Xaa-Pro-dipeptidyl-aminopeptidase from Lactococcus lactis catalyses kinetically controlled synthesis of peptide bonds involving proline.

Xaa-Pro-dipeptidyl-aminopeptidase (EC 3.4.14.5) from Lactococcus lactis (PepX) was used, for the first time, as a catalyst in kinetically controlled synthesis of peptide bonds involving proline. PepX had amidase and esterase activities in addition to peptidase activity. Thus amide and ester derivatives of X-Pro peptides could be employed as acyl donors. PepX showed a broad specificity for the residue in position P'1, accepting a large variety of amino acid amides, esters, peptides as well as free amino acids as nucleophiles. This also indicated that it was not necessary to protect the C-terminus of the nucleophile. The major factors controlling yield, e.g. pH, an excess of nucleophile, ionic strength and type of carboxyl protecting and activating groups, were evaluated. Under optimum reaction conditions (pH 8.5, high excess of nucleophile over acyl donor and moderate ionic strength) the selectivity of the reaction ranged from 5 to 99% depending on the structure of the nucleophile and the acyl donor. Our work contributes to the elucidatation of the mechanism of aminolysis reactions catalysed by an aminopeptidase.