耐热耐碱木聚糖酶在大肠杆菌中的高效分泌表达及酶学性质研究

High Level Expression of Recombinant Thermo-alkaliphilic Xylanase in E. coli and Its Enzymatic Characterization

目的研究枯草芽孢杆菌来源的木聚糖酶基因在大肠杆菌BL2l中的高效分泌表达并对其产木聚糖酶进行酶学性质分析。方法全基因合成枯草芽孢杆菌木聚糖酶基因序列并进行密码子优化,经大肠杆菌BL2l表达后获得基因工程菌株,通过SDS-PAGE电泳检测、硫酸铵分级沉淀和AKTA系统分离纯化,分析表达产物的酶学性质。结果重组木聚糖酶为胞外分泌性表达,相对分子质量约43×103;酶促反应最适温度为65℃,最适p H为10.0,最适条件下酶活最高可达1201.5 IU/ml。结论成功获得高效分泌表达重组木聚糖酶的基因工程菌株,该木聚糖酶具较好的耐热性和耐碱性。

Objective To construct an engineering strain in E. coli BL21 with high production of thermo-alkaliphilic xylanase and analyze the properties of the secreted xylanase. Methods The optimized sequence from Bacillus subtilis encoding thermo-alkaliphilic xylanase（xyn） was synthesized and introduced into Escherichia coli. The recombinant protein was identified by SDS-PAGE and purified by ammonium sulfate precipitation and AKTA system, then the enzymatic properties of the recombinant xylanase were analyzed. Results The recombinant xylanase was successfully expressed and secreted into the medium, and SDS-PAGE analysis revealed that the relative molecular mass of the protein is determined about 43×103. The optimum temperature and p H for enzymatic reaction were 65 ℃ and 10.0, respectively. The maximum enzymatic activity under optimal condition reached 1201.5 IU/ml. Conclusion The genetic engineering strain gene with high level of secretory expression of thermo-alkaliphilic xylanase is obtained.