Thermolysin is industrially used for the synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-L-aspartic acid (ZD) and L-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, 1168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009:145:103-13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 degrees C, the molecular activity k(cat) values of the variants were 1.6-3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K-m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 mu M, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis. (C) 2010 Elsevier Inc. All rights reserved.