UBC Theses and Dissertations
Gluconic acid oxidizing system of Pseudomonas aeruginosa Ramakrishnan, Thekkepat
Earlier work has shown that Pseudomonas aeruginosa 9027 can oxidize glucose to carbon dioxide and water by way of gluconic, 2-ketogluconic and pyruvic acids. However, it has been found that closely related organisms can phosphorylate gluconic acid. The object of the present work was to isolate the gluconate oxidizing enzyme, to solubilize it, purify it, determine the co-factor requirements and ascertain whether or not any energy was gained or lost by the system during the reaction. Cells harvested from a gluconic acid medium were disintegrated in a 10 kc. Raytheon sonic oscillator. The enzyme which was still attached to the cell particles was solubilized with sodium glycocholate and remaining particles were removed by the addition of 0.30 saturation ammonium sulphate. Nucleoproteins were then removed by the addition of protamine sulphate. Further fractionation with acid and alkaline ammonium sulphate purified the enzyme 200 fold. Finally the enzyme was absorbed on tricalcium phosphate and eluted with M/5 phosphate buffer of pH 7.0. The pH optimum of the purified enzyme was found to be 5.6 while in the whole cells the maximum activity was at pH 7.0. A hydrogen acceptor was necessary for linking the system to atmospheric oxygen; 2,6-dichlorophenolindophenol and pyocyanine were found to be the most efficient acceptors. Ferricyanide poisoned the system, while brilliant cresyl blue was inactive as a hydrogen acceptor. Reaction with methylene blue was slow. Diphosphopyridine nucleotide, triphosphopyridine nucleotide, flavin mononucleotide, flavin adenine dinucleotide, cytochrome c, adenosine diphosphate and adenosine triphosphate had no influence on the enzyme activity. Sodium fluoride, 2,4-dinitrophenol, azide, iodoacetate, arsenite or 8-hydroxyquinoline did not act as inhibitors. Cyanide, glutathione and cysteine activated the enzyme slightly. The enzyme is specific for gluconic acid. Glucose, glucuronic acid, 2-ketogluconic acid, pyruvic acid, saccharic acid, ribonic acid, arabonic acid, fructose, mannose, ribose-5-phosphate, glucose-6-phosphate or 6-phosphogluconic acid were not oxidized by the enzyme. No carbon dioxide was evolved during the oxidation of gluconic acid by the enzyme. The product on chromatographic analysis, was found to be 2-ketogluconic acid. The enzyme was routinely stored at -10°C in M/10 tris buffer, pH 7.0. Under these conditions it was stable for several weeks. At 4°C, under the same conditions, the enzyme may be kept for three to four days without any appreciable loss of activity. When dialyzed against distilled water, there was a gradual loss of activity after eight to ten hours, accompanied by precipitation. Dialysis against neutral buffers for as long as 24 hours in the cold produced no loss in activity. Instead of sodium glycocholate, "Cutscum" can be used, for solubilizing the enzyme. Purification can also be effected from the sonicate through the use of the ultracentrifuge. The supernatant left after one hour of centrifugation at 105,000 x G oxidized gluconic acid in the presence of pyocyanine and showed two peaks in the electrophoretic apparatus, one of which is believed to be due to protamine sulphate. Though no phosphorylation of the substrate was demonstrable as evidenced by the lack of activation by ATP and the lack of inhibition by fluoride, the problem was further investigated in the sonic extracts. No increase in acid was found either aerobically or anaerobically in P. aeruginosa as tested by the method of Colowick and Kalckar. Moreover, sonic extracts failed to reduce TPN in the presence of gluconate and an excess of phosphogluconic dehydrogenase isolated from Brewer's yeast. In contrast to these data, it was found that by either of the last two mentioned criteria, P. fluorescens A. 312 did phosphorylate gluconate. p. fluores-cens thus possesses an additional phosphorylated pathway for dissimilating glucose and this is absent in P. aeruginosa. No energy was found to be produced in the initial stages of glucose oxidation. The system could not be coupled to the "zwischenferment" reaction of glucose which requires ATP. Chromatographic analysis failed to show any ATP formed during the oxidation of gluconic acid. The significance of these findings in the light of the glucose metabolism by P. aeruginosa is discussed.
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