Enzymatic characterization of a novel recombinant 1,3-α-3,6-anhydro-L-galactosidase specific for neoagarobiose hydrolysis into monosaccharides.

Abstract

Two GH117 family α-neoagarobiose hydrolases (GH117A α-NABH and GH117B α-NABH) from the freshwater agar-degrading 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 α-NABH (364 amino acids, 40.9 kDa) showed 35% identity with that of GH117B α-NABH (392 amino acids, 44.2 kDa). GH117A α-NABH, but not GH117B α-NABH, could hydrolyze neoagarobiose (NA2) into monosaccharides 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The presence of GH117A α-NABH homologues in all of the agar-degrading bacteria aligned suggests that GH117A α-NABH hydrolyzing NA2 into L-AHG and D-galactose is an essential component of the agar-degrading enzyme machinery. For GH117A α-NABH-catalyzed hydrolysis, NA2 was the sole substrate among various neoagaro-oligosaccharides (NA2~NA18). GH117A α-NABH appeared to exist as a dimer, and optimal enzymatic temperature and pH were 35 °C and 7.5, respectively. GH117A α-NABH was stable up to 35 °C and at pH 7.5 and unstable beyond 35 °C and outside pH 7.0~7.5. The kinetic parameters Km, Vmax, kcat, and kcat/Km for NA2 were 16.0 mM, 20.8 U/mg, 14.2 s-1, and 8.9 × 102 s-1 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 ~ 192 mg L-AHG from 400 mg NA2 (~ 92% of the theoretical maximum yield). These results indicate that the recombinant GH117A α-NABH is NA2-specific and useful to produce L-AHG from NA2. KEY POINTS: • Recombinant GH117A α-NABH (364 aa, 40.9 kDa) purified from E. coli forms a dimer. • The enzyme hydrolyzes only NA2 among various neoagaro-oligosaccharides (NA2~NA18). • The enzyme completely hydrolyzes up to 5% NA2 into monomers under optimal conditions.