Characterization of an Eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia yezoensis.
Characterization of an Eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia yezoensis.
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DOI：
10.2174/2211550104666150915210434
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发表时间：
2015-08
期刊：
影响因子：
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通讯作者：
Ojima T
中科院分区：
文献类型：
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作者：
Inoue A;Mashino C;Uji T;Saga N;Mikami K;Ojima T
背景资料：藻酸盐裂解酶属于多糖裂解酶家族7（PL-7），是目前结构和功能研究最深入的藻酸盐裂解酶。然而，所有表征的PL-7藻酸盐裂解酶都来自原核细菌细胞。本文首次从条斑Pyropia yezoensis中鉴定出真核生物的PL-7褐藻胶裂解酶。研究方法：克隆了褐藻酸裂解酶PyAly的cDNA，并将其用于构建重组PyAly（rPyAly）在大肠杆菌中的表达系统。测定纯化的rPyAly以鉴定其酶性质。并研究了其在虾夷扇贝中的表达模式。结果：PyAly可能是一种分泌蛋白，N端信号肽25个残基，催化结构域216个残基。催化结构域的氨基酸序列显示出19-29%的同一性的细菌特征的藻酸盐裂解酶分类到家庭PL-7。重组PyAly蛋白，rPyAly，经E. coliBL 21（DE 3）中表达，在反应初期，海藻酸钠溶液的粘度显著降低。rPyAly的最佳底物为海藻酸钠的poly（M），最适温度为35 ℃，最适pH为8.0。反应后，由聚（M）生成不饱和的三-和四-取代基作为主要的最终产物。这些酶性质表明PyAly是属于PL-7的内溶性藻酸盐裂解酶。此外，我们发现PyAly基因被分成4个外显子和3个内含子。PyAly在配子体单倍体阶段也特异表达。结论：条斑紫菜中的PyAly是一种新型的PL-7褐藻胶裂解酶，具有内溶方式。PyAly是配子体特异表达的蛋白质，其结构基因由4个外显子和3个内含子组成。因此，PyAly是第一个酶法表征的真核PL-7藻酸盐裂解酶。
Background: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. Methods: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. Results: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. Conclusion: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.