A Novel Glycosyl Hydrolase Family 16 β-Agarase from the Agar-Utilizing Marine Bacterium JEA5: the First Molecular and Biochemical Characterization of Agarase in Genus .

The agarase gene was identified from a draft genome sequence of JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, , , and , in the genus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in . Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) β-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in . rGaa16A had maximum activity at 55°C and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM CaCl₂. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.