Characterization of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. lactis

Abstract

A dipeptidyl aminopeptidase catalysing hydrolysis of X-prolyl amidomethylcoumarin (AMC) substrates has been purified from Lactococcus lactis subsp. lactis H1. The active enzyme has a molecular mass of approximately 150 kDa, a subunit molecular mass of 82 to 83 kDa and is inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. The K m and k cat values for five different dipeptidyl AMC substrates (Gly-Pro-; Leu-Pro-; Lys-Pro-; Phe-Pro- and Glu-Pro-AMC) are similar except for the K m value for Glu-Pro-AMC, which is about threefold higher than that for the other substrates. The enzyme also catalyses hydrolysis of X-Ala-AMC substrates but with much lower k cat and higher K m values than the corresponding X-Pro-AMC substrates. The β -casein-derived heptapeptides Lys-Ala-Val-Pro-Tyr-Pro-Gln and Tyr-Pro-Phe-Pro-Gly-Pro-Ile were hydrolysed, but bradykinins with N-terminal sequences Arg-Pro-Pro- and Lys-Pro-Pro-were not. Dipeptidyl aminopeptidase specific activity is the same in a plasmid-free strain of L . lactis subsp. lactis H1 and in the wild-type, indicating that the enzyme is chromosomally encoded.