caa a22 4500 445318333 CHVBK 20180317142655.0 cr unu---uuuuu 170323e20110601xx s 000 0 eng 10.1007/s10126-010-9312-0 doi (NATIONALLICENCE)springer-10.1007/s10126-010-9312-0 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 [Elektronische Daten] [Yuji Hatada, Masahiro Mizuno, Zhijun Li, Yukari Ohta] A gene of unknown function from the genome of the agar-degrading deep-sea bacterium Microbulbifer thermotolerans JAMB-A94T 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 (55kDa) 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. Springer Science+Business Media, LLC, 2010 Deep-sea nationallicence ι-Carrageenan 4-β-D-glycanohydrolase nationallicence ι-Carrageenase nationallicence Microbulbifer thermotolerans nationallicence Sequence nationallicence Hatada Yuji Marine Biodiversity Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, 237-0061, Yokosuka, Japan aut Mizuno Masahiro Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University, 4-17-1 Wakasato, 380-8553, Nagano, Japan aut Li Zhijun Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599, Chapel Hill, NC, USA aut Ohta Yukari Marine Biodiversity Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, 237-0061, Yokosuka, Japan aut Marine Biotechnology Springer-Verlag 13/3(2011-06-01), 411-422 1436-2228 13:3<411 2011 13 10126 https://doi.org/10.1007/s10126-010-9312-0 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10126-010-9312-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hatada Yuji Marine Biodiversity Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, 237-0061, Yokosuka, Japan aut NATIONALLICENCE 700 1- Mizuno Masahiro Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University, 4-17-1 Wakasato, 380-8553, Nagano, Japan aut NATIONALLICENCE 700 1- Li Zhijun Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599, Chapel Hill, NC, USA aut NATIONALLICENCE 700 1- Ohta Yukari Marine Biodiversity Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, 237-0061, Yokosuka, Japan aut NATIONALLICENCE 773 0- Marine Biotechnology Springer-Verlag 13/3(2011-06-01), 411-422 1436-2228 13:3<411 2011 13 10126 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer