Hyper-production and Characterization of the ι-Carrageenase Useful for ι-Carrageenan Oligosaccharide Production from a Deep-sea Bacterium, Microbulbifer thermotolerans JAMB-A94T, and Insight into the Unusual Catalytic Mechanism

A gene of unknown function from the genome of the agar-degrading deep-sea bacterium Microbulbifer thermotolerans JAMB-A94(T) was functionally identified as a iota-carrageenase gene. This gene, designated as cgiA, is located together with two beta-agarase genes, agaA and agaO in a cluster. The cgiA gene product is 569 amino acids and shares 29% identity over 185 amino acids with the iota-carrageenase from Zobellia galactanivorans Dsij DSM 12802. Recombinant, cgiA-encoded iota-carrageenase (55 kDa) was hyper-produced in Bacillus subtilis. The recombinant enzyme shows maximal activity at 50A degrees C, the highest reported optimal temperature for a carrageenase. It cleaved beta-1,4 linkages in iota-carrageenan to produce a high ratio of iota-carrageenan tetramer, more than 75% of the total product, and did not cleave the beta-1,4 linkages in kappa- or lambda-carrageenan. Therefore, this enzyme may be useful for industrial production of iota-carrageenan oligosaccharides, which have demonstrated antiviral potential against diverse viruses. Furthermore, we performed site-directed mutagenesis on the gene to identify the catalytic amino acid residues. We demonstrated that a conserved Glu351 was essential for catalysis; however, this enzyme lacked a catalytic Asp residue, which is generally critical for the catalytic activity of most glycoside hydrolases.