2-Keto-D-Gluconate-Yielding Membrane-Bound Glucose Dehydrogenase From Arthrobacter Globiformis C224: Purification And Characterization

2-Keto-D-gluconic acid(2KGA) is the precursor for the synthesis of the food additives erythorbic acid and sodium erythorbate. Membrane bound glucose dehydrogenase(m GDH, EC 1.1.5.2) is the key oxidoreductase involved in the 2KGA biosynthesis from glucose by specifically catalyzing D-glucose to D-gluconate.The present study will focused on: 1) purifying and identifying the membrane bound glucose dehydrogenase(m GDH) from 2KGA producer Arthrobacter globiformis C224, 2) systematically evaluating of m GDH enzymatic properties, and 3) preliminary investigating its enzymatic reaction kinetics. The findings will provide a theoretical reference for further m GDH overexpression and even 2KGA high producing strains construction.The main conclusions of this study are as follows:(1) Using the ultrasonication, two-phase separation with Triton X-114, precipitations with acetone, polyethylene glycol 6000 and ethanol, and Hydroxyapatite Fast Flow chromatography, the m GDH with the specific activity of 88.08 U/mg and purification factor of 183.50 fold was purified from A. globiformis C224.(2) The m GDH from A. globiformis C224 had one subunit with molecular weight of about 90000 shown in sodium dodecyl sulfate-polyacryamide gel electrophoresis(SDS-PAGE) electrophoregram. Matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS) analysis proved that the protein purified from A. globiformis C224 was PQQ dependent m GDH, and the molecular weight of about 87000.(3) Enzymatic properties results of m GDH from A. globiformis C224 showed that m GDH had variable optimal p H depending on the electron acceptor, when 2,6-dichlorophenolindophenol-phenazine methosulfate(DCIP-PMS) or ferricyanide used as electron acceptor its optimal p Hs were 6.0 and 5.0, respectively. The optimum temperature of the m GDH was 45 ℃, it had a stable catalytic activity at a p H ranging from 6.0~7.0 and temperature of 20~40 ℃. m GDH also had a broad substrate specificity with D-glucose, maltose, D-xylose, D-galactose and D-arabinose as substrates. Mg2+ increased the m GDH activity while Mn2+, Zn2+, Cu2+, Fe3+, some organic solvents including methanol, ethanol, acetone and n-hexane inhibited its catalytic reaction in some extent. EDTA also showed the significantly inhibition on the m GDH activity indicating that this m GDH was a metallic ions dependent enzymes.(4) With the presence of electron acceptor ferricyanide, the maximum reaction rate and Michaelis constant for D-glucose of m GDH from A. globiformis C224 were 192.31 μmol/mg·min and 0.21 mmol/L at the temperature of 25 and p H of 5.0. The ℃maximum reaction rates Vmax and Km values of m GDH were 44.05 μmol/mg·min, 18.32 μmol/mg·min, 19.05 μmol/mg·min and 0.34 mmol/L, 0.46 mmol/L, 0.59 mmol/L, resepetivly, when D-xylose, D-galactose and maltose were used as the substrate. The analysis of the dual substrates kinetics and inhibition caused by products kinetics showed that the reaction followed a Ping-Pong Bi-Bi kinetic mechanism with PMS binding to the free enzyme to produce PMSH, and then to the second substrate D-glucose to produce the final product D-gluconate.