The characteristics and stabilization of a caffeine demethylase enzyme complex

Cell-free extracts (CFE) from Pseudomonas putida , cultured on caffeine as sole carbon and/or nitrogen source, contained proteins which demethylate caffeine. Caffeine metabolism correlated with production of two major proteins (43.5 and 36.6 kDa); we suggest that one or both of these are caffeine demethylase enzymes. HPLC analysis of caffeine degradation by P. putida showed that caffeine is converted to uric acid via 3–7-dimethylxanthine, 7-methylxanthine and xanthine. The stability of caffeine demethylase was greatly improved by the use of cryoprotectants and freeze drying to low moisture contents. Freeze-dried cell-free extracts maintained 83% of activity after 200 days of storage at 4 °C.