利用TAIL-PCR克隆Gluconobacter suboxydans葡萄糖脱氢酶基因及其生物信息学分析

doi:10.7506/spkx1002-6630-201401038

食品科学 ›› 2014, Vol. 35 ›› Issue (1): 194-198.doi: 10.7506/spkx1002-6630-201401038

利用TAIL-PCR克隆Gluconobacter suboxydans葡萄糖脱氢酶基因及其生物信息学分析

戴宝新1,2，冯惠勇1，李天明1，刘天佳1,3，刘静文1，仪宏1,*

1.河北科技大学生物科学与工程学院，河北石家庄 050018；2.石家庄以岭药业股份有限公司，河北石家庄 050018；
3.河北常山生化药业股份有限公司，河北石家庄 050018

收稿日期:2012-11-29 修回日期:2013-11-20 出版日期:2014-01-15 发布日期:2014-01-22
通讯作者: 仪宏 E-mail:yihonglaoshi@163.com

Cloning through TAIL- PCR and Bioinformatics Analysis of the Glucose Dehydrogenase Gene from Gluconobacter suboxydans

DAI Bao-xin1,2, FENG Hui-yong1, LI Tian-ming1, LIU Tian-jia1,3, LIU Jing-wen1, YI Hong1,*

1. College of Biological Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China;
2. Shijiazhuang Yiling Pharmaceutical Co. Ltd., Shijiazhuang 050018, China;
3. Hebei Changshan Biochemical Pharmaceutical Co. Ltd., Shijiazhuang 050018, China

Received:2012-11-29 Revised:2013-11-20 Online:2014-01-15 Published:2014-01-22
Contact: YI Hong E-mail:yihonglaoshi@163.com

摘要/Abstract

摘要：

以Gluconobacter suboxydans J菌株基因组DNA为模板，基于吡咯喹啉醌附着位点的保守区域设计引物，通过交错式热不对称PCR获得编码葡萄糖脱氢酶（glucose dehydrogenase，GDH）的全长基因（gdh），并对其序列进行生物信息学分析。结果表明：gdh基因全长2 268 bp，编码755个氨基酸，其蛋白序列与Gluconobacter属的GDH具有较高的同源性。GDH的分子质量约为81.72 ku，pI值约为5.14；GDH蛋白的二级结构由18.41%的α-螺旋、16.16%的延伸和65.43%的无规则卷曲3种结构模块组成；GDH N末端的AA 1～140区域有5个跨膜结构域。

关键词: 葡萄糖酸, 交错式热不对称PCR, 弱氧化醋酸杆菌, 葡萄糖脱氢酶, 生物信息学

Abstract:

Pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-dependent GDH, EC1.1.5.2), which catalyzes
the conversion of D-glucose to gluconic acid, is an important enzyme in the production of gluconic acid by fermentation
or enzymatic method. The aim of this study was to clone and characterize the gene enconding PQQ-dependent GDH from
Gluconobacter suboxydans. Primers were designed based on the conserved region of the PQQ-attaching sites, and the entire
gdh gene was obtained by thermal asymmetric interlaced-PCR (TAIL-PCR). The gene was then sequenced and analyzed
by bioinformatics methods. The sequence analysis suggested that its coding region consisted of 2268 bp nucleotides
encoding 755 amino acids. The deduced amino acid sequence showed a high level of similarity to the PQQ-dependent GDH
of Gluconobacter oxydans. The molecular weight of the encoded protein was 81.72 ku with an isoelectric point of 5.14.
The bioinformatic analysis suggested that its second structure consisted of 18.41% alpha helix, 16.16% extended strand
and 65.43% random coil and its N-terminal contained five transmembrane domains locating in the region between amino
acid residues 1 and 140. This study indicates that TAIL-PCR provides a simple and efficient method for the cloning of the
unknown genes. The bioinformatic analyses of GDH provide a foundation for further investigation on the characteristics and
catalytic mechanism of GDH and its potential applications in gluconic acid production.

Key words: gluconic acid, thermal asymmetric interlaced-PCR (TAIL-PCR), Gluconobacter suboxydans, glucose dehydrogenase, bioinformatics

中图分类号:

Q812

戴宝新1,2，冯惠勇1，李天明1，刘天佳1,3，刘静文1，仪宏1,*. 利用TAIL-PCR克隆Gluconobacter suboxydans葡萄糖脱氢酶基因及其生物信息学分析[J]. 食品科学, 2014, 35(1): 194-198.

DAI Bao-xin1,2, FENG Hui-yong1, LI Tian-ming1, LIU Tian-jia1,3, LIU Jing-wen1, YI Hong1,*. Cloning through TAIL- PCR and Bioinformatics Analysis of the Glucose Dehydrogenase Gene from Gluconobacter suboxydans[J]. FOOD SCIENCE, 2014, 35(1): 194-198.

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利用TAIL-PCR克隆Gluconobacter suboxydans葡萄糖脱氢酶基因及其生物信息学分析

Cloning through TAIL- PCR and Bioinformatics Analysis of the Glucose Dehydrogenase Gene from Gluconobacter suboxydans

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