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OALib Journal期刊
ISSN: 2333-9721
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基于宏基因组学壳聚糖酶挖掘研究
DOI: 10.3724/SP.J.1145.2014.01039, PP. 597-601
Keywords: 宏基因组文库,fosmid,壳聚糖酶,壳寡糖,大肠杆菌
Abstract:
壳聚糖酶（ec3.2.1.132）可有效地将壳聚糖降解为活性良好和应用前景广泛的低分子壳聚糖或壳寡糖，但目前存在专一性强的壳聚糖酶发酵水平低、种类单一、应用性能较差等问题.本研究从虾蟹堆积场淤泥采样，采用宏基因组文库方法，从其fosmid文库中筛选出壳聚糖酶基因chie.测序表明，chie由1362bp组成，编码453个氨基酸，预测分子量（mr）为42×103左右.将chie与pet-28a(+)载体连接，转化大肠杆菌escherichiacolirosetta-gami(de3)构建了壳聚糖酶重组菌.采用离子交换柱（deaesepharoseff）和凝胶色谱柱（superdextm75）纯化目的蛋白.酶学性质结果显示，该酶最适作用温度70℃，最适ph6.0，li+、sr2+、k+等离子对其具有一定激活作用，fe3+、pb2+、fe2+等离子则具有不同程度的抑制.在最适作用条件下，该酶比酶活为2158u/mg.本研究表明，重组菌e.colirosetta-gami(de3)/chie具有培养简便、酶发酵产量大等优点，结果可为利用基因工程菌生产壳聚糖酶奠定基础.图5表2参17
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