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

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.

Automated summary

The GH117A alpha-neoagarobiose hydrolase from Cellvibrio sp. KY-GH-1 was found to specifically hydrolyze NA2 into L-AHG and D-galactose, making it a key component in agar-degrading enzyme machinery. Its optimal enzymatic temperature and pH were determined, and the enzyme exhibited stability under certain conditions. The addition of specific compounds further enhanced the enzyme activity, allowing for efficient production of L-AHG from NA2.