Characterization of an exo-β-1,3-galactanase from Clostridium thermocellum
Characterization of an exo-β-1,3-galactanase from Clostridium thermocellum
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DOI：
10.1128/aem.72.5.3515-3523.2006
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发表时间：
2006-05-01
影响因子：
4.4
通讯作者：
Kaneko, Satoshi
中科院分区：
文献类型：
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作者：
Ichinose, Hitomi;Kuno, Atsushi;Kaneko, Satoshi
从热细胞梭状芽胞杆菌中分离到一个编码β -1,3-半乳糖酶的基因ct1,3 gal43a。该序列与黄孢Phanerochaete chrysosporium的外显-1,3-半乳糖酶（pc1,3gal43a）相似。该基因编码一个模块蛋白，由n端糖苷水解酶家族43 （GH43）模块、家族13碳水化合物结合模块（CBM.13）和c端dockerin结构域组成。在大肠杆菌中表达了GH43模块对应的基因，并对基因产物进行了表征。重组酶在pH 6.0和50℃条件下具有最佳的水解活性，只催化β -1,3-链半乳糖低聚糖和多糖的水解。水解产物的高效液相色谱分析表明，该酶以外显作用方式从β -1,3-半乳聚糖中产生半乳糖。当该酶作用于阿拉伯半乳糖蛋白（AGPs）时，该酶产生与半乳糖一起的低聚糖，这表明该酶能够容纳-1,6连接的半乳糖侧链。该酶的底物特异性与pc1,3gal43a非常相似，表明该酶是一种外显-1,3-半乳糖酶。CBM13的c端亲和凝胶电泳没有显示出对多糖的亲和性，包括β -1,3-半乳聚糖。然而，CBM13的正面亲和层析表明，CBM13特异性地与在非还原端含有β -1,3-半乳糖糖、β -1,4-半乳糖葡萄糖或β -1,4-半乳糖n -乙酰氨基葡萄糖片段的低聚糖相互作用。有趣的是，ct1,3gal43a C端的CBM13似乎干扰了β -1,3-半乳糖和α -(L)-阿拉伯糖醛酸苷酶处理的AGP的酶活性。
A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM.13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-(L)-arabinofuranosidase-treated AGP.