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J Bacteriol. 1963 May; 85(5): 1124-1131
Copyright © 1963, The Williams & Wilkins Company. All Rights Reserved.

GLYCOLIC ACID OXIDATION BY ESCHERICHIA COLI ADAPTED TO GLYCOLATE

^a Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois

ABSTRACT

FURUYA, AKIRA (University of Illinois College of Medicine, Chicago) AND JAMES A. HAYASHI. Glycolic acid oxidation by Escherichia coli adapted to glycolate. J. Bacteriol. 85:1124–1131. 1963.—A procedure is described for extraction and partial purification of glycolic acid oxidase from Escherichia coli adapted to grow on glycolate as the sole carbon source. Enzyme activity was assayed by oxygen uptake and by reduction of 2,6-dichlorophenol-indophenol. Glyoxylic acid was the product of glycolate oxidation by the enzyme. Enzyme activity, which diminishes rapidly on storage, shows a maximum at pH 6 to 7. We were unable to show any cofactor requirement. Compounds which inhibited glycolate oxidation and their order of inhibitory activity were: p-hydroxymercuribenzoate > sodium azide > iodoacetate and o-phenanthroline > ethylenediaminetetraacetic acid. Tests of enzyme specificity showed that the following compounds were oxidized, but at different rates: glycolate, D-lactate, L-lactate, DL--hydroxybutyrate, DL-malate, and DL-glycerate. Citrate, tartrate, and DL-ß-hydroxybutyrate were not oxidized. Potassium cyanide stimulated oxygen uptake when glycolate and lactate were oxidized. Whether the oxidations were due to different oxidases or to a single oxidase with a wide range of specificities was tested by observing the oxidation of glycolate, D-lactate, and L-lactate under various conditions. Ammonium sulfate fractionation of a crude extract did not change the relative ability to oxidize the three acids. However, the three oxidative capacities diminished at different rates during storage at 0 C for 6 days. The partially purified glycolic oxidase preparations were probably mixtures of several different oxidases.

¹ Present address: Tokyo Research Laboratory of Kyowa Fermentation Industry Co., Machidashi, Tokyo, Japan.

² Present address: Department of Biochemistry, Presbyterian-St. Luke's Hospital, Chicago, Ill.