Isolation and characterization of an intracellular serine protease from Rhodococcus erythropolis

Abstract

An intracellular serine protease from the bacterium Rhodococcus erythropolis was partially purified and characterized. The enzyme, which is present in the 100,000 g supernatant fraction of disrupted cells, was purified 160-fold by ammonium sulfate fractionation, and chromatography on DEAE-cellulose, Sephadex G-150, hydroxyapatite, and benzamidine-glycylglycine-Sepharose. The molecular weight of the protease estimated by gel filtration was 82,000. The subunit molecular weight of the protein was estimated to be 90,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the enzyme appears to be a monomer. The purified enzyme preparation hydrolyzed several arginine-containing synthetic substrates; benzoyl-arginine-4-methyl-7-coumarylamide was routinely used as substrate. Hemoglobin, casein, and azocasein were also hydrolyzed. No carboxypeptidase activity was detected. The pH optimum of the enzyme was 7.0–7.2. The protease was not affected by inhibitors of cysteine proteases (iodoacetate), aspartic proteases (pepstatin), or metallo-proteases (EDTA). Inhibition was observed with the serine protease inhibitor diisopropylfluorophosphate. Several trypsin inhibitors (tosyl-lysine chloromethyl ketone, antipain, leupeptin, 4-aminobenzamidine, ovomucoid, and gramicidin S) inhibited the Rhodococcus protease, but others did not (phenylmethylsulfonylfluoride, lima bean trypsin inhibitor, aprotinin, α-1-antitrypsin). Salts with monovalent cations (e.g., NaCl, KCl) activated the protease two-to threefold at ionic strengths of 0.2 to 0.5. MgCl 2 stimulated activity fourfold at ionic strengths of 0.05 to 0.15; CaCl 2 stimulated activity (1.5-fold) maximally at an ionic strength of 0.05 and inhibited above 0.15.