title: Characterization of antifungal activity of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1.
abstract: We characterized the antifungal activity of the Bacillus circulans subclass III MH-K1 chitosanase (MH-K1 chitosanase), which is one of the most intensively studied glycoside hydrolases (GHs) that belong to GH family 46. MH-K1 chitosanase inhibited the growth of zygomycetes fungi, Rhizopus and Mucor, even at 10 pmol (0.3 μg)/ml culture probably via its fungistatic effect. The amino acid substitution E37Q abolished the antifungal activity of MH-K1 chitosanase, but retained binding to chitotriose. The E37Q mutant was fused with green fluorescent protein (GFP) at its N-terminus and proved to act as a chitosan probe in combination with wheat-germ agglutinin (WGA), which is a chitin-specific binding lectin. The GFP-fused MH-K1 chitosanase mutant E37Q (GFP-E37Q) bound clearly to the hyphae of the Rhizopus and Mucor strains, indicating the presence of chitosan. In contrast, Cy5-labelled WGA (Cy5-WGA), but not GFP-E37Q, stained the hyphae of non-zygomycetes species, i.e. Fusarium oxysporum, Penicillium expansum, and Aspergillus awamori. When the mycelia of Rhizopus oryzae were treated with wild type MH-K1 chitosanase, they could not bind to GFP-E37Q but were stained instead by Cy5-WGA. We conclude that chitin is covered by chitosan in the cell walls of R. oryzae.
authors.affiliation: Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan.
authors.forename: Masayo
authors.initials: M
authors.lastname: Tomita
authors.forename: Ayami
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Mariko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Ifuku
authors.forename: Shiro
authors.initials: S
authors.lastname: Yamashoji
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
authors.forename: Akihiro
authors.initials: A
authors.lastname: Saito
mesh_terms: D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001230:Aspergillus / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D048271:Chitosan / Q000032:analysis; D004252:DNA Mutational Analysis; D005670:Fusarium / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009089:Mucor / Q000187:drug effects* / Q000254:growth & development; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010407:Penicillium / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012233:Rhizopus / Q000187:drug effects* / Q000254:growth & development; D013194:Staining and Labeling / Q000379:methods; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D001426:Bacterial Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014312:Trisaccharides; D049452:Green Fluorescent Proteins; C041161:chitotriose; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase