum

5972, 5982

7 - 11

-

0°C, 30 min, stable. Inactivated out of this range

15

-

30 min, stable

28

-

purified native enzyme, catalytic activity after about 72 h shows a loss of 40.4% in activity

30

-

half-life 300 min

30 - 40

-

35

-

30 min, rapid inactivation at 35°C or above

37

-

half-life 57 min

40

-

rapid loss of activity above, more than 25% loss of activity after 20 min

45

50

55

60

melting temperature, presence of 10 mM Mg2+

CaCl2 is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4°C or freezing-thawing

-

enzyme is stabilized by dithiothreitol

-

Stability maximum: 0.15 M NaCl

-

-20°C, purified native enzyme, 72 h, below 12% loss of activity

-

1 month at -15°C

-

4°C, in the presence of CaCl2 15% loss of activity after 5 days

-

4°C, purified native enzyme, 72 h, below 22% loss of activity

-

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

Flavobacteriia

-

5978

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

-

unstable if stored at 4ºC, stability is increased with bovine serum albumin addition

-

-

ammonium sulfate precipitation followed by DEAE-Sephacel and Sephacryl S-200 column chromatography

Niallia circulans

-

650632

by several chromatographic steps, including QAE-cellulose, DEAE-cellulose, Sephadex and hydroxyapatite

-

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)

Aspergillus oryzae

-

enzyme expressed in Escherichia coli as a fusion protein with GST at the N-terminus, one-step affinity purification

-

677819

immobilization

-

5981, 5982

immobilized metal ion affinity chromatography, ion exchange chromatography, gel filtration

-

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

-

large scale

-

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

-

partial

-

purification of a recombinant enzyme by ammonium sulfate precipitation followed by DEAE-Sephacel and Sephacryl S-200 column chromatography

Niallia circulans

Q8KKH9

650636

recombinant protein

Pseudomonas aeruginosa

A0A0H2ZDM9

747419

three steps osmotic shock procedure, followed by chromatography on SP-Sepharose and Source 30S resin

-

construction of recombinant Escherichia coli for over-production of soluble heparinase I by fusion to maltose-binding protein at high yield

-

663909

expressed in Escherichia coli BL21(DE3)

expressed in Pedobacter heparinus

-

expression as fusion protein, fused to the C-terminus of soluble partners

-

730873

expression in Escherichia coli

7 entries

expression in Escherichia coli as a fusion protein with GST at the N-terminus

-

677819

expression of heparinase I in Escherichia coli

-

715131

expression of the recombinant wild type and several mutants in Escherichia coli

-

His-tagged version expressed in Escherichia coli BL21(DE3)

-

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

-

inducible heparinase