Isolation and biochemical characterization of an endo-1,3-beta-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-beta-glucan.
A gene encoding 1,3-beta-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-beta-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-beta-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-beta linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble beta-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-beta-glucan, whereas the former had much stronger specific activity on insoluble 1,3-beta-glucans, suggesting that the C-terminal CBM enhances the activity of the S. sioyaensis 1,3-beta-glucanase against insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrate.
Hong
Tang-Yao
TY
Graduate Institute of Agricultural Biotechnology1, and Department of Plant Pathology2, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan40227.
Cheng
Chun-Wei
CW
Graduate Institute of Agricultural Biotechnology1, and Department of Plant Pathology2, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan40227.
Huang
Jenn-Wen
JW
Graduate Institute of Agricultural Biotechnology1, and Department of Plant Pathology2, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan40227.
Meng
Menghsiao
M
Graduate Institute of Agricultural Biotechnology1, and Department of Plant Pathology2, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan40227.
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005822:Genetic Vectors; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D005936:Glucans; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase