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Functional heterologous expression and characterization of mannuronan C5-epimerase from the brown alga Saccharina japonica

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Title: Functional heterologous expression and characterization of mannuronan C5-epimerase from the brown alga Saccharina japonica
Authors: Inoue, Akira Browse this author →KAKEN DB
Satoh, Aya Browse this author
Morishita, Mio Browse this author
Tokunaga, Yuko Browse this author
Miyakawa, Takuya Browse this author
Tanokura, Masaru Browse this author
Ojima, Takao Browse this author →KAKEN DB
Keywords: Alginate
Mannuronan C5-epimerase
Brown algae
Alginate biosynthesis
Cell wall
Issue Date: Jun-2016
Publisher: Elsevier
Journal Title: Algal research
Volume: 16
Start Page: 282
End Page: 291
Publisher DOI: 10.1016/j.algal.2016.03.030
Abstract: Brown algae produce alginate that has various ratios and diverse sequences of two uronic acids, beta-D-mannuronic acid and alpha-L-guluronic acid, compared with those of alginate produced by bacteria. This diversity of alginate in brown algae is caused by mannuronan C5-epimerases (MC5Es), which catalyze the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid. Although several bacterial MC5E enzymes have been well characterized, to date, there exists no information on the biochemical properties of eukaryotic MC5E. In this study, MC5E expression was detected in a brown alga Saccharina japonica sporophyte by immunoblot analysis. We also searched for MC5E mRNA from S. japonica by RT-PCR and revealed eight partial amino acid sequences, SjC5-I to -VIII. We focused on the highest frequency clone, SjC5-VI, and elucidated its full-length cDNA and putative gene structure. The translated SjC5-VI protein consists of 499 amino acids, with the N-terminal 21 amino acids predicted as a secretion signal sequence. Functional recombinant SjC5-VI (rSjC5-VI) was successfully expressed as a secreted protein using an insect-cell expression system, and we determined the optimal temperature, pH, and NaCl concentrations to be 35 degrees C, 7.0-8.2, and 300 mM, respectively, using the Ca2+-induced gel-formation assay. In addition, Ca2+ enhanced gelation by 1.7-fold following rSjC5-VI activity. Furthermore, H-1-NMR spectroscopy of rSjC5-VI-treated polyM revealed alternate epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid. To the best of our knowledge, this is the first report on the characterization of MC5E activity in eukaryotes. (C) 2016 Elsevier B.V. All rights reserved.
Rights: © 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
Type: article (author version)
Appears in Collections:水産科学院・水産科学研究院 (Graduate School of Fisheries Sciences / Faculty of Fisheries Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 井上 晶

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