Synthesis of aspartame precursor with an immobilized thermolysin in tert‐amyl alcohol

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N-(benzyloxycarbolyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert-amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparble to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70°C in tert-amyl alcohol in the absence of the subatrate, and up to 50°C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed. The substrate concentration dependence of the initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z-AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z-Asp as the substrate for over 300 h at 45°C and a space velocity of 1 h−1 without any loss of acivity. © 1994 John Wiley & Sons, Inc.