CATABOLISM OF CAFFEINE AND PURIFICATION OF A XANTHINE OXIDASE RESPONSIBLE FOR METHYLURIC ACIDS PRODUCTION IN PSEUDOMONAS PUTIDA L

Abstract

ABSTRACTCaffeine catabolism and a xanthine oxidase involved in the alkaloid breakdown werestudied in Pseudomonas putida L, a strain displaying high ability to grow on this substrate.Cells cultured with unlabelled caffeine and 14 C labeled caffeine and xanthine showed thatthis alkaloid was broken-down via theobromine/paraxanthine -> 7-methylxanthine ->xanthine -> uric acid -> allantoin -> allantoic acid. Methyluric acids were formed fromthe oxidation of theobromine, paraxanthine and 7-methylxanthine, although no bacterialgrowth was observed on these compounds, indicating that this might be due to a widesubstrate specificity of xanthine oxidase. This was confirmed by activity staining in PAGEwhere activity was observed with theophylline and 3-methylxanthine, which are notinvolved in the alkaloid breakdown. A single band of activity was detected without additionof NAD + , showing an oxidase form of the enzyme. The enzyme optimum temperatureand pH were 30 o C and 7.0, respectively. The determined K