Ichinose Hitomi, Yoshida Makoto, Fujimoto Zui, Kaneko Satoshi
Food Biotechnology Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
Appl Microbiol Biotechnol. 2008 Sep;80(3):399-408. doi: 10.1007/s00253-008-1551-x. Epub 2008 Jul 30.
A gene encoding an alpha-L-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L-arabinofuranoside but did not hydrolyze PNP-alpha-L-arabinopyranoside, PNP-beta-D-xylopyranoside, or PNP-beta-D-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside and methyl 3-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.
从阿维链霉菌中克隆出一个编码α-L-阿拉伯呋喃糖苷酶的基因，命名为SaAraf43A。推导的氨基酸序列显示其具有模块化结构，由一个N端糖苷水解酶家族43模块和一个C端家族42碳水化合物结合模块（CBM42）组成。重组酶在pH 6.0和45℃时表现出最佳活性，在30℃下pH 5.0 - 6.5范围内稳定。该酶能水解对硝基苯酚（PNP）-α-L-阿拉伯呋喃糖苷，但不能水解PNP-α-L-阿拉伯吡喃糖苷、PNP-β-D-木吡喃糖苷或PNP-β-D-半乳吡喃糖苷。去分支的1,5-阿拉伯聚糖能被该酶水解，但阿拉伯木聚糖、阿拉伯半乳聚糖、阿拉伯胶和阿拉伯聚糖则不能。在阿拉伯呋喃二糖苷的合成区域异构体中，只有甲基5-O-α-L-阿拉伯呋喃糖基-α-L-阿拉伯呋喃糖苷能被该酶水解，而甲基2-O-α-L-阿拉伯呋喃糖基-α-L-阿拉伯呋喃糖苷和甲基3-O-α-L-阿拉伯呋喃糖基-α-L-阿拉伯呋喃糖苷则不能。这些数据表明该酶仅切割α-1,5-连接的阿拉伯呋喃糖基键。对阿拉伯呋喃五糖水解产物的分析表明，该酶以外切方式释放阿拉伯糖。这些结果表明该酶肯定是一种外切-1,5-α-L-阿拉伯呋喃糖苷酶。C端的CBM42虽然能结合阿拉伯聚糖和阿拉伯木聚糖，但对阿拉伯半乳聚糖和去分支的阿拉伯聚糖没有任何亲和力，这表明CBM42与分支的阿拉伯呋喃糖基残基结合。去除该模块会降低酶对去分支阿拉伯聚糖的活性。尽管CBM42偏好结合阿拉伯呋喃糖基侧链，但它在增强催化模块对去分支阿拉伯聚糖的水解作用中发挥着作用。
