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Antonie Van Leeuwenhoek. 2013 Nov;104(5):737-48. doi: 10.1007/s10482-013-9982-5. Epub 2013 Jul 27.

Characterization of antifungal activity of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1.

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  • 1Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan.

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.

PMID:
23892828
DOI:
10.1007/s10482-013-9982-5
[PubMed - indexed for MEDLINE]
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