PURIFICATION AND CHARACTERIZATION OF X-PROLYL DIPEPTIDYL PEPTIDASE FROM LACTOBACILLUS-CASEI SUBSP CASEI LLG

X-Prolyl dipeptidyl peptidase, which hydrolysed X-Pro-Y almost specifi cally, has been purified to homogeneity from crude cell-free extracts of Lactobacillus casei subsp. casei LLG using fast protein liquid chro matography equipped with preparative and analytical anion exchange col umns. The enzyme was purified to 274-fold by ammonium sulphate fractio nation, and by two successive ion-exchange chromatographies with a rec overy of 34%. The purified enzyme appeared as a single band on both na tive-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulp hate (SDS)-PAGE and had a molecular mass of 79 kDa. The pH and the tem perature optima by the purified enzyme were 7.0 and 50 degrees C, resp ectively. X-PDP was a serine-dependent enzyme, as both diisopropylfluo rophosphate and phenylmethylsulphonylfluoride caused complete inhibiti on of the enzyme activity. The Michaelis constant (K-m) and maximum re action velocity (V-max) values were 0.2 mM and 43 mM per milligram, re spectively.