Purification and Characterization of a Dipeptidase from Lactobacillus delbrueckii subsp bulgaricus

A metal-dependent dipeptidase has been purified from a cell-free extract of Lactobacillus deibrueckii subsp, bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography, metal chelating affinity chromatography with immobilized Cu2+. and repeated FPLC anion exchange chromatography. The molecular mass of the purified enzyme was estimated to be 51 kD by SDS-polyacrylamide gel electrophoresis as well as by gel filtration, which indicates that it does not consist of subunits. The enzyme was most active at pH 7 and 50 degrees C. Reducing agents, like dithiothreitol and beta-mereaptoethanol. increased enzyme activity while metal chelating agents had an inhibitory effect. Enzyme activity, inhibited by EDTA and EGTA, could be partially restored by Co2+ and Mn2+. The enzyme was most active on dipeptides containing an aminoterminal hydrophobic amino acid such as Leu-Leu and Leu-Gly. Kinetic studies indicated that the dipeptidase had a higher affinity for the first substrate mentioned. The K-m-values for both substrates were about 0.56 and 1.23 mM, with turnover numbers of 870 and 480 s(-1), respectively.