黑曲霉木聚糖酶基因xynB在大肠杆菌中的表达及重组木聚糖酶的特性

从黑曲霉(Aspergillus niger) mafic-005中克隆得到木聚糖酶基因xynB。序列分析表明，该基因全长745 bp，含有一个67 bp内含子，编码225个氨基酸，理论分子量为24 kD(GenBank登录号:DQ174549)，与已知黑曲霉xynB基因的同源性均较高。将该基因定向插入大肠杆菌(Escherichia coli )表达载体pET-28a (+)上，并转化E. coli BL21 获得重组菌株。经过IPTG诱导，xynB基因获得特异性表达。经SDS-PAGE分析，重组蛋白分子量约为30 kD。该重组蛋白经镍NTA琼脂糖凝胶FF纯化后达到电泳纯。酶学性质分析表明，重组木聚糖酶最适温度为40 ℃，最适pH值为5.0，在酸性和常温条件下具有良好的稳定性。

Expression of Xylanase Gene xynB from Aspergillus niger in Escherichia coli and Characterization of Its Recombinant Xylanase

The gene xynB encoding β-1, 4-xylanase was cloned from Aspergillus niger mafic-005. The sequencing results showed that the full-length gene contained 745 bp, including an intron of 67 bp, and encoding a protein of 225 amino acids which had a presumed molecular mass of 24 kD(GenBank accession No. DQ174549). Compared this xylanase xynB gene with others from A. niger in GenBank, the homology was high. The xynB gene was inserted into the expression vector of pET-28a(+) and transformed into Escherichia coli BL21. The recombinant protein was expressed successfully in E. coli by inducing with IPTG, and purified by Ni-NTA Sepharose FF. The molecular mass of the recombinant protein was about 30 kD according to SDS-PAGE. Characterization of the recombinant enzyme indicated that the optimum temperature and pH of the recombinant xylanase were 40℃ and 5.0, respectively. The recombinant protein showed an extreme stability in the acidic solution and ambient temperature.