Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis. |
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Authors: | T Gonzalès A Awadé C Besson J Robert-Baudouy |
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Affiliation: | Laboratoire de Génétique Moléculaire des Microorganismes, URA CNRS 1486, Villeurbanne, France. |
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Abstract: | 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. |
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