Purification and characterization of the X-prolyl-dipeptidyl aminopeptidase (PepX) from Streptococcus macedonicus and cloning of the pepX gene

摘要

The X-prolyl-dipeptidyl aminopeptidase from Streptococcus macedonicus ACA-DC 191 was purified by anion exchange and hydrophobic interaction chromatography. A single band of a molecular mass of about 84 000 g.mol(-1) appeared in SDS-PAGE; by gel filtration it was shown that the native enzyme was dimeric. The enzyme showed optimum activity on glycyl-prolyl-4-nitroanilide at pH 7.0, with a K-M = 0.42 mmol.L-1 and a V-max = 12.8 mumol.mg(-1).min(-1). It was active over a temperature range of 10-60 degreesC. Over 60 degreesC, the enzyme activity declined rapidly. The peptidase was completely inactivated by PMSF, DTNB and Cu2+, while metal chelators had no effect on enzyme activity. By using the PCR technique with synthetic primers, the pepX gene was amplified, cloned and sequenced. This 2 289 nucleotide gene encodes a protein of 763 amino acids with a molecular mass of 86 866 g.mol(-1). The deduced amino acid sequence analysis of the pepX gene shows a high identity with PepX enzymes from other lactic acid bacteria and contains a motif around the active site serine (G-K-S-Y-L-G) that is well conserved among the PepX enzymes.