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

Madyastha, KM and Sridhar, GR and Bhat, Vadiraja B and Sudha, Madhavi Y (1999) Purification and Partial Characterization of Caffeine Oxidase—A Novel Enzyme from a Mixed Culture Consortium. In: Biochemical and Biophysical Research Communications, 263 (2). pp. 460-464.

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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 $11.4 \hspace{2mm}\mu M$. various analogues of theobromine were also effective substrates for caffeine oxidase. The activity was inhibited by o-phenanthroline, $H_2O_2$, 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.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Elsevier.
Keywords: Mixed culture;Purification of caffeine oxidase;C-8 oxidation;Substrate specificity;Partial characterization
Department/Centre: Division of Chemical Sciences > Organic Chemistry
Date Deposited: 24 Oct 2007
Last Modified: 19 Sep 2010 04:40
URI: http://eprints.iisc.ernet.in/id/eprint/12183

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