RRC ID 47540
Author Hatada Y, Mizuno M, Li Z, Ohta Y.
Title 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.
Journal Mar Biotechnol (NY)
Abstract 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.
Volume 13(3)
Pages 411-22
Published 2011-6-1
DOI 10.1007/s10126-010-9312-0
PMID 20686828
MeSH Alteromonadaceae / chemistry Alteromonadaceae / enzymology* Amino Acid Sequence Bacterial Proteins / genetics* Base Sequence Biotechnology / methods* Carrageenan / biosynthesis* Catalysis Genes, Bacterial / genetics* Glycoside Hydrolases / genetics* Hot Temperature Models, Molecular* Molecular Sequence Data Mutagenesis, Site-Directed Sequence Analysis, DNA Sequence Homology Species Specificity
IF 2.798
Times Cited 26
General Microbes JCM 14709