Expression of pectate lyase A from Aspergillus nidulans in Bacillus subtilis
Expression of pectate lyase A from Aspergillus nidulans in Bacillus subtilis
复制标题
DOI：
10.1007/s11274-008-9784-5
复制
发表时间：
2008-06
影响因子：
4.1
通讯作者：
Qing-xin Zhao;Runrong Ding;Yijun Kang;Jian Chen
中科院分区：
文献类型：
--
作者：
Qing-xin Zhao;Runrong Ding;Yijun Kang;Jian Chen
果胶酸裂解酶催化脱酯化果胶的消除性裂解，所述脱酯化果胶是许多高等植物初生细胞壁的主要组分。将编码无信号肽的构巢曲霉菌裂合酶A（PelA）基因与C端的6个His标签融合到含有Sac B信号肽和p43启动子的表达载体VBSzqx中。将VBSzqx-pelA质粒转化枯草芽孢杆菌（Bacillussubtilis），获得了一种安全、无内毒素的表达宿主菌，具有在食品工业中大规模生产外源蛋白的潜力。PelA成功表达，并使用Ni 2 +-次氮基三乙酸盐-琼脂糖柱有效纯化。在烧瓶中摇动，培养基在30°C、200 rpm和起始pH 7.0下产生最大酶产量620 U ml− 1培养基。在30°C、pH 7.0、500 rpm和2.0 l min− 1气流速率下，在2.5 l发酵罐中达到更高的酶产量（985 U ml− 1培养基）。表达的PelA在pH8.5和50°C下表现出最佳活性。重组PelA的Vmax和Km分别为77 μmol min− 1 mg − 1和0.50 mg ml−1。
Pectate lyases catalyze the eliminative cleavage of de-esterified pectin, which is a major component of the primary cell walls of many higher plants. The gene encodingAspergillus nidulanspectate lyase A (PelA) without signal peptide was fused with a six-His tag at its C-terminus in the expression vector VBSzqx containing Sac b signal peptide and p43 promotor. The VBSzqx-pelA plasmid was transformed intoBacillus subtilis, an expression host that is safe, free of any endotoxin, has a potential for large-scale production of foreign proteins in food industry. PelA was successfully expressed, and effectively purified using a Ni2+-nitrilotriacetate-agarose column. Shaken in flasks, the medium produced the maximum enzyme production of 620 U ml−1medium at 30°C, 200 rpm, and starting pH 7.0. Higher enzyme production (985 U ml−1medium) was reached in 2.5-l fermentor at 30°C, pH 7.0, 500 rpm, and 2.0 l min−1airflow rate. The expressed PelA exhibited its optimal activity at pH 8.5 and 50°C. TheVmaxandKmof recombinant PelA were 77 μmol min−1mg−1and 0.50 mg ml−1.