GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall

• Published in: Journal of Applied Glycoscience 2022/08/22, Vol.69(3), pp.49-56
• Main Authors: Otsuka, Yuitsu, Sato, Koki, Yano, Shigekazu, Kanno, Haruki, Suyotha, Wasana, Konno, Hiroyuki, Makabe, Koki, Taira, Toki
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Antifungal activity; Binding; Carbohydrates; Cell surface; Chitinase; Domains; Fluorescence; fungal cell-wall; Fungi; Fungicides; Fusion protein; Gene expression; Glucan; Glucans; glycoside hydrolase family 16; Green fluorescent protein; Lysobacter; Protein B; Proteins; Regular Paper; Selective binding; Selectivity; Substrates; Surface structure; β-1,3-glucanase; β-Glucan
• ISSN: 1344-7882; 1880-7291
• DOI: 10.5458/jag.jag.JAG-2022_0002

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.