Cloning, Expression, and Characterization of a Glycoside Hydrolase Family 118 β-Agarase from Agarivorans sp. JA-1

Abstract

We report a glycoside hydrolase (GH)-118 beta-agarase from a strain of Agarivorans, in which we previously reported recombinant expression and characterization of the GH- 50 beta-agarase. The GH comprised an open reading frame of 1,437 base pairs, which encoded a protein of 52,580 daltons consisting of 478 amino acid residues. Assessment of the entire sequence showed that the enzyme had 97% nucleotide and 99% amino acid sequence similarities to those of GH-118 beta-agarase from Pseudoalteromonas sp. CY24, which belongs to a different order within the same class. The gene corresponding to a mature protein of 440 amino acids was inserted, recombinantly expressed in Escherichia coli, and purified to homogeneity with affinity chromatography. It had maximal activity at 35 degrees C and pH 7.0 and had 208.1 units/mg in the presence of 300 mM NaCl and 1 mM CaCl2. More than 80% activity was maintained after 2 h exposure to 35 degrees C; however, < 40% activity remained at 45 degrees C. The enzyme hydrolyzed agarose to yield neoagarooctaose as the main product. This enzyme could be useful for industrial production of functional neoagarooligosaccharides.