Purification and Characterization of a Dipeptidase from Streptococcus cremoris Wg2

Abstract

A dipeptidase was purified to homogeneity from a crude cell extract of Streptococcus cremoris Wg2 by DEAE-Sephacel column chromatography followed by preparative disc gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 49,000. The dipeptidase is capable of hydrolyzing a range of dipeptides, but not peptides with longer chains. The enzyme was shown to be a metallo-Mn 2+ enzyme with a pH optimum of 8 and a temperature optimum of 50�C. The enzyme is strongly inhibited by thiol-reducing reagents but not by sulfhydryl reagents. Kinetic studies indicated that the enzyme has a relatively low affinity for leucyl-leucine and alanyl-alanine ( K m , 1.6 and 7.9 mM, respectively) but can hydrolyze these substrates at very high rates ( V max , 3,700 and 13,000 μmol/min per mg of protein, respectively).