耐热木聚糖酶基因xyn11^(EM)在大肠杆菌中的表达

Expression of the Thermotolerant Xylanase Gene xyn11^(EM) in Escherichia coli

将人工合成经密码子优化的11家族耐热木聚糖酶基因xyn11^(EM)克隆至表达质粒p ET-28a(+)中,获得重组质粒p ET-28a-xyn11^(EM)。将其转化Escherichia coli BL21(DE3),构建表达耐热木聚糖酶的重组工程菌E.coli BL21/xyn11^(EM)。用IPTG诱导表达重组木聚糖酶Xyn11^(EM)(re Xyn11^(EM)),酶活性可达47.5 U/m L。SDS-PAGE分析显示,re Xyn11^(EM)的表观相对分子质量为24 800。re Xyn11^(EM)的最适反应温度为70℃,在70℃以下稳定。最适反应p H为6.5~7.0,在p H5.0~8.0范围内稳定。大多数金属离子和EDTA对该重组酶的活性影响不大。re Xyn11^(EM)的Km和Vmax值分别为7.2 mg/m L和54.7 U/mg。结果表明xyn11^(EM)成功在E.coli中实现了异源表达,其良好的热稳定性具有较好的工业应用潜力。

A codon-optimized gene （named xyn11^EM）, which encodes a thermotolerant xylanase belonging to the glycoside hydrolase family 11, was synthesized, and cloned into the expression plasmid pET-28a （＋）. The recombinant Escherichia coli （designated E. coli BL21/xyn11^EM）, expressing the thermostable xylanase,was constructed by transforming the recombinant plasmid, pET-28a-xyn11^EM,into E. coli BL21 （DE3）. The recombinant E. coli BL21/xyn11^EM was induced with IPTG to express reXyn11^EM. The reXyn11^EM activity reached 47.5 U/mL. The apparent molecular weight of reXyn11^EM was estimated to be 24 800 by SDS-PAGE analysis. Its optimal temperature and pH were 70 ℃ and 7.0,respectively. It was stable at a temperature of 70℃ or below, and at a pH range of 5.0-8.0. Its activity was not significantly affected by most of metal ions tested and EDTA. The Km and of reXyn11^EM toward birchwood xylan were 7.2 mg/mL and 54.7U/mg. The excellent thermostability of reXyn11^EM make it has great potential in industrial application.