1887

Abstract

A gene encoding 1,3-β-glucanase was isolated from based on an activity plate assay. Analysis of the deduced amino acid sequence of the gene revealed that the matured 1,3-β-glucanase has two functional domains separated by a stretch of nine glycine residues. The N-terminal domain shares sequence similarity with bacterial endo-1,3-β-glucanases classified in glycosyl hydrolase family 16 (GHF 16), while the C-terminal domain is a putative carbohydrate-binding module (CBM) grouped into CBM family 6. To characterize the function of each domain, both the full-length and the CBM-truncated versions of the protein were expressed in and purified to homogeneity. Biochemical data suggest that the glycosyl hydrolase domain preferentially catalyses the hydrolysis of glucans with 1,3-β linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble β-glucans (1,3-, 1,3–1,4- and 1,4- linkages) but not to xylan, a primary binding target for most members of CBM family 6. The full-length and the CBM-truncated proteins had similar specific activity (units per mol of hydrolase domain) on soluble 1,3-β-glucan, whereas the former had much stronger specific activity on insoluble 1,3-β-glucans, suggesting that the C-terminal CBM enhances the activity of the 1,3-β-glucanase against insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrate.

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2002-04-01
2020-01-19
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