Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido, Japan.
Sci Rep. 2019 Mar 20;9(1):4937. doi: 10.1038/s41598-019-41351-6.
Despite the progress in massive gene analysis of brown algal species, no alginate-degrading enzyme from brown alga has been identified, impeding the understanding of alginate metabolism in brown alga. In the current study, we identified and characterized alginate lyase from Saccharina japonica using a protein-based approach. First, cDNA library was prepared from the S. japonica sporophyte. Expression screening was then performed; the encoding gene was identified and cloned; and the recombinant enzyme was purified and characterized. Alginate lyase production in algal tissues was evaluated by western blotting. The identified alginate lyase, SjAly (359 amino acids, with a predicted N-terminal secretion signal of 27 residues), is encoded by an open reading frame comprising seven exons. Recombinant SjAly exhibited endolytic alginate lyase activity, specifically toward stretches of consecutive β-D-mannuronic acid units. The optimum temperature, pH, and NaCl concentration were 30 °C, pH 8.0, and 100 mM, respectively. SjAly exhibited pronounced activity below 20 °C, the S. japonica growth temperature. SjAly was highly expressed in the blade but not the stipe and rhizoid. The data indicate that S. japonica possesses at least one active alginate lyase. This is the first report of a functional alginate lyase from brown alga, the major natural alginate producer.
尽管在大型褐藻物种的基因分析方面取得了进展，但仍未鉴定出褐藻来源的海藻酸盐降解酶，这阻碍了对褐藻中海藻酸盐代谢的理解。在本研究中，我们采用基于蛋白质的方法从裙带菜中鉴定和表征了海藻酸钠裂解酶。首先，从裙带菜孢子体中制备 cDNA 文库。然后进行表达筛选；鉴定并克隆编码基因；并纯化和表征重组酶。通过 Western blot 评估海藻组织中海藻酸钠裂解酶的产生。鉴定出的海藻酸钠裂解酶 SjAly（359 个氨基酸，预测 N 端分泌信号为 27 个残基）由一个包含七个外显子的开放阅读框编码。重组 SjAly 表现出内切型海藻酸钠裂解酶活性，特别是对连续的β-D-甘露糖单元链的作用。最适温度、pH 值和 NaCl 浓度分别为 30°C、pH8.0 和 100mM。SjAly 在 20°C 以下表现出明显的活性，这是裙带菜的生长温度。SjAly 在叶片中高度表达，但在茎和根中不表达。数据表明，裙带菜至少拥有一种活性海藻酸钠裂解酶。这是首次从褐藻这一主要的天然海藻酸盐产生者中报道功能性的海藻酸钠裂解酶。
