Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the Enzyme. The Enzyme was purified from lysozyme-lysate of the Bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the Enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The Enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the Enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the Enzyme is a subunit oligomer, presumably trimer. The Enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified Enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various Peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.