Protoplast Preparation from Laminaria japonica with Recombinant Alginate Lyase and Cellulase
Protoplast Preparation from Laminaria japonica with Recombinant Alginate Lyase and Cellulase
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DOI：
10.1007/s10126-010-9290-2
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发表时间：
2011-04-01
影响因子：
3
通讯作者：
Ojima, Takao
中科院分区：
文献类型：
--
作者：
Inoue, Akira;Mashino, Chieco;Ojima, Takao
利用杆状病毒表达系统表达功能性重组鲍鱼藻酸盐裂解酶(RHdAly)和β-1，4-内切葡聚糖酶(RHdEG66)作为分泌型蛋白。在30A时，rHdAly和rHdEG66的比活力分别为2490U/mg和18.2U/mg，与天然酶相当。纯化的rHdAly和rHdEG66在35A℃和最适pH分别为8.7和5.9时比活力最高。这些特性也可与天然酶相媲美。用rHdAly和rHdEG66从海带分离原生质体。在含有rHdAly和rHdEG66的人工海水中培养，可获得极低数量的原生质体(1×10(3)个/g鲜重)。而用蛋白酶K处理的叶片，原生质体最高可达5×10(6)个/g鲜重。由于分离出的原生质体平均直径为11.6微米，因此这些细胞大多来自表皮，而不是皮质层。我们的结果表明，至少有三种酶，海藻酸裂解酶，纤维素酶和蛋白酶，是有效地从日本乳杆菌中分离原生质体所必需的。本研究中的原生质体分离方法比以前的方法更有用，因为它优先产生已知能够再生的表皮层的原生质体。
Functional recombinant abalone alginate lyase (rHdAly) and beta-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30A degrees C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35A degrees C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (< 1 x 10(3) protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 x 10(6) protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 mu m, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.