一种新型木聚糖酶基因在毕赤酵母中的表达

Expression of a Novel Xylanase Gene in Pichia pastoris

为了获得新型木聚糖酶的高效分泌表达,在工程酶项目组前期原核表达了木聚糖酶基因xyn A的基础上,将xyn A基因(Gen Bank登录号:U57819)编码耐热性木聚糖酶成熟蛋白的部分序列克隆到p PICZαA表达载体上,转化毕赤酵母X33,成功构建真核表达体系,再分批加入0. 5%(V/V)甲醇诱导毕赤酵母工程菌株产胞外木聚糖酶。结合木聚糖酶活力检测、SDS-PAGE电泳和Western Blot分析胞外木聚糖酶表达情况。结果表明,该XynA含有典型的糖苷水解酶11类催化结构域(GH11),成熟蛋白的预测分子量为38. 6 ku,等电点为6. 86; 7个毕赤酵母基因工程菌株X33/p PICZαA-xyn A分泌的木聚糖酶活力均≥300 U/m L,最高达487. 2 U/m L;胞外木聚糖酶在SDS-PAGE和Western Blot检测的特征谱带分子量约为45 ku,均比预期分子量(38. 6 ku)略大。一种新型木聚糖酶获得了高效分泌表达,为进一步分离纯化,研究其性质、结构和功能奠定了基础。

To obtain a new extracellular xylanase in high yield, the xynA （GenBank number：U57819） can be expressed in different ways. A xynA was early expressed in prokaryotic microbiology. Subsequently, we cloned the partial xynA encoding mature xylanase into expression vector pPICZαA, and then transformed into Pichia pastoris X33, so eukaryotic expression system was successfully constructed to produce heat-resistant xylanase without signal peptide. 0.5% （V/V）methanol was added in batch to induce the engineering Pichia pastoris which produced extracellular xylanase. The extracellular xylanase was analyzed by the xylan-degrading activity, SDS-PAGE and Western Blot. The results showed that the XynA contained typical catalytic structure domain of glycosidic hydrolyzyme 11 （GH11）, and the predicted molecular weight of mature protein was 38.6 ku, and the isoelectric point was 6.86. Xylanase activities secreted by the seven genetic-engineering Pichia pastoris X33/pPICZαA-xynA were more than 300 U/mL, and the highest xylanase activity was up to 487.2 U/mL;the molecular weights of extracellular xylanases on SDS-PAGE and Western Blot were about 45 ku by the characteristic strips, bigger than the predicted molecular weight （38.6 ku）. Therefore, a novel extracellular xylanase has been effectively expressed and may lay a foundation for its separation and purification to study the property, structure and function.