一种来源于Bispora betulina的酸性木聚糖酶基因克隆及其酶学性质研究

Gene Cloning,Expression and Characterization of an Acidic Xylanase from Bispora betulina

利用简并PCR和TAIL-PCR技术从嗜酸性真菌Bispora betulina中克隆得到一个酸性木聚糖酶基因xyn11BB。该基因全长1 207 bp,含有三段内含子、一段外显子和一个终止密码子,编码由297个氨基酸组成的蛋白质,推测蛋白质含有一个CBM1结构域。将不带原基因信号肽编码序列的cDNA基因以正确阅读框架克隆到表达载体pPIC9上,并在毕赤酵母(Pichia pastoris)GS115中诱导表达,其表达活性达121.15 U/mL。重组蛋白经硫酸铵分级沉淀和阴离子交换柱纯化后达到电泳纯。对其酶学性质分析表明,重组木聚糖酶最适温度为50℃,最适pH为4.5,在酸性条件下具有良好的活性和稳定性,可作为饲料用酶的备选。

An acidic xylanase-encoding gene was cloned from Bispora betulina by degenerate PCR and TAIL-PCR.The complete gene sequence consists of 1 207 bp with three introns,one exon and a stop coden.It encodes a protein with 297-amino acid polypeptide,which contains a cellulose binding module 1（CBM1）.The cDNA gene without the signal peptide-encoding sequences was ligated into the expression vector pPIC9 and transformed into the strain Pichia pastoris GS115.After the induction of methanol,extracellular recombinant xylanase in the supernatant of the recombinant P.pastoris strain reached 121.15 U/mL.The recombinant enzyme was purified to homogeneity by ammonium sulfate precipitation and anion exchange chromatography.The optimal pH and temperature of purified recombinant enzyme were pH 4.5 and 50℃,respectively.It exhibited high activity and stability at acidic pH range.It could be used as an alternative feed enzyme.