Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 188, Issue 4, Pages 897-913Publisher
SPRINGER
DOI: 10.1007/s12010-019-02951-0
Keywords
Pseudomonas plecoglossicida; 2-Keto-d-gluconic acid (2KGA); Gluconate dehydrogenase; Heterologous expression
Funding
- National Natural Science Foundation of China [31571885]
- Innovation Group Construction Program of Jiangxi Province [20142BCB24024]
- Science & Technology Program of Jiangxi Province [[2015] 64]
- Science & Technology Program of Dexing city [[2015] 44]
- National First-class Discipline Program of Light Industry Technology and Engineering [LITE2018-11, LITE2018-18]
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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 similar to 27, 65, and 47kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71U/mg at optimum pH and temperature of 6.0 and 35 degrees C. The K-m and V-max values of calcium d-gluconate were 0.631mM and 0.734mM/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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