Purification and enzymatic characterization of membrane-bound d-gluconate dehydrogenase from Arthrobacter globiformis

• A membrane-bound GADH was firstly purified from the 2KGA producer Arthrobacter globiformis.
• The GADH was characterized as FAD-dependent and containing cytochrome c.
• FAD-GADH had pH/thermo-stable activities and strict specificity for d-gluconate.
• Our finding benefits for improving 2KGA efficient production by A. globiformis.

Arthrobacter globiformis C224 is an industrial 2-keto-gluconic acid (2KGA) producer and currently used for erythorbic acid production in China. In the present study, a membrane-bound gluconate dehydrogenase (GADH) with specific activity of 326.06 U/mg and purification fold of 412 was purified from A. globiformis using a five-step procedure including ultrasonication, phase separation with Triton X-114, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The GADH was identified as to be flavin adenine dinucleotide (FAD)-dependent and consisted of three subunits with molecular mass of 66,000 Da, 44,000 Da and 23,000 Da. The optimal pH and temperature of A. globiformis GADH were 5.0 and 40 °C, respectively. It had the stable activity at pH of 5.0–7.0 or below 50 °C, and the strict substrate specificity for d-gluconate with the Km of 3.15 mmol L−1, 1.04 mmol L−1 at pH 5.0 and pH 6.0, respectively. The GADH activity also was significantly influenced by metal ions, organic solvents, and organic acids. Our study will benefit for better understanding of 2KGA production process by A. globiformis C224.

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