Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.
Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.
Gonzalès
T
T
Laboratoire de Génétique Moléculaire des Microorganismes, URA CNRS 1486, Villeurbanne, France.
Awadé
A
A
Besson
C
C
Robert-Baudouy
J
J
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002384:Catalysis; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D011751:Pyroglutamyl-Peptidase I / Q000096:biosynthesis / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013297:Streptococcus pyogenes / Q000276:immunology
D011994:Recombinant Proteins; D011751:Pyroglutamyl-Peptidase I; D000645:Ammonium Sulfate