The alpha-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 alpha-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized alpha-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 alpha-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for alpha-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45A degrees C; exhibited strong resistance to digestion by trypsin, alpha-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-alpha-d-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).