Purification and characterization of a dipeptidase from Lactobacillus helveticus SBT 2171

Abstract

A metal-dependent dipeptidase was purified to homogeneity from a cell extract of Lactobacillus helveticus SBT 2171 by fast protein liquid chromatography. The enzyme was purified 237-fold from the extract, with a yield of 1.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 50,000. The dipeptidase hydrolyzes a range of only dipeptides. Dipeptides containing proline, glutamic acid, and aspartic acid are not hydrolyzed. The enzyme was shown to be a metalloenzyme with a pH optimum of 8.0 and a temperature optimum of 55°C. Dithiol-reducing reagents exert strong inhibition on enzyme activity. Kinetic studies indicated that the enzyme has a relative average affinity for leucyl-leucine (K(m), 0.5 mM). The negative immunoresponse of the purified enzyme with monoclonal antibodies raised against a dipeptidase from Lactococcus lactis subsp. cremoris Wg2 shows that both enzymes can be immunologically distinguished.