Isolation and biochemical characterization of an endo-1,3-β-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-β-glucan
Isolation and biochemical characterization of an endo-1,3-β-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-β-glucan
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DOI：
10.1099/00221287-148-4-1151
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发表时间：
2002-04-01
期刊：
影响因子：
2.8
通讯作者：
Meng, MS
中科院分区：
文献类型：
--
作者：
Hong, TY;Cheng, CW;Meng, MS
利用活性平板法从Sioyaensis链霉菌中分离到一个编码1，3-β-葡聚糖酶的基因。对该基因推导的氨基酸序列的分析表明，成熟的1，3-β-葡聚糖酶具有由9个甘氨酸残基的延伸分开的两个功能结构域。N-末端结构域与分类在糖基水解酶家族16（GHF 16）中的细菌内切-1，3-β-葡聚糖酶共享序列相似性，而C-末端结构域是分类到CBM家族6中的推定的碳水化合物结合模块（CBM）。为了表征每个结构域的功能，在大肠杆菌中表达蛋白质的全长和CBM截短版本并纯化至均一。生物化学数据表明，糖基水解酶结构域优先催化具有1，3-β键的葡聚糖的水解，并具有内溶作用模式。结合试验表明，C-末端CBM结合各种不溶性β-葡聚糖（1，3-、1，3-、1，4-和1，4-键），但不结合木聚糖，木聚糖是CBM家族6大多数成员的主要结合靶标。全长和CBM截短的蛋白对可溶性1，3-β-葡聚糖具有相似的比活性（单位/摩尔水解酶结构域），而前者对不溶性1，3-β-葡聚糖具有更强的比活性，这表明C-末端CBM增强了S. Sioyaensis 1，3-β-葡聚糖酶对不溶性底物的作用，推测是通过增加水解酶结构域和底物之间的相遇事件的频率。
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.