Kinetics of enzymatic solid-to-solid peptide synthesis: Intersubstrate compound, substrate ratio, and mixing effects

A systematic study of thermolysin-catalyzed solid-to-solid peptide synthesis using Z-Gln and Leu-NH₂ as model substrates was carried out. The aim was to extend the kinetic knowledge of this new reaction system involving highly concentrated substrate mixtures with little water (10% to 20% w/w). Preheating of the substrates, and ultrasonic treatment, as described in the literature, had no significant effect on our system. The formation of a third compound, the salt of the two substrates, was discovered during melting point experiments. This was associated with a very strong dependence of kinetics on the exact substrate ratio (e.g., two-fold higher initial rate with 60% Leu-NH₂ and 40% Z-Gln than with the equimolar substrate ratio). A model was developed to show how the composition and pH of the liquid phase depends on the substrate ratio, and seemed to explain the experimental rates. In addition, the influences of different mixing and water distribution methods are described. Finally, we can now summarize the major effects of the reaction system as a starting point for further research and scale-up studies.