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 ι-carrageenase gene. This gene, designated as cgiA, is located together with two β-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 ι-carrageenase from Zobellia galactanivorans Dsij DSM 12802. Recombinant, cgiA-encoded ι-carrageenase (55 kDa) was hyper-produced in Bacillus subtilis. The recombinant enzyme shows maximal activity at 50°C, the highest reported optimal temperature for a carrageenase. It cleaved β-1,4 linkages in ι-carrageenan to produce a high ratio of ι-carrageenan tetramer, more than 75% of the total product, and did not cleave the β-1,4 linkages in κ- or λ-carrageenan. Therefore, this enzyme may be useful for industrial production of ι-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.
Hatada
Yuji
Y
Marine Biodiversity Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, 237-0061, Japan.
Mizuno
Masahiro
M
Li
Zhijun
Z
Ohta
Yukari
Y
D034241:Alteromonadaceae / Q000737:chemistry / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002351:Carrageenan / Q000096:biosynthesis*; D002384:Catalysis; D005798:Genes, Bacterial / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics*; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013045:Species Specificity
D001426:Bacterial Proteins; D002351:Carrageenan; D006026:Glycoside Hydrolases; C019624:agarase; C019846:kappa-carrageenase protein, Alteromonadaceae