Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris
Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris
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DOI：
10.1080/10242420600768771
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发表时间：
2006-09-01
影响因子：
1.8
通讯作者：
Zhang, Xian-En
中科院分区：
文献类型：
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作者：
Zhang, Gui-Min;Hu, Yong;Zhang, Xian-En
采用鸟枪法从短小芽孢杆菌中克隆了编码碱性木聚糖酶的基因xynHB。将该基因克隆到表达载体pHBM 905 A中，并在毕赤酵母GS115中表达。在含有RBB-木聚糖的平板上选择分泌木聚糖酶的转化体。在25 ℃条件下，培养上清的酶活可达644 U mL（-1），最适分泌时间为4 d。SDS-PAGE显示32.2 kDa和29.6 kDa的两条带，均大于基于其氨基酸序列预测的22.4 kDa的质量。酶谱分析表明，两条酶带均能水解木聚糖。通过内切糖苷酶H的去糖基化显示，两者都来自相同的蛋白质，但含有不同程度的糖基化（30和25%）。该酶的最适pH为6 -9，最适温度为50 ℃。
The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL(-1) and the optimal secretion time was 4 days at 25 degrees C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6-9 and 50 degrees C, respectively.