食品科学 ›› 2017, Vol. 38 ›› Issue (14): 9-16.doi: 10.7506/spkx1002-6630-201714002

• 生物工程 • 上一篇    下一篇

微紫青霉酸性木聚糖酶xynA基因的克隆与序列分析

侯洁,李琴,熊科,徐友强,李秀婷   

  1. (1.北京食品营养与人类健康高精尖创新中心,北京工商大学,北京 100048;2.北京市食品添加剂工程技术研究中心,北京工商大学,北京 100048;3.北京工商大学食品学院,北京 100048)
  • 出版日期:2017-07-25 发布日期:2017-09-06
  • 基金资助:
    国家自然科学基金面上项目(31371723)

Cloning and Bioinformatics Analysis of Acidophilic xynA Gene from Penicillium janthinellum

HOU Jie, LI Qin, Xiong Ke, XU Youqiang, LI Xiuting,   

  1. (1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China; 2. Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing 100048, China; 3. School of Food and Chemical Engineering, Beijing Technology & Business University, Beijing 100048, China)
  • Online:2017-07-25 Published:2017-09-06

摘要: 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。

关键词: 微紫青霉, 酸性木聚糖酶, 基因克隆, 生物信息学分析

Abstract: Acidic xylanases have extensive application in feed and wine industries. The whole sequence of the gene xynA encoding acidic xylanase was amplified from Penicillium janthinellum MA21601 by genome walking. A cDNA sequence was obtained through the elimination of introns by overlapping PCR and analyzed by bioinformatics. The whole sequence was about 720 bp in length with only one intron of 63 bp. The cDNA sequence was 657 bp long and putatively encoded a protein which contained a 28-amino acid (aa) signal peptide and a 190-aa mature peptide. The molecular weight of the protein was estimated to be about 20.61 kD, which had an isoelectric point of 7.0. Bioinformatics analysis showed that XynA was a hydrophilic protein without disulfide bond. The amino acid sequence comparison of XynA with other fungal GH11 acidophilic xylanases indicated that the XynA had an identified specific recognition site of Asp, which displayed a β-jelly-roll architecture as a conserved region which was the characteristic of the GH11 family xylanases. The recombinant xylanase was successfully expressed in Escherichia coli with a specific activity of up to 220.5 U/mg.

Key words: Penicillium janthinellum, acidophilic xylanase, gene cloning, bioinformatics analysis

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