Selecting optimal conditions for Alcalase CLEA-OM for synthesis of dipeptides in organic media

In protease-catalyzed peptide synthesis, the availability of water is essential, as a compromise must be made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions. We studied the coupling of the carbamoylmethyl ester of N-protected phenylalanine and phenylalanine amide in tetrahydrofuran catalyzed by Alcalase CLEA-OM at a range of water activity (aw) values, including the coupling in the presence of molecular sieves (i.e. at very low aw values). The hydrolytic side reaction (in the present system only the hydrolysis of substrate occurs) was found to dominate above an aw value of about 0.2. To prevent hydrolysis, the presence of molecular sieves was found to be necessary.

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► Increasing aw promotes dipeptide synthesis, but also – and more strongly – substrate hydrolysis.
► The pH of washing buffer has an effect on the dipeptide synthesis catalyzed by Alcalase CLEA-OM.
► The reaction pH neither affects dipeptide synthesis nor substrate hydrolysis.
► Optimal amounts of molecular sieves prevent substrate hydrolysis but allow enzymatic activity.