Synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

摘要

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, I168A, 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°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 μM, 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.