Purification and characterization of Bacillus subtilis JM-3 protease from anchovy sauce

Bacillus subtilis JM-3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 +/- 3C for 3 years. The activity of the B. subtilis protease was highest in the 40-60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K-m and V(max)values were 1.75 mu g/mL and 318 mu M 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM-3 protease was classified as an acid protease. The relative activities of the B. subtilis JM-3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM-3 protease was benzyloxycarbonyl-glycine-p-nitrophenyl ester followed by bovine serum albumin. p-Toluene-sulfonyl-L-lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N-ethylmaleimide did not inhibit this enzyme. The B. subtilis JM-3 protease was therefore presumed to be a trypsin-like serine protease.