Abstract
A cell-free system has been developed from Coffea arabica that is a rich source of the N-methyltransferase activity which catalyses the transfer of the methyl group from S-adenosyl-L-methionine to methylxanthines producing caffeine. Purification of the enzyme by anion-exchange chromatography results in low activity yields. Part of the reason was found to be inhibition of the N-methyltransferase activity by KCI and NaCl which are used to elute the protein during anion-exchange chromatography. The enzyme was found to have a half life at -4 degrees C of approximately 90 min. An extensive study of a wide range of protease inhibitors failed to show any effective stabilisation of activity suggesting that the losses are not due to the action of endogenous proteases in the extract. The stability of the enzyme has been shown to be improved substantially with the incorporation of 20% (v/v) glycerol or 20% (v/v) ethylene glycol in the buffers used. Incorporation of 20% (v/v) glycerol in buffers during anion-exchange chromatography resulted in 54 - 78% yield of N-methyltransferase activity and a ca. 10-20 fold purification.