Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8326843 | International Journal of Biological Macromolecules | 2018 | 35 Pages |
Abstract
The membrane-bound glucose dehydrogenase (mGDH) is a rate-limiting enzyme for the industrial production of 2-keto-d-gluconic acid (2KGA) from glucose. In this study, mGDH was firstly purified from a 2KGA industrial producing strain Pseudomonas plecoglossicida JUIM01. The purified mGDH exhibited a specific activity of 16.85â¯U/mg and was identified as monomeric membrane-bound PQQ-dependent dehydrogenase with a molecular mass of ~87â¯kDa. The Km and Vmax value of d-glucose were 0.042â¯mM and 14.620â¯Î¼M/min, and the optimal pH and temperature were of 6.0 and 35â¯Â°C with favorable acid resistance and poor heat tolerance. Ca2+/Mg2+ showed a significantly positive effect on mGDH activity with 20% increase, whereas EDTA/EGTA had a negative influence, and Ca2+ was essential for enzyme activity. Furthermore, a 2412â¯bp-length gcd was amplified by genome walking technique and heterologously expressed in Escherichia coli. Bioinformatics analysis and heterologous expression further confirmed it as a mGDH encoding gene. mGDH contained binding sites of Ca2+, cofactor PQQ and polypeptide binding sites concluded from alignment results of mGDHs from different genera. This study would lay the foundation for improving 2KGA productivity through further strain modification.
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Authors
Da-Ming Wang, Lei Sun, Wen-Jing Sun, Feng-Jie Cui, Jin-Song Gong, Xiao-Mei Zhang, Jin-Song Shi, Zheng-Hong Xu,