Molecular characterization of a novel 1,3-α-3,6-anhydro-L-galactosidase, Ahg943, with cold- and high-salt-tolerance from Gayadomonas joobiniege G7.

1,3-alpha-3,6-anhydro-L-galactosidase (alpha-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the alpha-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other alpha-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other alpha-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6anhydro-L-galactose, indicating that it is an exo-acting alpha-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing alpha-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20 degrees C, respectively. In particular, rAhg943 could maintain enzyme activity at 10 degrees C (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca(2+)ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K-m and V-max of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 mu M/min (3 U/mg), respectively. Therefore, Ahg943 is a unique alpha-neoagarooligosaccharide hydrolase that has coldand high-salt-adapted features, and possibly exists as a monomer.