A unique β-agarase, AgaA, from a marine bacterium, Vibrio sp strain PO-303

The agaA gene encoding beta-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40 degrees C and pH 7.5, with a specific activity of 16.4 units mg(-1), a K-m of 1.10 mg ml(-1), and a V-max of 22.5 mu mol min(-1) mg(-1) for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.