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Literature summary for 3.2.1.14 extracted from

  • Itoh, T.; Sugimoto, I.; Hibi, T.; Suzuki, F.; Matsuo, K.; Fujii, Y.; Taketo, A.; Kimoto, H.
    Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains (2014), Biosci. Biotechnol. Biochem., 78, 624-634.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene ChiW-SLHd, DNA and amino acid sequence determination and analysis, phylogenetic analysis, N-terminally His-tagged enzyme, the enzyme's N-terminus lacks the signal peptide and SLH domains , overexpression of active enzyme ChiW and mutants in Escherichia coli strain BL21 Paenibacillus sp.

Protein Variants

Protein Variants Comment Organism
E1177Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paenibacillus sp.
E691/E1177Q site-directed mutagenesis, inactive mutant Paenibacillus sp.
E691Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paenibacillus sp.

Inhibitors

Inhibitors Comment Organism Structure
2-mercaptoethanol 15% inhibition Paenibacillus sp.
4-nitrophenyl N,N'-diacetylchitobiose substrate inhibition at the first catalytic domain with E691 Paenibacillus sp.
4-O-N-acetyl-beta-D-glucosaminyl-N-acetyl-D-glucosamine 21% inhibition Paenibacillus sp.
Ca2+ 12% inhibition at 5 mM Paenibacillus sp.
Co2+ 9% inhibition at 5 mM Paenibacillus sp.
Cu2+ 28% inhibition at 5 mM Paenibacillus sp.
D-glucose 18% inhibition Paenibacillus sp.
DTT 21% inhibition Paenibacillus sp.
EDTA 17% inhibition Paenibacillus sp.
Fe3+ 59% inhibition at 5 mM Paenibacillus sp.
glutathione GSH, 6% inhibition Paenibacillus sp.
Mg2+ 20% inhibition at 5 mM Paenibacillus sp.
N-acetyl-D-glucosamine 24% inhibition Paenibacillus sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information modified Michaelis-Menten kinetic with substrate inhibition Paenibacillus sp.
0.14
-
4-nitrophenyl N,N'-diacetylchitobiose recombinant enzyme, pH 5.5, 37°C Paenibacillus sp.

Localization

Localization Comment Organism GeneOntology No. Textmining
cell surface
-
Paenibacillus sp. 9986
-

Metals/Ions

Metals/Ions Comment Organism Structure
additional information no effect by 5 mM Mn2+ Paenibacillus sp.

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
130000
-
recombinant truncated His-tagged enzyme, gel filtration Paenibacillus sp.
150000
-
1 * 150000, about, chitinase ChiW, sequence calculation, 1 * 130000, recombinant truncated His-tagged enzyme, specifically cleaved between Asn282 and Ser283 during the purification step, the resulting two polypeptide fragments were non-covalently bound, SDS-PAGE Paenibacillus sp.

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
chitin + H2O Paenibacillus sp.
-
?
-
?

Organism

Organism UniProt Comment Textmining
Paenibacillus sp. K7ZLW6 produces several different chitinases
-

Purification (Commentary)

Purification (Comment) Organism
recombinant soluble His-tagged enzyme ChiW, lacking the the signal peptide and SLH domains in the N-terminus, 1.6fold from Escherichia coli strain BL21 to homogeneity by nickel affinity and anion exchange chromatography Paenibacillus sp.

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.71
-
purified recombinant enzyme, pH 5.5, 37°C, substrate 4-nitrophenyl-N,N'-diacetyl-D-chitobiose Paenibacillus sp.

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl N,N'-diacetylchitobiose + H2O synthetic substrate Paenibacillus sp. 4-nitrophenol + N,N'-diacetylchitobiose
-
?
chitin + H2O
-
Paenibacillus sp. ?
-
?
chitin + H2O insoluble, powdered chitin and colloidal chitin, and chitin nanofiber Paenibacillus sp. N,N'-diacetylchitobiose + ? final product ?
ethylene glycol chitin + H2O
-
Paenibacillus sp. ?
-
?
additional information thin layer chromatography analysis of the reaction products, substrate specificity, overview. No activity with glycol chitosan Paenibacillus sp. ?
-
?

Subunits

Subunits Comment Organism
monomer 1 * 150000, about, chitinase ChiW, sequence calculation, 1 * 130000, recombinant truncated His-tagged enzyme, specifically cleaved between Asn282 and Ser283 during the purification step, the resulting two polypeptide fragments were non-covalently bound, SDS-PAGE Paenibacillus sp.
More the enzyme contains an anchoring domains in the N-terminus and two catalytic domains at the C-terminal region, presence of a beta-stranded-rich structure in the N-terminus Paenibacillus sp.

Synonyms

Synonyms Comment Organism
ChiW
-
Paenibacillus sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
Paenibacillus sp.

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
20 70 activity range, profile overview Paenibacillus sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
40
-
purified recombinant enzyme, stable up to Paenibacillus sp.
55
-
purified recombinant enzyme, 50% activity remaining Paenibacillus sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.99
-
4-nitrophenyl N,N'-diacetylchitobiose recombinant enzyme, pH 5.5, 37°C Paenibacillus sp.

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
assay at Paenibacillus sp.

pH Range

pH Minimum pH Maximum Comment Organism
4.5 8.5 activity range, profile overview Paenibacillus sp.

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.089
-
4-nitrophenyl N,N'-diacetylchitobiose recombinant enzyme, pH 5.5, 37°C Paenibacillus sp.

General Information

General Information Comment Organism
evolution the enzyme is a typical glycoside hydrolase family 18 chitinase Paenibacillus sp.
additional information Glu691 and Glu1177 in the two GH-18 domains are catalytic residues, two catalytic domains act synergistically to degrade chitin. The reaction mechanism of ChiW is suggested to be similar to the mechanism of GH-18 chitinases, in which one glutamate residue functions as a general acid/base catalyst and a GlcNAc C-2 acetamide group of the chitin substrate at subsite -1 carries out a nucleophilic attack on the GlcNAc C-1 carbon atom generating an oxazoline ion intermediate. Other important residues of the chitinase subfamily A enzymes for saccharide binding and catalytic reaction are also highly conserved in the ChiW GH-18 domains: Trp568 (Trp1055), Trp905 (Trp1396), Trp652 (Trp1138), and Trp772 (Trp1258) for saccharide binding, Tyr766 (Tyr1252), Asp687 (Asp1173), and Asp689 (Asp1175) for the catalytic reaction Paenibacillus sp.