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

Abstract

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., 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 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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 316–321, 1999.

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…As noted in the Introduction, in our previous study of Z-Gln-Leu-NH 2 synthesis by thermolysin, we attributed the increased rates with excess of Leu-NH 2 to an increase in the pH of the liquid phase (Erbeldinger et al, 1999). Therefore, we also investigated the addition of KHCO 3 in this reaction, in this case starting with the hydrochloride of Leu-NH 2 .…”

“…Lines show trend. based on substrate ratio (Erbeldinger et al, 1999). In the current system with Z-Asp and Phe-OMe и HCl as substrates, the pH of the liquid phase saturated with substrates must be very low without base addition, leading to very poor thermolysin activity.…”

“…We start from the indication that an important effect of unequal substrate amounts is to control the pH or acid-base conditions in the medium, and hence enzymatic activity (Erbeldinger et al, 1999). Relatively low quantities of buffers added in some studies have little effect, because the high substrate concentrations dominate the acid-base balance.…”

“…Effect of unequal substrate ratio of Z-Gln and Leu-NH 2 on initial rate (᭺) as shown inErbeldinger et al (1999) (total quantity of substrates 0.2 mmol, 0.5 mg Thermolysin, and 10 L H 2 O per sample). Results at least in duplicate.…”

Modeling the Kinetics of Enzymic Reactions in Mainly Solid Reaction Mixtures

“…In these unstirred reaction mixtures, mass transfer will be more likely to be rate limiting, although there may be cases in which surface processes are important. (Indeed, there is experimental evidence that nucleation can limit overall kinetics in the early stages of an enzymic reaction in such systems; Erbeldinger et al (5)).…”