Key Laboratory for Feed Biotechnology of Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Dec;88(6):1297-309. doi: 10.1007/s00253-010-2809-7. Epub 2010 Aug 17.
The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-D-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).
α-半乳糖苷酶基因 galA17 是从共生菌 Flavobacterium sp. TN17 中克隆得到的，该共生菌分离自枯叶蛱蝶幼虫的肠道。该基因全长 2205bp，编码一个包含 734 个残基的多肽（GalA17），含有一个假定的 28 个残基的信号肽和一个属于糖苷水解酶家族 36（GH 36）的催化结构域。GalA17 的推导氨基酸序列与 Pedobacter sp. BAL39 的一种假定的 α-半乳糖苷酶（EDM38577；66.6%同源性）和 Carnobacterium piscicola BA 中已鉴定的 α-半乳糖苷酶（AAL27305；30.1%同源性）最为相似。系统发育分析表明，GalA17 与具有两个假定催化基序 KWD 和 SDXXDXXXR 的共生细菌的 GH 36 α-半乳糖苷酶相似，基于定点突变，D480、S548、D549 和 R556 对 α-半乳糖苷酶活性是必需的。纯化的重组 GalA17 在 pH5.5 和 45°C 下表现出明显的最佳活性；对胰蛋白酶、α-糜蛋白酶、胶原酶和蛋白水解酶 K 的消化具有很强的抗性；并能有效地水解几种合成和天然底物（对硝基苯-α-D-半乳糖吡喃糖苷、棉子糖、密二糖、水苏糖、豆粕、瓜尔豆胶和刺槐豆胶）。
