Synthesis of Aspartame Precursor Using Protease Suspended in Microaqueous Molten Amino Acids Mixture

Enzymatic synthesis of the aspartame precursor, N -(benzyloxycarbonyl)- l -aspartyl- l -phenylalanine methyl ester (Z-AspPheOMe) was performed with highly concentrated molten substrates. A mixture composed of molten N -(benzyloxycarbonyl)- l -aspartic acid (Z-Asp) and l -phenylalanine methyl ester (PheOMe) mixtures of 20 M could be prepared at 50°C. This Z-Asp/PheOMe mixture was applied to the enzymatic synthesis of Z-AspPheOMe using free thermolysin. Synthesis of Z-AspPheOMe was observed in the range of 100-150 w l of NaOH solution (12.5 M) addition to a reaction mixture consisting of 1.0 mmol Z-Asp and 1.0 mmol PheOMe at 50°C. The enzymatic activity increased with increasing water addition, and reached a maximum at 100 w l in addition to the reaction mixture of 1.0 mmol Z-Asp, 1.0 mmol PheOMe and 125 w l of the NaOH solution. In this reaction system, the conversion at the reaction equilibrium was about 60%, the initial reaction rate calculated on the basis of the enzyme weight was 2.2 w mol/g s, and the productivity calculated on the basis of the reaction mixture volume was 300 mol/m 3 h.