PepX from Lactobacillus helveticus: Automated multi-step purification and determination of kinetic parameters with original tripeptide substrates

Abstract A X-prolyl-dipeptidyl aminopeptidase (PepX, EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 was automatically purified to homogeneity after cultivation in MRS broth. The purification of PepX lasted only 6 h and the PepX activity yield was 56%. The specific activity was determined at 370 nkat H-Ala-Pro- p NA  mg −1 and the purification factor was 162-fold. A gas chromatographic assay was established for the determination of the PepX activity using original tripeptide substrates. The kinetic parameters of purified PepX were analyzed using different original tripeptide substrates with the structure Xxx-Pro-Yyy. Alanine at position P 2 (Xxx) or P 1 ′ (Yyy) was retained, while the corresponding amino acid at P 1 ′ or P 2 (serine, tyrosine, aspartic acid, or arginine) was varied. We analyzed the influence of the position of the different amino acids (P 2 or P 1 ′) and the side chain group (serine: polar and uncharged; tyrosine: hydrophobic; aspartic acid: negatively charged; arginine: positively charged) on the PepX activity and the kinetic parameters. Significant differences were observed by comparing the kinetic parameters of PepX using original tripeptides as substrates with the chromogenic peptide H-Ala-Pro- p NA as a substrate; for instance, a K m -value of 0.54 mM and a V max -value of 4.76 nkat mL −1 were determined using the tripeptide Ser-Pro-Ala as a substrate. The kinetic parameters for the chromogenic peptide H-Ala-Pro- p NA were 1.53 mM and 2.61 nkat mL −1 for K m and V max , respectively.