Abstract
Alginates are heteropolysaccharides comprising β-D-mannuronate (M) and α-L-guluronate (G) units. Natural sources of alginate include brown algae and certain bacteria. Brown algal alginates possess a greater degree of sequence diversity than do those produced by bacteria, and this may be due to the presence of multiple mannuronan C5-epimerase (MC5E) isozymes in brown algae, although only three have been enzymatically studied to date. In this study, a novel MC5E candidate protein, CoC5-1, from the brown alga Cladosiphon okamuranus, for which no enzymatic function has been previously determined, was expressed as a glycosylated protein in insect cells. The action of recombinant CoC5-1 on polymannuronate (polyM) increased the amount of gel formed in the presence of Ca2+, indicating that CoC5-1 possesses MC5E activity. The optimum temperature, pH, and NaCl concentration were 40 °C, pH 8.0, and 100 mM, respectively. The thermal stability of CoC5-1 was higher than that of the Saccharina japonica MC5E SjC5-VI. CoC5-1 remained active after incubation at 50 °C for 1 h, whereas SjC5-VI was completely inactivated. 1H-nuclear magnetic resonance (NMR) analysis revealed that the M:G ratio of the polyM substrate was altered from 9.0 to 2.1 by CoC5-1. This is the first report of MC5E enzymatic activity in brown algae of the family Chordariaceae.
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Acknowledgements
This study was supported by the Japan Society for the Promotion of Science KAKENHI program (grant numbers 16H04977 and 19H03039), the Center of Innovation Program of the Japan Science and Technology Agency (grant number JPMJCE1301), the Center of Innovation NEXT Program of the Japan Science and Technology Agency (grant number JPMJPF2108), and the Hokkaido University NMR Facility as a program of the “NMR Platform” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
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Sakagami, M., Ohnishi, Y., Kumaki, Y. et al. Enzymatic characterization of a mannuronan C5-epimerase from the subtropical brown alga Cladosiphon okamuranus. Fish Sci 89, 823–835 (2023). https://doi.org/10.1007/s12562-023-01720-7
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DOI: https://doi.org/10.1007/s12562-023-01720-7