Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

Abstract

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl- l-aspartic acid with l-phenylalanine methyl ester to form N-benzyloxycarbonyl- l-aspartyl- l-phenylalanine methyl ester (Z · Asp · Phe · OMe), the precursor to the sweet-tasting compound l-aspartyl- l-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl- l-glycyl- l-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z · Asp · Phe · OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.