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.
Laboratoire de Génétique Moléculaire des Microorganismes, URA CNRS 1486, Villeurbanne, France.
T
T
Gonzalès
A
A
Awadé
C
C
Besson
J
J
Robert-Baudouy
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