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A Membrane-Bound Gluconate Dehydrogenase from 2-Keto-d-Gluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01: Purification, Characterization, and Gene Identification

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Abstract

The membrane-bound gluconate dehydrogenase (mGADH) is a critical enzyme for 2-keto-d-gluconic acid (2KGA) production in Pseudomonas plecoglossicida JUIM01. The purified native flavin adenine dinucleotide-dependent mGADH (FAD-mGADH) was consisted of a gamma subunit, a flavoprotein subunit, and a cytochrome c subunit with molecular mass of ~ 27, 65, and 47 kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71 U/mg at optimum pH and temperature of 6.0 and 35 °C. The Km and Vmax values of calcium d-gluconate were 0.631 mM and 0.734 mM/min. The metal ions Mg2+ and Mn2+ showed slight positive effects on FAD-mGADH activity. On the other hand, a 3868-bp-length gad gene cluster was amplified and expressed in Escherichia coli BL21(DE3). The recombinant protein showed the same molecular weight and enzyme activity as the native FAD-mGADH, which confirmed it as a FAD-mGADH encoding gene. The flavoprotein subunit and the cytochrome c subunit containing a putative FAD-binding motif and three possible heme-binding motifs concluded from alignment results of mGADHs. This study characterized the native and recombinant FAD-mGADH and would provide the basis for further genetic modification of Pseudomonas plecoglossicida JUIM01 with the intention of 2KGA productivity improvement.

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Acknowledgements

Many thanks go to Ph.D. Xue Cai (Jiangnan University) for her help regarding the language polish of this manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (31571885), the Innovation Group Construction Program of Jiangxi Province (20142BCB24024), the Science & Technology Program of Jiangxi Province (No. [2015]64), Science & Technology Program of Dexing city (No. [2015]44), and the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-11 and LITE2018-18).

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  1. Da-Ming Wang and Lei Sun are the co-first authors and have the equal contributions.

Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Da-Ming Wang, Lei Sun, Jin-Song Gong, Xiao-Mei Zhang, Jin-Song Shi & Zheng-Hong Xu

  2. Parchn Sodium Isovitamin C Co., Ltd., Dexing, 334221, People’s Republic of China

    Da-Ming Wang, Wen-Jing Sun & Feng-Jie Cui

  3. School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, People’s Republic of China

    Wen-Jing Sun & Feng-Jie Cui

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Wang, DM., Sun, L., Sun, WJ. et al. A Membrane-Bound Gluconate Dehydrogenase from 2-Keto-d-Gluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01: Purification, Characterization, and Gene Identification. Appl Biochem Biotechnol 188, 897–913 (2019). https://doi.org/10.1007/s12010-019-02951-0

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