UBC Theses and Dissertations
A study of some enzyme systems of PSEUDOMONAS AERUGINOSA Warburton, Roger Hartley
Pyruvate has been determined at 16, 28 and 40 hours in a culture of Pseudomonas aeruginosa when grown in a glucose medium. Since throughout this entire period the organism possessed the enzyme system capable of rapidly oxidizing pyruvate, it was concluded that pyruvate was being formed and dissimilated continuously and was therefore an intermediate in the oxidation of glucose. It was found that glucose oxidation was not inhibited by 0.02M sodium fluoride and that pyruvate formation and utilization continued unimpaired in the presence of the inhibitor. This would indicate that enolase is not concerned in the formation of pyruvate by P. aeruginosa and therefore the Embden-Meyerhof scheme of glucose degradation does not function in this organism. To study the initial reaction in the degradation of glucose by glucose oxidase of P. aeruginosa a preparation of dried cells was employed using a 24 hour old culture and drying them over phosphorus pentoxide. The dried cells although capable of oxidizing glucose to 2 ketogluconic at pH7.2, were unable to oxidize glucose further than gluconic acid at pH7.5. It was found that the enzyme was not inhibited by malonate, iodoacetate or arsenate but that the system was impaired by cyanide and sodium azide. The inhibition by cyanide would indicate that cytochrome oxidase or a second cyanide sensitive carrier is involved. The action of proteolytic enzymes, light and temperature were determined. The enzyme trypsin was found to destroy the glucose oxidase activity while the effect of pepsin and papain could not be established. Strong light was found to interfere with the full enzyme activity and a temperature of 55°C. completely destroyed the enzyme. Temperatures of 37°C. and 45°C. after 60 minutes reduced the oxygen uptake for the enzyme by as much as 50%. The separation of the enzyme into two fractions, co-enzyme and apo-enzyme, was effected by ammonium sulphate precipitation and dialysis against distilled water. It was found that these two fractions alone, or when combined, could not mediate the reaction glucose to gluconic acid. The co-enzyme was therefore concluded to be dialysable. Addition of known compounds to the fraction showed magnesium and manganese as ions capable of restoring the activity of the enzyme. Magnesium was found to be the more active of the two substances in restoring the enzymes activity. This would indicate that magnesium was the co-enzyme and was capable of being replaced by manganese.
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