Cloning, expression, and characterization of a glycoside hydrolase family 50 b-agarase from a marine Agarivorans isolate

The gene for a thermostable b-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to b-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant b-agarase was purified to homogeneity. It had maximal activity at 40C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl sub(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.