Purification and Partial Characterization of Caffeine Oxidase—A Novel Enzyme from a Mixed Culture Consortium

• Published in: Biochemical and biophysical research communications Vol. 263; no. 2; pp. 460 - 464
• Main Authors: Madyastha, K.M., Sridhar, G.R., Vadiraja, Bhat.B., Madhavi, Y.Sudha
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24-09-1999
• Subjects: 1,3,7-Trimethyluric acid; C-8 oxidation; Caffeine - metabolism; caffeine oxidase; Klebsiella; Klebsiella - enzymology; mixed culture; Oxidation-Reduction; Oxygenases - antagonists & inhibitors; Oxygenases - isolation & purification; Oxygenases - metabolism; partial characterization; purification of caffeine oxidase; Rhodococcus; Rhodococcus - enzymology; Substrate Specificity; Theobromine - metabolism; Theophylline - metabolism; Uric Acid - analogs & derivatives; Uric Acid - metabolism; Xanthine Oxidase - metabolism; partial characterization; purification of caffeine oxidase; mixed culture; C-8 oxidation; substrate specificity
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1999.1401

Cell-free extract prepared from a mixed culture consisting of strains belonging to the genera Klebsiella and Rhodococcus grown in the presence of caffeine contains a novel enzyme, caffeine (1,3,7-trimethylxanthine) oxidase which catalyzes the oxidation of caffeine at the C-8 position to produce 1,3,7-trimethyluric acid. The enzyme was purified to homogeneity by a combination of ion-exchange and hydrophobic column chromatographies. Both native and SDS/PAGE of the purified enzyme showed a single protein band and the subunit molecular mass of the protein was determined to be 85 kDa. Dichlorophenol indophenol and cytochrome c served as good electron acceptors but NAD and NADP did not. Caffeine served as the best substrate with an apparent Km of 11.4 μM. various analogues of theobromine were also effective substrates for caffeine oxidase. The activity was inhibited by o-phenanthroline, H2O2, and methanol, but salicylate, thiol-group blocking reagents, and sodium arsenite, the known xanthine oxidase inhibitors, did not inhibit the reaction. The spectral characteristics of the purified enzyme suggest that it is a flavoprotein containing non-heme iron.