Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases
Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases
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DOI：
10.1007/s10811-007-9199-y
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发表时间：
2008-10-01
影响因子：
3.3
通讯作者：
Ojima, Takao
中科院分区：
文献类型：
--
作者：
Inoue, Akira;Kagaya, Mayu;Ojima, Takao
海带原生质体释放与高产量使用鲍鱼藻酸盐裂解酶HdAly与纤维素酶和螯合剂的组合。添加浓度高于10 mM的EDTA到已与HdAly和纤维素酶Onozuka预孵育的海带菌体中，显着提高了原生质体的产量。EDTA比EGTA更有效，这表明除了Ca2+之外，螯合二价金属离子如Mg2+和Sr2+是海带原生质体高产的关键因素。原生质体的平均直径为27 μ m，这表明大多数原生质体来自皮层细胞，而不是表皮层细胞。重组HdAly（rHdAly）在Sf9昆虫细胞表达系统中由cDNA克隆产生。rHdAly具有与天然HdAly基本相同的酶性质和原生质体产生能力。研究了利用天然和重组HdAlys高产海带原生质体的最佳条件。
Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg2+ and Sr2+ in addition to Ca2+ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 mu m, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.