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

Cell-free extract prepared from a mixed culture consisting of strains belon ging to the genera Klebsiella and Rhodococcus grown in the presence of-caff eine contains a novel enzyme, caffeine (1,3,7-trimethylxanthine) oxidase wh ich 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 combinati on of ion-exchange and hydrophobic column chromatographies. Both native and SDS/PAGE of the purified enzyme showed a single protein band and the subun it molecular mass of the protein was determined to be 85 kDa. Dichloropheno l indophenol and cytochrome c served as good electron accepters but NAD and NADP did not. Caffeine served as the best substrate with an apparent K-m o f 11.4 mu M. various analogues of theobromine were also effective substrate s for caffeine oxidase. The activity was inhibited by o-phenanthroline, H2O 2, and methanol, but salicylate, thiol-group blocking reagents, and sodium arsenite, the known xanthine oxidase inhibitors, did not inhibit the reacti on. The spectral characteristics of the purified enzyme suggest that it is a flavoprotein containing non-heme iron. (C) 1999 Academic Press.