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

doi:10.7506/spkx1002-6630-201401038

Abstract

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

CLC Number:

Q812

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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