Enzymological Aspects of Caffeine Demethylation and Formaldehyde Oxidation byPseudomonas putidaC1

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Abstract
The enzymatic demethylation of caffeine (1,3,7-trimethylxanthine) by P. putida C1 was investigated; an inducible enzyme system was observed. This enzyme shows an optimum pH of .apprx. 6.0 and the optimum temperature is in the range of 22-24.degree. C. The enzyme is absolutely dependent on NADH or NADPH as a cosubstrate and is activated by Co2+. The formaldehyde generated by the demethylation of caffeine is oxidized by an NAD-dependent formaldehyde dehydrogenase, which is independent of Mg2+ and glutathione. The enzyme was purified from cell-free extracts of P. putida C1 by DEAE-cellulose, Sephadex G-150 and Sephadex A-50 chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and was most active at a pH between 8.5-9.0. The MW was estimated to be .apprx. 250,000 by the gel filtration method. Kinetic analysis gave Km values of .apprx. 0.2 mM for formaldehyde and 0.5 mM for NAD.