Inoue Akira, Mashino Chieco, Uji Toshiki, Saga Naotsune, Mikami Koji, Ojima Takao
Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho Hakodate, Hokkaido 041-8611, Japan.
Curr Biotechnol. 2015 Aug;4(3):240-248. doi: 10.2174/2211550104666150915210434.
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.
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.
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 35C 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.
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.
在所有的海藻酸裂合酶中，属于多糖裂合酶家族7（PL-7）的海藻酸裂合酶在结构和功能方面研究得最为深入。然而，所有已表征的PL-7海藻酸裂合酶均来自原核细菌细胞。在此，我们报道首次从海洋红藻条斑紫菜中鉴定出真核PL-7海藻酸裂合酶。
克隆编码海藻酸裂合酶PyAly的cDNA，并用于构建大肠杆菌中的重组PyAly（rPyAly）表达系统。对纯化的rPyAly进行检测以鉴定其酶学性质。还研究了其在条斑紫菜中的表达模式。
PyAly可能是一种分泌蛋白，由一个25个残基的N端信号肽和一个216个残基的催化结构域组成。催化结构域的氨基酸序列与已表征的归为PL-7家族的细菌海藻酸裂合酶的氨基酸序列具有19%-29%的同一性。通过冷诱导表达系统在大肠杆菌BL21(DE3)中产生的重组PyAly蛋白rPyAly在反应早期显著降低了海藻酸盐溶液的粘度。rPyAly最适宜的底物是海藻酸的聚甘露糖醛酸（poly(M)），最佳温度和pH分别为35℃和8.0。反应后，从聚甘露糖醛酸产生的不饱和三糖和四糖是主要终产物。这些酶学性质表明PyAly是一种属于PL-7的内切海藻酸裂合酶。此外，我们发现PyAly基因被分为4个外显子和3个内含子。PyAly也在配子体单倍体阶段特异性表达。
本研究表明，海洋红藻条斑紫菜中的PyAly是一种新型的以内切方式作用的PL-7海藻酸裂合酶。PyAly是一种配子体特异性表达的蛋白，其结构基因由四个外显子和三个内含子组成。因此，PyAly是首个经酶学表征的真核PL-7海藻酸裂合酶。
Zotero 插件
