Kinetics of Alcalase-catalyzed dipeptide synthesis in near-anhydrous organic media

The coupling kinetics of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran were investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads; these immobilized enzymes were hydrated prior to use. Near-anhydrous conditions (i.e. extremely low water activity) were maintained by a carefully chosen amount of molecular sieve powder. Kinetic characteristics were determined from reaction time courses up to full conversion at various initial concentrations of substrate and product. These progress curve data were fitted with different kinetic models to determine which of these models best approximates the kinetic properties of the immobilized Alcalase with respect to the coupling under study. An appropriate model of the coupling reaction is necessary in order to design a reactor and predict its performance adequately leading to the practical implementation of biocatalyic peptide synthesis. The kinetics of the coupling were found to be complex and to obey a two-substrate kinetic model with two competitive product inhibition terms. To reduce the effect of the product inhibition on immobilized Alcalase, a reactor should be designed in which at least glycolamide is selectively removed as this was found to be the strongest inhibitor.

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► Coupling kinetics catalyzed by covalently immobilized Alcalase (Cov) were studied.
► The reactions were performed in tetrahydrofuran in the presence of molecular sieves.
► An 2-substrate kinetic model with 2 competitive product inhibition terms was found.
► To minimize product inhibition on Cov, glycolamide should be selectively removed.