Chitosanase, a Novel Enzyme

Monaghan, Richard L.; Eveleigh, D. E.; Tewari, Ram P.; Reese, Elwyn T.

doi:10.1038/newbio245078a0

Cited by 60 publications

(32 citation statements)

References 16 publications

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“…While this work was in progress, the three-dimensional structure of the Streptomyces sp. N174 chitosanase was solved, 3 following an earlier report on its crystallization (30). In the current structural model, Glu-22 and Asp-40 are proposed as catalytic residues corroborating the conclusions drawn from the present work.…”

Section: Resultssupporting

confidence: 73%

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Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Boucher

Fukamizo

Honda

et al. 1995

Journal of Biological Chemistry

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The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and k cat , while K m was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity.

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Section: Resultssupporting

confidence: 73%

“…Chitosanases were described in prokaryotes, fungi (3), and more recently in plants (4). The distinction between chitinases and chitosanases became elucidated with the works of several groups that analyzed the structure of the hydrolysis products generated from chitosan by various enzymes of bacterial origin.…”

mentioning

confidence: 99%

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Boucher

Fukamizo

Honda

et al. 1995

Journal of Biological Chemistry

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“…G-I that we isolated also mainly released GlcNAc 2 from the non-reducing end of N-acetylchitooligosaccharides and p-nitophenyl-chitooligosaccharides. The enzyme reactions that continued for many hours produced mainly GlcNAc2, but there was some GlcNAc 3 and GlcNAc. Colloidal chitin was hydrolyzed to the same products as N-acetyl-chitooligosacchairdes, and other N-acetyl-chitooligosacchaides were not detected.…”

Section: Discussionmentioning

confidence: 99%

Purification and Mode of Action of Chitosanolytic Enzyme fromEnterobactersp. G-1

Yamasaki

Hayashi

Ohta

et al. 1993

Bioscience, Biotechnology, and Biochemistry

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“…EC 3.2.1.14) and lysozymes (e.g. EC 3.2.1.17) (Monaghan et al, 1973). Up to now, chitosanases have been characterized mostly from bacterial (Pelletier & Syguch, 1990, and references therein) and fungal sources (Reyes et al, 1985).…”

Section: Discussionmentioning

confidence: 99%

Colloidal gold-complexed chitosanase: a new probe for ultrastructural localization of chitosan in fungi

Grenier

Benhamou

Asselin

1991

Journal of General Microbiology

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A chitosanase was purified from the intercellular fluid extract of chemically stressed barley (Hordeum vulgare) leaves. Purification was achieved by preparative PAGE involving separation under native conditions at pH 4.3 (Reisfeld system) followed by denaturing PAGE in the presence of SDS. One basic chitosanase with a molecular mass of 19 kDa could hydrolyse chitosan and glycol chitosan of crustacean origin in addition to fungal chitosan isolated after treatment of Mucor rnucedo cells with sodium hydroxide followed by extraction in acetic acid. This enzyme did not exhibit activity towards P-1,4-polymers such as chitin, cellulose or peptidoglycan (from Micrococcus luteus) after SDS-PAGE. The chitosanase was conjugated to colloidal gold at pH 9.5 and applied to fungal tissue sections or to isolated glycol chitosan, chitosan, cellulose or chitin. The chitosanase-gold complex reacted intensely with glycol chitosan and chitosan but did not bind to chitin or cellulose. The enzyme-gold complex also labelled chitosan isolated from M. rnucedo. However, it did not react with M. rnucedo cells in tissue sections. Labelling with the chitosanase-gold complex was easily detected over cell walls of Fusariurn oxysporurn f. sp. radicis-lycopersici spores and hyphae as well as over cell walls of Ophiostoma ulrni, Aspergillus niger and Colletotrichurn lindernuthianum. No binding was observed with Pythiurn ultirnum.

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Chitosanase, a Novel Enzyme

Cited by 60 publications

References 16 publications

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Purification and Mode of Action of Chitosanolytic Enzyme fromEnterobactersp. G-1

Colloidal gold-complexed chitosanase: a new probe for ultrastructural localization of chitosan in fungi

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