Purification and partial characterization of caffeine oxidase-a novel enzyme from a mixed culture consortium

Abstract

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 K_m 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.