Information on EC 3.2.1.157 - iota-carrageenase

Cgi

Alteromonas fortis

-

Cgi82A

CgiA

cgiA2

cgiB

-

CgiB_Ce

-

cgiF

-

753336, 753830

endo-type iota-carrageenase

GH82 iota-carrageenase

iota-carrageenase

iotase

Alteromonas fortis

-

iota-carrageenan degradation

zobellia_4265

6 entries

Endohydrolysis of (1->4)-beta-D-linkages between D-galactose 4-sulfate and 3,6-anhydro-D-galactose-2-sulfate in iota-carrageenans

6 entries

MetaCyc

iota-carrageenan 4-beta-D-glycanohydrolase (configuration-inverting)

The main products of hydrolysis are iota-neocarratetraose sulfate and iota-neocarrahexaose sulfate. iota-Neocarraoctaose is the shortest substrate oligomer that can be cleaved. Unlike EC 3.2.1.81, beta-agarase and EC 3.2.1.83, kappa-carrageenase, this enzyme proceeds with inversion of the anomeric configuration. iota-Carrageenan differs from kappa-carrageenan by possessing a sulfo group on O-2 of the 3,6-anhydro-D-galactose residues, in addition to that present in the kappa-compound on O-4 of the D-galactose residues.

74191-25-6

-

iota-/nu-carrageenan + H2O

hydrolyzed iota/nu-carrageenan

Alteromonas fortis

-

703077

the smallest hybrid is an octasaccharide with a iota-iota-nu-iota structure, the second fraction is composed of two decasaccharides with iota-iota-iota-nu-iota and iota-[iota/nu]iota-iota structures, the third fraction is a mixture of dodecasaccharides which contains at least a iota-iota-iota-iota-nu-iota oligosaccharide

-

?

iota-carrageenan + H2O

?

14 entries

iota-carrageenan + H2O

hydrolyzed iota-carrageenan

iota-carrageenan + H2O

iota-carrabiose + iota-carratetraose

-

-

?

iota-carrageenan + H2O

iota-carrahexaose + iota-carratetraose

iota-carrageenan + H2O

iota-carrahexaose + iota-carratetraose + iota-carrabiose

iota-carrageenan + H2O

iota-carratetraose

iota-carrageenan + H2O

iota-carratetraose + + iota-carrabiose

iota-carrageenan + H2O

iota-carratetraose + iota-carrabiose

9 entries

iota-carrageenan + H2O

iota-neocarratetraose sulfate + iota-neocarrahexaose sulfate

iota-carrageenan + H2O

neo-iota-carratetraose

iota-carrageenan + H2O

neo-iota-carratetraose + ?

CgiF is an endo-type iota-carrageenase that hydrolyzes beta-1,4-linkages of iota-carrageenan, yielding neo-iota-carratetraose as the main product

-

?

iota-carrageenan + H2O

neo-iota-carratetraose + neo-iota-carrahexaose

Alteromonas fortis

-

703075

most abundant products

-

?

additional information

?

-

19 entries

iota-carrageenan + H2O

?

12 entries

iota-carrageenan + H2O

hydrolyzed iota-carrageenan

Alteromonas fortis

-

703077

-

?

iota-carrageenan + H2O

iota-carrabiose + iota-carratetraose

-

-

?

iota-carrageenan + H2O

iota-carrahexaose + iota-carratetraose

iota-carrageenan + H2O

iota-carrahexaose + iota-carratetraose + iota-carrabiose

iota-carrageenan + H2O

iota-carratetraose

iota-carrageenan + H2O

iota-carratetraose + + iota-carrabiose

iota-carrageenan + H2O

iota-carratetraose + iota-carrabiose

iota-carrageenan + H2O

neo-iota-carratetraose

additional information

?

-

Ag+

-

activates by 20% at 1 mM

Al3+

-

activates by 10% at 1 mM

Ba2+

Ca2+

Co2+

-

activates by 108% at 1 mM

Cs+

-

in the presence of 0.1 M CsCl, 63% of maximal activity is observed

Fe2+

K+

5 entries

KCl

-

activates at 0.1 M, inhibitory at over 0.3 M

Li+

Mg2+

Mn2+

Na+

NaCl

Ni2+

128% activation at 1 mM

Zn2+

-

activates by 44% at 1 mM

additional information

2-mercaptoethanol

-

23% inhibition at 1 mM

Al3+

Ca2+

-

6% inhibition at 1 mM

Cd2+

-

26% inhibition at 1 mM

Cu2+

EDTA

Fe2+

-

18% inhibition at 1 mM

Fe3+

Hg2+

K+

KCl

-

activates at 0.1 M, inhibitory at over 0.3 M

Li+

-

Mn2+

Na+

-

activates at up to 700 mM, inhibitory above

NaCl

-

activates at 0.1 M, inhibitory at over 0.3 M

NH4+

-

PMSF

-

47.5% inhibition at 1 mM

SDS

Tween 20

-

30% inhibition at 1 mM

Zn2+

6 entries

Al3+

-

activates by 80% at 1 mM

CTAB

-

activates by 20% at 1 mM

DTT

-

activates by 10% at 1 mM

Triton X-100

-

activates by 24% at 1 mM

Tween 80

-

activates by 16% at 1 mM

0.00112 - 8.5

iota-carrageenan

-

additional information

-

Michaelis-Menten kinetics

-

4.1 - 726.6

iota-carrageenan

-

3.42 - 85.18

iota-carrageenan

-

11.55

-

substrate iota-carrageenan, pH 8.0, 30Â°C, recombinant enzyme

1207.8

purified recombinant enzyme, pH 7.6, 30Â°C

124.55

-

purified recombinant enzyme, pH 8.0, 45Â°C

1870

pH 6.5, 45Â°C, with iota-carrageenan; purified enzyme, pH 6.5, 45Â°C

365.9

-

purified native enzyme, pH 7.0, temperature not specified in the publication

7.8

-

culture supernatant, at pH 7.5 and 50Â°C

80.6

8068

uncultured bacterium

-

purified native enzyme, pH 8.0, temperature not specified in the publication

6.5

7

7.5

7.6

-

753336, 753830

8

5 entries

5 - 10

-

activity range, recombinant enzyme, profile overview

5 - 10.6

activity range, profile overview

5 - 11

activity range, overview

7 - 10

-

in a range of pH 7.0-10.0, the enzyme retains more than 80% of the original activity

25

-

753336, 754224

30

-

40

45

50

-

in 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl and 1 mM CaCl2

55

-

15 - 65

activity range, overview

30 - 60

40 - 60

-

5 - 50

activity range, CgiF is a cold-adapted iota-carrageenase with 36.5% and 57% of maximal activity at 10Â°C and 15Â°C, respectively, maximal activity at 30Â°C, profile overview

4.79

sequence calculation

Alteromonas fortis

-

UniProt

gene cgiB_Ce; isolated from the red alga Grateloupia livida collected from Qingdao coastal waters of China Yellow Sea, gene cgiB_Ce

UniProt

-

UniProt

-

716089, 753336, 755018, 755283

Microbulbifer thermotolerans JAMB-A94

UniProt

-

Microbulbifer thermotolerans JAMB-A94T

UniProt

-

716089, 753336

Microbulbifer thermotolerans JAMBA94T

UniProt

-

UniProt

-

-

Pseudoalteromonas carrageenovora ASY5

-

-

Pseudoalteromonas carrageenovora CICC 23819

-

-

uncultured bacterium

-

-

-

753336, 754224

UniProt

Wenyingzhuangia fucanilytica CZ1127

-

UniProt

Zobellia galactanivorans CCUG 47099

-

Zobellia galactanivorans CIP 106680

UniProt

-

Zobellia galactanivorans Dsij

UniProt

-

Zobellia galactanivorans DSM 12802

UniProt

-

Zobellia galactanivorans NCIMB 13871

UniProt

-

culture condition:iota-carrageen-grown cell

UniProt

-

additional information

evolution

metabolism

physiological function

iota-carrageenases play a role in marine iota-carrageenan degradation, and their enzymatic hydrolysatesare thought to be excellent antioxidants

additional information

residues R243, R303, and R353 are involved in substrate binding, residues E245, D247, E310, and H281 are catalytic residues. Glu245 acts as a catalytic proton donor in CgiA, and Asp247 acts as the general base that activates the catalytic water molecule. Three other residues are indirectly involved in the activity: Gln222 simultaneously binds the catalytic water molecule and a chloride ion, playing the essential function of structuring the water network in the active site. His281 participates in iota-carrageenan binding and is likely involved in proton trafficking with the proton donor Glu245, and Glu310 stabilizes the substrate intermediate conformation. Active site structure and molecular docking, overview

Alteromonas fortis

46300

-

48071

x * 48071, sequence calculation; x * 48071, sequence calculation, x * 54000, recombinant His-tagged enzyme, SDS-PAGE

50000

-

gel filtration

662105

50600 - 55800

-

51000

-

53400

-

54000

55000

?

monomer

additional information

enzyme structure analysis and comparisons, overview

concentration to 6 mg/ml using a dialyzing concentrator. The structure of iota-carrageenase bound to iota-carrageenan fragments is solved at 2.0 A resolution

Alteromonas fortis

662612

hanging-drop vapour-diffusion method using polyethylene glycol (MW = 6000) as a precipitant. Crystals belong to space group P2(1), with unit-cell parameters a = 56.75 A, b = 91.04 A, c = 125.01 A, beta = 93.4Â°. The unit cell contains two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Crystals diffracted to 2.0 A resolution on a synchrotron beamline

Alteromonas fortis

-

660570

native and selenomethionyl-iota-carrageenase. Single crystals are obtained with polyethylene glycol, and the presence of Ca2+ appears to be crucial for crystallization. High quality crystals, typically 0.25 * 0.25 * 0.15 mM in dimension are grown with 0.1 M sodium cacodylate at pH 6.5, 15-17% polyethylene glycol and 200 mM calcium acetate. Crystallization of Se-Met-iota-carrageenase is performed under similar conditions except for the addition of 1 mM dithiothreitol and the replacement of sodium cacodylate by imidazole to avoid the reaction between cacodylate and dithiothreitol. Crystal structure at 1.6 A resolution

Alteromonas fortis

-

662109

X-ray diffraction structure determination and analysis at 1.60 A resolution, PDB ID 1H80

D247A

Alteromonas fortis

-

the enzymatic activity is completely abolished

E245Q

Alteromonas fortis

-

the enzymatic activity is completely abolished

E310Q

Alteromonas fortis

-

the Km value is reduced by a factor of about 4, the reduction in kcat by 50fold results in a substantial decrease in the overall enzyme efficiency (about 10fold)

H281A

Alteromonas fortis

-

the mutant shows 7fold decreased Km and 180fold decreased kcat value compared to the wild type enzyme with 4% relative efficiency

Q222E

Alteromonas fortis

-

the enzymatic activity is abolished

Q222K

Alteromonas fortis

-

the enzymatic activity is abolished

D359N

10

purified recombinant His-tagged enzyme, 45Â°C, 24 h, 60% activity remaining

731522

11

purified recombinant His-tagged enzyme, 45Â°C, 24 h, inactivation

731522

5 - 10

-

purified recombinant enzyme, over 60% activity remaining

755283

5 - 10.6

-

purified enzyme, 24 h, over 70% activity remaining

755018

5 - 9

-

purified recombinant His-tagged enzyme, stable at

754224

6 - 9

purified recombinant His-tagged enzyme, 45Â°C, 24 h, stable

731522

6 - 9.6

stable at

731522

7 - 10

-

purified enzyme, over 80% activity remaining

755018

0 - 50

over 80% activity remaining at 0-30Â°C, 5-10% at 50Â°C

30

-

purified recombinant His-tagged enzyme, stable up to, 97% and 84% of the initial activity is retained after 24 h at 4Â°C and 25Â°C

40

45

-

the enzyme is stable up to 45Â°C in 50 mM Tris/HCl, pH 7.5, containing 0.1 M NaCl and 1 mM CaCl2 for 1 h. Incubation at 45Â°C inactivates the enzyme completely within 5 min at either condition of low NaCl concentration (less than 10 mM) or presence of 10 mM EDTA

50

enzyme contains three calcium binding sites involved in stabilizing the enzyme structure

Alteromonas fortis

-

662109

4Â°C, purified recombinant His-tagged enzyme, 84% of initial activity remains after 30 days storage at 4Â°C without any protective additives

-

-

ammonium sulfate precipitation, butyl-Toyopearl column chromatography, heparin affinity column chromatography, and Superdex 200 gel filtration

-

native enzyme 10.3fold by gel filtration

-

native enzyme 31fold by gel filtration

-

native enzyme by hydrophobic interaction chromatography

uncultured bacterium

-

Ni-Sepharose column chromatography, Source 15S column chromatography, gel filtration

Alteromonas fortis

-

purified by affinity chromoatography

Alteromonas fortis

-

681078

recombinant enzyme from Escherichia coli strain BL21(DE3) culture supernatant by affinity chromatography; recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography

recombinant His-tagged enzyme from Brevibacillus choshinensis strain HPD31-SP3 by dialysis, nickel affinity chromatography, and gel filtration

-

recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography

recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography

-

expressed in Bacillus subtilis

-

expressed in Escherichia coli BL21(DE3) cells

Alteromonas fortis

-

703075, 703077

expressed in Escherichia coli Origami (DE3) pLysS, BL21 (DE3), C41 (DE3), and C43 (DE3) cells

Alteromonas fortis

-

gene cgi82A, recombinant expression of His6-tagged enzyme in Escherichia coli

-

gene cgiA, sequence comparisons and phylogenetic analysis

gene cgiA2, sequence comparisons and phylogenetic analysis; gene cgiA, sequence comparisons and phylogenetic analysis

gene cgiB, sequence comparisons and phylogenetic analysis

-

gene cgiB_Ce, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression in Escherichia coli strain BL21(DE3); gene cgiB_Ce, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)

gene cgiF, sequence comparisons and phylogenetic analysis

gene cgiF, sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha

gene encoding iota-carrageenase flanked by a C-terminal hexahistidine tag and a N-terminal PelB signal peptide for targeting the gene product into Escherichia coli periplasm. The recombinant plasmid, referred to as pETIAf, is used to transform Escherichia coli BL21(DE3) strain harbouring pLysS plasmid

Alteromonas fortis

-

660570

overexpressed in Escherichia coli BL21(DE3)

Alteromonas fortis

-

681078

synthetic gene CGIOP, locus MF459059 in GenBank, codon-optimized gene encoding the iota-carrageenase without a signal peptide, cloned from gene cgiA of Microbulbifer thermotolerans strain JAMB-A94, construction of the integrative vector pBCGA, recombinant expression of extracellular His-tagged enzyme in Brevibacillus choshinensis strain HPD31-SP3, real-time PCR expression analysis, copy numbers of the CGIOP gene in different lines are approximately 5-6, respectively

-

analysis

Alteromonas fortis

-

usage of the enzyme for the analysis of the distribution of the carrabiose moieties in hybrid carrageenan chains, hybrid iota-/alpha-carrageenan, by Pseudoalteromonas atlantica a 4S-iota carrageenan sulfatase that converts iota-carrabiose into alapha-carrabiose units

732432

industry

CgiF is an excellent candidate for industrial applications in production of iota-carrageen oligosaccharides from seaweed polysaccharides

synthesis