Performance of Alcalase formulations in near dry organic media: Effect of enzyme hydration on dipeptide synthesis

The use of different Alcalase formulations for protease-catalyzed dipeptide synthesis was investigated by studying the coupling of the carbamoylmethyl ester of N-protected phenylalanine with phenylalanine amide in tetrahydrofuran in the presence of molecular sieves (i.e. under near dry conditions). Hydration prior to drying (with anhydrous tert-butanol and anhydrous tetrahydrofuran) of the Alcalase formulations resulted in a significant increase in rate of the subsequent dipeptide synthesis. Repeated use, in the presence of molecular sieves, without intermediate rehydration led to inactivation of the enzyme. For three enzyme formulations this inactivation could be counteracted by intermediate rehydration. Inactivation of another enzyme formulation was only partially reversible by hydration. Alcalase immobilized onto dicalite with glutaraldehyde was found to be the most active in dipeptide synthesis, i.e. the formulation that initially produces the largest amount of product per gram of total formulation per unit of time. Due to its small particle size and its lack of operational stability, this formulation may nevertheless not be the best choice for the synthesis of dipeptides in neat organic media on a large scale. The most promising enzyme formulation for this is Alcalase covalently immobilized onto macroporous acrylic beads due to its reasonable activity, its seemingly good operational stability, and its size and uniform shape.