Long-term continuous synthesis of aspartame precursor in a column reactor with an immobilized thermolysin

N-(Benzyloxycarbonyl)-L-asparty-L-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25 degrees C with the substrates (N-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl2 with or without the substrate at 25 degrees C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h-1.