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

Enzymatic peptide synthesis can be carried out efficiently in solid-to-solid reaction mixtures with 10% (w/w) water added to a mixture of substrates. The final reaction mass contains =80% (by weight) of product. This article deals with acid-base effects in such reaction mixtures and the consequences for the enzyme. In the Thermoase-catalyzed synthesis of Z-Asp-Phe-OMe, the reaction rate is strongly dependent on the amount of basic salts added to the system. The rate increases 20 times, as the KHCO₃ or K₂CO₃ added is raised 2.25-fold from an amount equimolar to the Phe-OMe · HCL starting material. With further increases in KHCO₃ addition, the initial rate remains at the maximum, but with K₂CO₃ it drops sharply. Addition of NaHCO₃ is less effective, but rates are faster if more water is used. With >1.5 equivalents of basic salt, the final yield of the reaction decreases. Similar effects are observed when thermolysin catalyzes the same reaction, or Z-Gln-Leu-NH₂ synthesis. These effects can be rationalized using a model estimating the pH of these systems, taking into account the possible formation of up to ten different solid phases. © 2001 John Wiley & Sons, Inc.