N-benzyloxycarbonyl-L-aspartic acid ethyl ester-D-alanine amide, a derivative of alitame, was synthesized from a eutectic mixture of the substrates N-benzyloxycarbonyl-L-aspartic acid diethyl ester and D-alanine amide using cu-chymotrypsin. The hydrophilic solvents DMSO and MEA wen found to be the best adjuvants for formation of a eutectic substrate mixture. A low eutectic temperature of 7 degreesC was obtained for the substrate mixture containing 9% DMSO, 18% MEA, and 12% water. Under these conditions a conversion yield of 70.3% (mol/mol) was obtained at 37 degreesC. The optimum molar ratio of the acyl acceptor D-alanine amide and the acid donor N-benzyloxycarbonyl-L-aspartic acid diethyl ester was 1:1.