微紫青霉酸性木聚糖酶xynA基因的克隆与序列分析

Cloning and Bioinformatics Analysis of Acidophilic xynA Gene from Penicillium janthinellum

基于酸性木聚糖酶在饲料及酿酒行业良好的应用前景,通过基因组步移的方法克隆得到微紫青霉酸性木聚糖酶的全长基因xynA,然后采取重叠延伸PCR技术进行内含子的切除获得xynA的cDNA序列,并对其进行了生物信息学分析。序列分析结果显示xynA基因全长720 bp,内含子63 bp,cDNA全长657 bp。推测该木聚糖酶编码信号肽28个氨基酸,成熟肽190个氨基酸;预测该蛋白为分子质量20.61 kD、等电点7.0的亲水性蛋白,且分子内不含二硫键。与其他真菌来源的GH11族耐酸性木聚糖酶进行序列比对,结果显示该酶的相应位置具有特征天冬氨酸残基Asp,且具有糖苷水解酶11族的保守区域特征以及典型的"右手半握"状结构,重组木聚糖酶基因xynA能够在大肠杆菌中成功表达,比酶活力达220.5 U/mg。

Acidic xylanases have extensive application in feed and wine industries.The whole sequence of the gene xynAencoding acidic xylanase was amplified from Penicillium janthinellum MA21601by genome walking.A cDNA sequencewas obtained through the elimination of introns by overlapping PCR and analyzed by bioinformatics.The whole sequencewas about720bp in length with only one intron of63bp.The cDNA sequence was657bp long and putatively encodeda protein which contained a28-amino acid(aa)signal peptide and a190-aa mature peptide.The molecular weight of theprotein was estimated to be about20.61kD,which had an isoelectric point of7.0.Bioinformatics analysis showed that XynAwas a hydrophilic protein without disulfide bond.The amino acid sequence comparison of XynA with other fungal GH11acidophilic xylanases indicated that the XynA had an identified specific recognition site of Asp,which displayed aβ-jellyrollarchitecture as a conserved region which was the characteristic of the GH11family xylanases.The recombinant xylanasewas successfully expressed in Escherichia coli with a specific activity of up to220.5U/mg.