Two GH117 family alpha-neoagarobiose hydrolases (GH117A alpha-NABH and GH117B alpha-NABH) from the freshwater agardegrading Cellvibrio sp. KY-GH-1 were expressed and purified as recombinant His-tagged proteins using an Escherichia coli expression system to compare activities. The amino acid sequence of GH117A alpha-NABH (364 amino acids, 40.9 kDa) showed 35% identity with that of GH117B alpha-NABH (392 amino acids, 44.2 kDa). GH117A alpha-NABH, but not GH117B alpha-NABH, could hydrolyze neoagarobiose (NA2) into monosaccharides 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The presence of GH117A alpha-NABH homologues in all of the agar-degrading bacteria aligned suggests that GH117A alpha-NABH hydrolyzing NA2 into L-AHG and D-galactose is an essential component of the agar-degrading enzyme machinery. For GH117A alpha-NABH-catalyzed hydrolysis, NA2 was the sole substrate among various neoagaro-oligosaccharides (NA2-NA18). GH117A alpha-NABH appeared to exist as a dimer, and optimal enzymatic temperature and pH were 35 degrees C and 7.5, respectively. GH117A alpha-NABH was stable up to 35 degrees C and at pH 7.5 and unstable beyond 35 degrees C and outside pH 7.07.5. The kinetic parameters K-m,V-max, k(cat), and k(cat)/K-m for NA2 were 16.0 mM, 20.8 U/mg, 14.2 s(-1), and 8.9 x 10(2) M-1, respectively. Combined addition of 5 mM MnSO4 and 10 mM tris(2-carboxyethyl)phosphine enhanced the enzyme activity by 2.4-fold. The enzyme-mediated hydrolysis of 5.0% NA2 into monosaccharide and purification of L-AHG from hydrolysis products by Sephadex G-10 column chromatography recovered similar to 192 mg L-AHG from 400 mg NA2 (similar to 92% of the theoretical maximum yield). These results indicate that the recombinant GH117A alpha-NABH is NA2-specific and useful to produce L-AHG from NA2.