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Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture

¹ Faculty of Medicine, Kagawa University;
² Kagawa Prefectural College of Health Sciences

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38-kDa) and light (16-kDa or 15-kDa) chains, and that the two light chains differ in the N-terminal cleavage site. Such a difference in the light chain did not affect the enzymatic activity toward N-benzoyl-L-arginine p-nitroanilide (Bz-L-arginine pNA), as demonstrated on assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp degraded Bz-DL-arginine pNA more preferentially than Bz-DL-lysine pNA, it degraded the latter more efficiently than Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in the substrate specificity. Clp caused an increase in vascular permeability when injected intradermally in mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.

³ E-mail: microbio{at}med.kagawa-u.ac.jp