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Isolation and biochemical characterization of an endo-1,3-ß-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-ß-glucan

Graduate Institute of Agricultural Biotechnology¹, and Department of Plant Pathology², National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan40227

Author for correspondence: Menghsiao Meng. Tel: +886 4 22840328. Fax: +886 4 22853527. e-mail: mhmeng{at}dragon.nchu.edu.tw

A gene encoding 1,3-ß-glucanase was isolated from Streptomyces sioyaensis based on an activity plate assay. Analysis of the deduced amino acid sequence of the gene revealed that the matured 1,3-ß-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-ß-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 Escherichia coli and purified to homogeneity. Biochemical data suggest that the glycosyl hydrolase domain preferentially catalyses the hydrolysis of glucans with 1,3-ß linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble ß-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-ß-glucan, whereas the former had much stronger specific activity on insoluble 1,3-ß-glucans, suggesting that the C-terminal CBM enhances the activity of the S. sioyaensis 1,3-ß-glucanase against insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrate.

Keywords: endo-1,3-ß-glucanase, ß-1,3-glucanase, laminarinase, carbohydrate-binding module

Abbreviations: CBM, carbohydrate-binding module; GHF, glucosyl hydrolase family; LC/MS, liquid chromatography/mass spectrometry

The GenBank accession number for the sequence reported in this paper is AF21741.

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