I. Isolation and Characterization of Four Proteases from Clostridium Botulinum type A.

Abstract

Four proteases (designated Proteases I, II, III, and IV) were obtained from Clostridium Botulinum type A. Purification steps were precipitation with ammonium sulfate, DEAE-Sephadex A-25,Sephadex G-75 and affinity column chromatography. The affinity column was prepared from CNBr-activated Sepharose 4B gel by coupling with L-arginine methyl ester (AME). The molecular weights of these enzymes were estimated to be 40,000 (protease I), 25,000 (protease II), 31,000 (protease III) and 20,000 (protease IV) by gel filtration and by SDS-polyacrylamide gel electrophoresis. The activities for these proteases were inhibited by Cu^<++>, Sn^<++>, EDTA and PCMB. The activities of four enzymes were activated by Ca^<++>. Proteases III and IV showed similar chracteristic for metal ions and inhibitors and they were activated by Mg^<++>, cysteine, DFP and PMSF. Optimum pH for casein hydrolysis was 8.0 for protease I and 8.5 for proteases II, III and IV. Proteases II and III were stable at 40 C for 60 min but proteases I and IV did not. A number of previous reports (2,3,6,9,12,13) have documented the isolation and characterization of Clostridium botulinum proteases (Inukai. 1963,DasGupta. 1971,DasGupta & Sugiyama. 1973,Ohishi. 1977,Nakane. 1978). Inukai et al (6) isolated three proteases from Clostridium botulinum type A. Tjaberg (12,13) isolated two protease produced by C. botulinum type A. Nakane (9) isolated four enzymes from a proteolytic mutant of C. botulinum type E. We attempted the isolation of four proteolytic enzymes (designated Proteases I, II, III and IV) and an esterolytic enzyme (designated Protease V) from C. botulinum type A. In this study, we report the isolation and characterization of four proteolytic enzymes, which differes from the previous findings.