II. Isolation and Some properties of Protease V from Clostridium Botulinum type A.

Abstract

A protease (Protease V ) with esterase activity for α-N-benzoyl-L-arginine ethyl ester (BAEE) was purified from Clostridium botulinum type A strain 190 by ammonium sulfate fraction, DEAE-Sephadex A-25,affinity chromatography and Sephadex G-50. The affinity column was prepared from Sepharose 4B gel by coupling with L-arginine methyl ester (AME). Protease V showed the greatest affinity for column at pH 7.0 and was eluted by the concentration of salt. The enzyme had a molecular weight of 67,000 as found by Sephadex G-75 gel filtration and by SDS-polyacrylamide gel electrophoresis. The enzyme had both activities for esterase and for amidase. The optlmum pH of esterase activity for BAEE and of amidase activity for α-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) was 7.0. The enzyme required dithiothreitol (DTT) for esterase activity and was activated by addition of I × l0^<-3>M Ca^&lt : ++>, Sn^<++> , Mg^<++> and K^+ . Cysteine, Cu^<++> , Fe^<++> and PCMB reduced the esterase activity by 40〜50%, but DFP did not. Several proteases have been isolated from cultures of C. botulinum (DasGupta, 1971,DasGupta & Sugiyama, 1972,Tjaberg, 1973,Ohishi, 1977,Nakane, 1978). DasGupta & Sugiyama (2,3) isolated a trypsin-like enzyme produced by C. botulinum type B. Ohishi et al (10) purified a sulfydryl-dependent protease (SHP) from a proteolytic strain of C. botulinum type F. Nakane (8) isolated four proteases (proteases I , II , III , and IV ) from a proteolytic mutant of C. botulinum type E. One of them, protease II hydrolyzed α-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and BAEE but not azocasein. The present report describes an isolation and characterization of a protease with esterase activity (designated protease V ) from C. botulinum type A.