嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

Expression of a pullulanase gene from the thermophilic Bacillus subtilis and characterization of the recombinant enzyme

摘要:

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达, 对重组酶进行纯化和酶学性质研究, 根据枯草芽孢杆菌的普鲁兰酶蛋白序列, 设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达。对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究, 并测定重组普鲁兰酶的底物特异性。将普鲁兰酶基因pluA克隆及分析序列后, 发现基因长度为2.2 kb, 编码718个氨基酸, 在大肠杆菌中异源表达。通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶, SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD。酶学性质研究表明, 该酶的最适温度为40 °C, 在温度不高于45 °C条件下稳定; 最适pH为6.0, 同一温度下pH 6.0?9.0范围内处理30 min可以保持80%以上的酶活力, 此酶对普鲁兰糖有很强的底物特异性。此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值。

Abstract:

The pullulanase gene of thermophilic Bacillus subtilis WY-34 was cloned in E. coli to characterize the recombinant pullulanase. The pluA gene encoding pullulanase from the thermophilic B. subtilis WY-34 was overexpressed in E. coli. The properties of recombinant pullulanase were studied. Also substrate specificity of the recombinant pullulanase was evaluated in this paper. The pluA gene was 2.2 kb in length and encoded 718 amino acid protein. The recombinant pullulanase was purified to homogeneity using Ni-IDA agarose chromatography, resulting in a specific activity of 93.2 U/mg. SDS-PAGE and gel permeation chromatography analysis showed that the molecular weight of the protein is approximately 76.2 kD and 74.3 kD respectively. The purified enzyme was optimally active at 40 °C and pH 6.0, stable at 45 °C and retained more than 80% relative activity with in pH 6.0?9.0. It exhibited high substrate specificity toward pullulan. The properties of recombinant pullulanase may be useful for application of pullulanase in industry.