An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium
An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium
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DOI：
10.1074/jbc.m501024200
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发表时间：
2005-07-08
影响因子：
4.8
通讯作者：
Kaneko, S
中科院分区：
文献类型：
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作者：
Ichinose, H;Yoshida, M;Kaneko, S
来自金孢原毛平革菌 (Phanerochaete chrysosporium) 的外切 β-1,3- 半乳聚糖酶基因已在毕赤酵母中进行克隆、测序和表达。外切-β-1,3-半乳聚糖酶的完整氨基酸序列表明，该酶由与糖苷水解酶家族 43 相似的 N 端催化模块和 C 端区域中与碳水化合物结合模块家族 6 (CBM6) 相似的附加未知功能域组成。基于SDS-PAGE估计重组酶的分子量为55kDa。该酶仅对作为底物的β-1,3-连接半乳糖基寡糖和多糖表现出反应性，但不水解β-1,4-连接半乳寡糖、β-1,6-连接半乳寡糖、果胶半乳聚糖、落叶松阿拉伯半乳聚糖、阿拉伯聚糖、阿拉伯树胶、脱支阿拉伯聚糖、昆布多糖、可溶性桦木木聚糖，或可溶性燕麦拼写为木聚糖。该酶也不水解β-1,3-半乳糖基半乳糖胺、β-1,3-半乳糖基氨基葡萄糖或β-1,3-半乳糖基阿拉伯呋喃糖苷，这表明它特异性裂解两个半乳糖基残基的内部β-1,3-键。水解产物的高效液相色谱分析表明，该酶以外切方式从β-1,3-半乳聚糖产生半乳糖。然而，没有检测到对硝基苯基β-吡喃半乳糖苷的活性。当与阿拉伯半乳聚糖蛋白一起孵育时，该酶产生寡糖和半乳糖，这表明它能够绕过 β-1,6-连接的半乳糖基侧链。 C-末端CBM6对CBM6的已知底物如木聚糖、纤维素和β-1,3-葡聚糖没有表现出任何亲和力，尽管当通过亲和电泳分析时它结合了β-1,3-半乳聚糖。使用几种末端含半乳糖寡糖作为分析物对 CBM6 部分进行正面亲和层析，清楚地表明 CBM6 与含有 β-1,3-半乳二糖部分的寡糖特异性相互作用。当半乳糖寡聚物的聚合度增加时，CBM6的结合亲和力没有表现出明显的变化。
An exo-beta- 1,3- galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta- 1,3- galactanase indicated that the enzyme consists of an N- terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydratebinding module family 6 ( CBM6) in the C- terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS- PAGE. The enzyme showed reactivity only toward beta- 1,3- linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta- 1,4- linked galacto- oligosaccharides, beta- 1,6- linked galacto- oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta- 1,3- galactosyl galactosaminide, beta- 1,3- galactosyl glucosaminide, or beta- 1,3- galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta- 1,3- linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta- 1,3- galactan in an exo- acting manner. However, no activity toward p- nitrophenyl beta- galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta- 1,6- linked galactosyl side chains. The C- terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta- 1,3- glucan, although it bound beta- 1,3- galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta- 1,3- galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.