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

作　　者：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] 

机构地区：[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

出　　处：《食品科学》2014年第1期194-198,共5页Food Science

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。Pyrroloquinoline quinone-dependent glucose dehydrogenase （PQQ-dependent GDH, EC1.1.5.2）, which catalyzesthe conversion of D-glucose to gluconic acid, is an important enzyme in the production of gluconic acid by fermentationor enzymatic method. The aim of this study was to clone and characterize the gene enconding PQQ-dependent GDH fromGluconobacter suboxydans. Primers were designed based on the conserved region of the PQQ-attaching sites, and the entiregdh gene was obtained by thermal asymmetric interlaced-PCR （TAIL-PCR）. The gene was then sequenced and analyzedby bioinformatics methods. The sequence analysis suggested that its coding region consisted of 2268 bp nucleotidesencoding 755 amino acids. The deduced amino acid sequence showed a high level of similarity to the PQQ-dependent GDHof 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 strandand 65.43% random coil and its N-terminal contained five transmembrane domains locating in the region between aminoacid residues 1 and 140. This study indicates that TAIL-PCR provides a simple and efficient method for the cloning of theunknown genes. The bioinformatic analyses of GDH provide a foundation for further investigation on the characteristics andcatalytic mechanism of GDH and its potential applications in gluconic acid production.

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 

分 类 号：Q812[生物学—生物工程]