Purification and characterization of X-prolyl dipeptidyl peptidase from Lactobacillus casei subsp. casei LLG.

X-Prolyl dipeptidyl peptidase, which hydrolysed X-Pro-Y almost specifically, has been purified to homogeneity from crude cell-free extracts of Lactobacillus casei subsp. casei LLG using fast protein liquid chromatography equipped with preparative and analytical anion exchange columns. The enzyme was purified to 274-fold by ammonium sulphate fractionation, and by two successive ion-exchange chromatographies with a recovery of 34%. The purified enzyme appeared as a single band on both native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate (SDS)-PAGE and had a molecular mass of 79 kDa. The pH and the temperature optima by the purified enzyme were 7.0 and 50 degrees C, respectively. X-PDP was a serine-dependent enzyme, as both diisopropylfluorophosphate and phenylmethylsulphonylfluoride caused complete inhibition of the enzyme activity. The Michaelis constant (Km) and maximum reaction velocity (Vmax) values were 0.2 mM and 43 mM per milligram, respectively.