Aspartame precursor (N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine ester), an artificial sweetener, was synthesized enzymatically from N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PM) as substrates with thermolysin as an enzyme in an aqueous monophase system. A reversible Theorell-Chance mechanism is proposed to explain the synthesis, and its kinetic parameters were determined at pH 6.5 and 40 degrees C. The rate equation of the enzymatic reaction, which has never been solved with respect to the concentration of the product, was solved with effective approximations. The equation obtained was more practical than the general integrated form of rate equations, and the calculated results could satisfactorily explain the experimental results. The synthesis of aspartame precursor can thus be said to be based on the reversible Theorell-Chance mechanism. It is considered that this effective method of approximation can be applied to other enzymatic reactions.