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 synthesi s 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 highl y concentrated substrate mixtures with little water (10% to 20% w/w). Prehe ating of the substrates, and ultrasonic treatment, as described in the lite rature, had no significant effect on our system. The formation of a third c ompound, the salt of the two substrates, was discovered during melting poin t experiments. This was associated with a very strong dependence of kinetic s on the exact substrate ratio (e.g., twofold 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 o n the substrate ratio, and seemed to explain the experimental rates. In add ition, the influences of different mixing and water distribution methods ar e described. Finally, we can now summarize the major effects of the reactio n system as a starting point for further research and scale-up studies. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 316-321, 1999.